identifier
stringlengths 11
32
| pdf_url
stringlengths 17
4.62k
⌀ | lang
stringclasses 120
values | error
stringclasses 1
value | title
stringlengths 2
500
⌀ | source_name
stringlengths 1
435
⌀ | publication_year
float64 1.9k
2.02k
| license
stringclasses 3
values | word_count
int64 0
1.64M
| text
stringlengths 1
9.75M
|
|---|---|---|---|---|---|---|---|---|---|
https://openalex.org/W2077795443
|
https://escholarship.org/content/qt93j0d05v/qt93j0d05v.pdf?t=n4fgx4
|
English
| null |
Sensitivity of tracer transports and stratospheric ozone to sea surface temperature patterns in the doubled CO<sub>2</sub> climate
|
Journal of geophysical research
| 2,002
|
cc-by
| 12,533
|
Peer reviewed Peer reviewed UC Irvine
Faculty Publications Copyright Information
This work is made available under the terms of a Creative Commons Attribution License,
availalbe at https://creativecommons.org/licenses/by/4.0/ UC Irvine
Faculty Publications
Title
Sensitivity of tracer transports and stratospheric ozone to sea surface temperature
patterns in the doubled CO
2
climate
Permalink
https://escholarship.org/uc/item/93j0d05v
Journal
Journal of Geophysical Research, 107(D24)
ISSN
0148-0227
Author
Rind, David
Publication Date
2002
DOI
10.1029/2002JD002483
Copyright Information
This work is made available under the terms of a Creative Commons Attribution License
availalbe at https://creativecommons.org/licenses/by/4.0/
Peer reviewed UC Irvine
Faculty Publications
Title
Sensitivity of tracer transports and stratospheric ozone to sea surface temperature
patterns in the doubled CO
2
climate
Permalink
https://escholarship.org/uc/item/93j0d05v
Journal
Journal of Geophysical Research, 107(D24)
ISSN
0148-0227
Author
Rind, David
Publication Date
2002
DOI
10.1029/2002JD002483
Copyright Information
This work is made available under the terms of a Creative Commons Attribution License
availalbe at https://creativecommons.org/licenses/by/4.0/
Peer reviewed UC Irvine
Faculty Publications
Title
Sensitivity of tracer transports and stratospheric ozone to sea surface temperature
patterns in the doubled CO
2
climate
Permalink
https://escholarship.org/uc/item/93j0d05v
Journal
Journal of Geophysical Research, 107(D24)
ISSN
0148-0227
Author
Rind, David
Publication Date
2002
DOI
10.1029/2002JD002483
Copyright Information
This work is made available under the terms of a Creative Commons Attribution Lice
availalbe at https://creativecommons.org/licenses/by/4.0/
Peer reviewed UC Irvine
Faculty Publications
Title
Sensitivity of tracer transports and stratospheric ozone to sea surface temperature
patterns in the doubled CO
2
climate
Permalink
https://escholarship.org/uc/item/93j0d05v
Journal
Journal of Geophysical Research, 107(D24)
ISSN
0148-0227
Author
Rind, David
Publication Date
2002
DOI
10.1029/2002JD002483
Copyright Information
This work is made available under the terms of a Creative Commons Attribution Lice
availalbe at https://creativecommons.org/licenses/by/4.0/
Peer reviewed Michael Prather Earth Systems Science Department, University of California, Irvine, Irvine, California, USA Received 26 April 2002; revised 30 July 2002; accepted 1 August 2002; published 28 December 2002. [1]
Two sets of sea surface temperature/sea ice changes are used to test the sensitivity of
tracer transport to the pattern of warming in the doubled CO2 climate. One set (2CO2WT)
has greater tropical and high latitude sea surface temperature changes than the other
(2CO2), although both fall within the range of plausible response. Simulations were done
both with and without interactive ozone. Results show that the SST pattern affects the
circulation change throughout the troposphere and middle atmosphere; the ozone
interaction affects primarily the upper stratosphere, but through wave-mean flow
interaction has effects that extend down into the upper troposphere. Both experiments
feature increased tropospheric/stratospheric exchange at low latitudes and greater vertical
mixing within the troposphere; only the WT experiments result in increased interhemispheric transport and a more direct circulation in the high latitude stratosphere. Ozone increases in the upper stratosphere and decreases in the lower stratosphere in all the
simulations, with greater transport of high latitude ozone into the troposphere in the WT
runs. At sea level there is a more positive phase of the Arctic Oscillation (AO)-type
oscillation, and this is also true at 100 mbar, but there is no significance in the middle
troposphere and the sign is different in the middle stratosphere. Many of these results
differ from those generated in older versions of the GISS GCMAM despite the same SST
forcing due to differences in control run characteristics, which has implications for model
intercomparison experiments. INDEX TERMS: 0341 Atmospheric Composition and Structure: p
p
Middle atmosphere—constituent transport and chemistry (3334); 0368 Atmospheric Composition and
Structure: Troposphere—constituent transport and chemistry; 1620 Global Change: Climate dynamics (3309);
3334 Meteorology and Atmospheric Dynamics: Middle atmosphere dynamics (0341, 0342); KEYWORDS:
tracer transports and climate, stratosphere and climate change, climate change and ozone, future SSTs and
tracer transports Citation:
Rind, D., J. Lerner, J. Perwitz, C. McLinden, and M. Prather, Sensitivity of tracer transports and stratospheric ozone to sea
surface temperature patterns in the doubled CO2 climate, J. Geophys. Res., 107(D24), 4800, doi:10.1029/2002JD002483, 2002. Copyright 2002 by the American Geophysical Union.
0148-0227/02/2002JD002483 Sensitivity of tracer transports and stratospheric ozone to sea surface
temperature patterns in the doubled CO2 climate David Rind, Jean Lerner, and Judith Perlwitz
Goddard Institute for Space Studies at Columbia University, New York, New York, USA David Rind, Jean Lerner, and Judith Perlwitz
Goddard Institute for Space Studies at Columbia University, New York, New York, USA David Rind, Jean Lerner, and Judith Perlwitz
Goddard Institute for Space Studies at Columbia University, New York, New York, USA Chris McLinden
Air Quality Research Branch, Meteorological Service of Canada, Downsview, Ontario, Canada Chris McLinden
Air Quality Research Branch, Meteorological Service of Canada, Downsview, Ontario, Canada Powered by the California Digital Library
University of California Powered by the California Digital Library
University of California eScholarship.org JOURNAL OF GEOPHYSICAL RESEARCH, VOL. 107, NO. D24, 4800, doi:10.1029/2002JD002483, 200 2.
Model and Experiments These limitations are removed here, as we use two of the sea
surface temperature/sea ice changes in the same (newer)
version of the model with on-line tracers, and compare their
effects directly. [4] These comparisons were based on the overt meteoro-
logical responses, and they did not explicitly calculate what
the effect would be on the tracer transports themselves. Furthermore, the latest experiment had used a newer version
of the GISS Global Climate/Middle Atmosphere Model
(GCMAM), so model dependence could not be separated
from the effect of different sea surface temperature patterns. These limitations are removed here, as we use two of the sea
surface temperature/sea ice changes in the same (newer)
version of the model with on-line tracers, and compare their
effects directly. [5] To the extent that altered depictions of tropospheric
climate change produce different dynamical responses
extending into the stratosphere, the effect of the doubled
CO2 climate on stratospheric ozone and its transport into
the troposphere might also be different. Our previous
investigations of the response of ozone to increased CO2
[Rind et al., 1998; Shindell et al., 1998] all used the older
version of the GCMAM [Rind et al., 1988]; how would it
differ with the newer model, and how would it depend on
the sea surface temperature changes? Furthermore, the
older approach did not allow the ozone changes to be
advected, a limitation which precluded investigation of
changes in stratospheric/tropospheric transport. These
questions are addressed briefly in this paper, and more
extensively in a companion paper (C.A. McLinden et al.,
manuscript in preparation, 2002) as the linearized ozone
photochemistry scheme (LINOZ) [McLinden et al., 2000],
including online transports, is incorporated into these
experiments. [9] Although the second-order moments advection
scheme [Prather, 1986] captures finer resolution effects
than would be apparent from the given 4 5 horizontal
resolution, this coarse grid is not likely to capture the
physical intricacies of stratosphere/troposphere exchange
associated with fine-scale tropopause folds. Overall, the
troposphere/stratospheric exchange is similar in this model
to the previous 4 5 versions of the stratospheric model,
which were shown to be produce generally realistic magni-
tudes of transport [Rind et al., 1999]. The model’s large-
scale and eddy processes appear to be producing overall [6] Finally, the resulting changes in ozone distributions
themselves might alter atmospheric dynamics. That issue is
investigated by running the doubled CO2/LINOZ simula-
tions in two ways. 1.
Introduction used in that experiment, the subtropical residual circulation
intensified in the upper troposphere/lower stratosphere,
increasing the rate of tropical transfer of tropospheric air
mass and constituents into the stratosphere by about 30%. Transport from the stratosphere to troposphere at higher
latitudes also increased as the high latitude residual circu-
lation intensified, so that the stratospheric residence time of
bomb 14C decreased by about 11%. Within the troposphere,
interhemispheric transport was reduced by 5% due to
decreased Hadley Cell intensity; greater poleward transport
occurred within the northern hemisphere due to eddy/Ferrel
Cell changes; and greater vertical transport took place from
low levels to the middle and upper troposphere via
increased penetrating and overall convective mass fluxes. [2] The possibility of increased atmospheric CO2 affect-
ing atmospheric dynamics has many ramifications, includ-
ing effects such as changes in the phase of the Arctic
Oscillation (AO) [e.g., Shindell et al., 1999; Fyfe et al.,
1999; Monahan et al., 2000; Perlwitz et al., 2000], and
altered distributions of tropospheric and stratospheric trace
gases. In a recent publication [Rind et al., 2001], we
investigated the impact of the doubled CO2 climate on
various tracer transports. In the doubled CO2 climate change 25 - 1 25 - 1 ACL RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL
25 - 2 In
the discussion (section 7) we comment on which results
depended on the SST field, and which were affected by the
stratospheric ozone changes; we also include a discussion of
potential inter-model differences by comparison to the
results given by Rind et al. [1998, 2001]. A summary of
the main results is given in the conclusions (section 8). ACL
25 - 2 [3] In that publication, and in work by Rind et al. [2002],
the generality of these doubled CO2 results were assessed
by comparing the meteorological changes in the latest
simulation to those generated with different sea surface
temperature changes [Rind et al., 1990, 1998]. The two
effects that were the most robust were the increase in the
subtropical residual circulation, and the increase in pene-
trating convection to the upper troposphere, although the
magnitude of their increase depended on the degree of
tropical warming. Increases in transport from the surface
to the middle troposphere depended on the convection
parameterization, while changes in the Hadley circulation
and the residual circulation in the polar stratosphere
depended on the latitudinal distributions of sea surface
temperature change. Hadley Cell changes with increasing
CO2 have been known to vary from model to model (e.g.,
Dai et al. [2001] with the NCAR CCM found the Hadley
Cell weakened by 10% by 2090, Ramstein et al. [1998] with
the LMD model also found a 10% weakening with doubled
CO2, while Senior [1995] found an increased Hadley cell
with the Hadley Centre model run at 250 km resolution, and
no change at 500 km resolution). atmospheric and tracer transport response depend on the
altered sea surface temperatures, and which are affected by
the ozone response. p
[7] Following the introduction and model/experiment
description (section 2), the different foci of this paper are
presented sequentially: meteorological effects of the differ-
ent SSTs (section 3), the effect on tracer transports (section
4), and on ozone (section 5), followed by the dynamical
influence of the interactive ozone response (section 6). In
the discussion (section 7) we comment on which results
depended on the SST field, and which were affected by the
stratospheric ozone changes; we also include a discussion of
potential inter-model differences by comparison to the
results given by Rind et al. [1998, 2001]. A summary of
the main results is given in the conclusions (section 8). [7] Following the introduction and model/experiment
description (section 2), the different foci of this paper are
presented sequentially: meteorological effects of the differ-
ent SSTs (section 3), the effect on tracer transports (section
4), and on ozone (section 5), followed by the dynamical
influence of the interactive ozone response (section 6). 2.
Model and Experiments [8] The model used for these simulations is the GISS
GCMAM, model II0 with some alterations. In previous
studies, several specific model weaknesses were identified
with the previous 31-layer version of the model [Douglass
et al., 1999; Pawson et al., 2000]. Chief among these was a
weaker than observed subtropical barrier to tracer transport
in the stratosphere, insufficient vertical resolution in the
lower stratosphere, reduced stratospheric winter variability,
and reduced vertical transport to the upper stratosphere. In
comparison to the previous version [e.g., Pawson et al.,
2000], the model now has 53 layers in the vertical, in
conjunction with 4 5 resolution. The increased vertical
layering provides for 500 m resolution in the middle to
upper troposphere, 0.5 to 1 km resolution in the lower
stratosphere, sufficient to resolve important gradients in that
region [Douglass et al., 1999], and 2 to 2.5 km in the upper
stratosphere. This layering can be compared with the 1.5 to
2 km resolution between 2 and 20 km used in the previous,
31-layer version of the model. The fourth order momentum
scheme [Rind and Lerner, 1996] used in the 31-layer model
was found to produce a tendency for spurious polar east-
erlies in the upper troposphere, which had the effect of
limiting interannual variability, and also altering planetary
wave refraction so as to reduce the stratospheric jet inten-
sity. It is replaced in this model by a second order momen-
tum scheme, which cured both of these problems. These
changes also ameliorated the leakiness of the subtropical
barrier, and the smaller than observed transport into the
upper stratosphere. However, the problems still remain to
some extent and may quantitatively affect the results; the
mean age of air at 20 km varies from 1.5 years in the tropics
to 3.5 years at 60N, compared with 1 year in the tropics to
4 years at 60N in observations [Andrews et al., 2001]. [4] These comparisons were based on the overt meteoro-
logical responses, and they did not explicitly calculate what
the effect would be on the tracer transports themselves. Furthermore, the latest experiment had used a newer version
of the GISS Global Climate/Middle Atmosphere Model
(GCMAM), so model dependence could not be separated
from the effect of different sea surface temperature patterns. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL
25 - 2 ACL
25 - 2 ACL
25 - 3 replicas of real-world processes without all the details; the
climate change effect of such differences can only be
gauged by future experiments with finer resolution models. [10] For the doubled CO2 experiments, we continue the
approach used by Rind et al. [2001] and input altered sea
surface temperature/sea ice fields from previous equilibrium
experiments. The atmospheric CO2 is doubled as well. The
first set of experiments (called ‘‘2 CO2’’) uses the same
doubled CO2 changes in sea surface temperatures as given
by Rind et al. [2001], which were originally generated by a
nine-layer version of the GISS model II0. The control runs
and the experiments are run both with and without the
ozone changes interacting with the radiation field (see
Table 1). The second set of experiments uses the sea surface
temperatures/sea ice field generated by an equilibrium run
of the older version of the GISS GCMAM [Rind et al.,
1998]. In both cases, fixed dynamical ocean transport in
conjunction with a mixed layer ocean (a ‘‘q-flux ocean’’)
was used in the original simulations to allow the sea surface
temperatures to change. ACL
25 - 3 ACL
25 - 3 Table 1. Simulations Used in This Study
SSTs/Sea Ice
Ozone
Control
current
noninteractive
Control (I)
current
interactive
2CO2
Rind et al. [2001]
noninteractive
2CO2 (I)
Rind et al. [2001]
interactive
2CO2WT
Rind et al. [1998]
noninteractive
2CO2WT(I)
Rind et al. [1998]
interactive 2CO2WT is slightly higher than the IPCC nominal range of
1.5–4.5C (as shown in Table 2). [13] In addition to ozone, the other online tracers used
were those discussed by Rind et al. [2001]: CO2, SF6,
Rn222, CFC-11, CH4, N2O, and bomb 14C. The sources/
sinks of each of these tracers is given by Rind et al. [2001];
the stratospheric photochemical sinks for CFC-11, CH4 and
N2O were calculated in a consistent manner with the
LINOZ photochemistry. [14] The control runs and experiments were integrated for
12 years with results reported for 10 year averages. In
addition, we investigated whether the differences seen
during five years in the first half of the simulations were
similar to those during the last half, and discuss only those
which had consistent changes for the two time periods. A
complete list of the control runs and doubled CO2 simu-
lations is given in Table 1. replicas of real-world processes without all the details; the
climate change effect of such differences can only be
gauged by future experiments with finer resolution models. [10] For the doubled CO2 experiments, we continue the
approach used by Rind et al. [2001] and input altered sea
surface temperature/sea ice fields from previous equilibrium
experiments. The atmospheric CO2 is doubled as well. The
first set of experiments (called ‘‘2 CO2’’) uses the same
doubled CO2 changes in sea surface temperatures as given
by Rind et al. [2001], which were originally generated by a
nine-layer version of the GISS model II0. The control runs
and the experiments are run both with and without the
ozone changes interacting with the radiation field (see
Table 1). The second set of experiments uses the sea surface
temperatures/sea ice field generated by an equilibrium run
of the older version of the GISS GCMAM [Rind et al.,
1998]. In both cases, fixed dynamical ocean transport in
conjunction with a mixed layer ocean (a ‘‘q-flux ocean’’)
was used in the original simulations to allow the sea surface
temperatures to change. 2.
Model and Experiments In the first, the model is run with a
climatological ozone field for atmospheric radiation purpo-
ses, and in those experiments the on-line ozone is a passive
tracer. In the second, we substitute the LINOZ-produced
ozone values in the radiation routine, so the doubled CO2
ozone changes interact with the climate. The complete set of
experiments allows us to separate which features of the RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL
25 - 3 3.
Meteorological Effects of the Different Sea
Surface Temperature Patterns [15] The different sets of sea surface temperatures help
generate different meteorological responses. We concentrate
on those phenomena deemed responsible for changes in
transports, and the dynamics discussion is focused on the
Northern Hemisphere. To indicate the robustness of the
conclusions, and/or the impact of the interactive ozone, we Figure 1a. The marine surface temperature (input sea
surface temperature plus calculated sea ice temperature)
anomaly in the two experiments ((top) 2CO2 and (bottom)
2CO2WT). [11] The marine surface (sea surface plus sea ice) temper-
ature differences for the individual experiments are shown
in Figure 1, with the resulting surface air temperature
changes (for the noninteractive experiments) shown in
Figure 1b. Since the sea surface temperatures from Rind
et al. [1998] featured a warmer tropical temperature
response, experiments which used those values are called
2CO2 WT (for warmer tropics), although the runs also
feature warmer high latitude temperatures. g
p
[12] Which SST pattern is more realistic? The differences
are largely associated with a change in the cloud cover
parameterization and cloud feedbacks [Rind et al., 2002]. The tropical warming in the two simulations in general
varies between 2.5 and 4C (Figure 1a). Yao and Del Genio
[1999] show that such variations can be generated by
differences in the parameterized cloud feedback within the
range of current uncertainty. The smallest response occurs
when thick anvil clouds arise in association with tropical
convection (reducing solar insolation reaching the surface)
and no cloud optical thickness feedback is allowed. The
greatest warming occurs with the reverse situation: Optical
thickness is predicted (and decreases in the tropics as
climate warms due to water depletion by precipitation),
while anvil clouds are not included. Yao and Del Genio
[1999] argue that the optical thickness feedback is sup-
ported by observations, while the realism of the anvil
detrainment feedback is more difficult to assess. That would
suggest that a higher tropical sensitivity might be more
realistic if one had to choose between the two, although
numerous processes (including other cloud parameteriza-
tions) affect the overall and tropical sensitivity. At this point
no conclusion can be definitive. The overall warming in Figure 1a. The marine surface temperature (input sea
surface temperature plus calculated sea ice temperature)
anomaly in the two experiments ((top) 2CO2 and (bottom)
2CO2WT). RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL Figure 1b. 3.
Meteorological Effects of the Different Sea
Surface Temperature Patterns As in Figure 1a except for surface air
temperature changes calculated in the experiments. cooled considerably. Given the magnitude of the imbalance,
we estimate that this model has an equilibrium climate
sensitivity for doubled CO2 of about 2.65C, or about
0.66C/Wm2. [17] Of relevance for interhemispheric transport in the
troposphere, the Hadley Cell during Northern Hemisphere
winter is intensified in the 2CO2WT runs, with little change
in 2CO2. Of relevance for intrahemispheric transport, plan-
etary longwave energy increases substantially in the
2CO2WT runs, although eddy kinetic energy in general
shows only small changes; again there is little change in
either of these quantities in the 2CO2 simulations. Concern-
ing vertical mixing within the troposphere, penetrative
convective mass flux increases in all the doubled CO2
simulations, more so in 2CO2WT, though total convective
mass flux decreases somewhat in all the experiments, again
more so in the WT runs. With increased penetrative con-
vection, there is the possibility of reducing the time-aver-
aged instability, decreasing more shallow convection. [18] Shown in Figure 2 are the temperature changes in the
different experiments, with tropospheric warming and stra-
tospheric cooling as expected. The prime difference is that
the WT experiments have greater warming in the tropical
upper troposphere (and at the tropical tropopause), consis-
tent with their warmer sea surface temperatures. In addition,
the WT runs have greater warming at high latitudes during
winter (Figure 1b). Given in Figures 3a and 3b are the
resulting changes in zonal wind and Eliassen-Palm (EP)
fluxes (the zonally averaged wave energy fluxes normalized
by the zonal wind). The WT runs have greater relative west
wind increases from 45S to 45N in the upper troposphere
and lower stratosphere; over this range of latitudes, all of the
doubled CO2 simulations show relative equatorward prop-
agation of EP fluxes in conjunction with these wind
changes. At the highest latitudes, however, only 2CO2
shows west wind increases in the troposphere during North-
ern Hemisphere winter, as the greater warming in the WT
runs leads to greater relative east winds, and poleward wave
refraction. The decrease in high latitude temperature gra-
dient imposed in WT at the surface (Figure 1b) extended up
to some 6.5 km altitude, reducing the zonal wind shear
throughout that region. Figure 1b. As in Figure 1a except for surface air
temperature changes calculated in the experiments. often show results from both the noninteractive and inter-
active runs. 3.
Meteorological Effects of the Different Sea
Surface Temperature Patterns [16] Given in Table 2 are the annual, global average
quantities of the relevant parameters (except for the peak
stream function values, which are shown for the two
solstice seasons). As an indication of the consistency of
the results, we also show the average difference in the
respective quantities over the two different 5-year periods
(years 2–6 versus years 8–12). The 2CO2WT runs are
considerably warmer than the 2CO2 simulations. The neg-
ative value of the net radiation at the model top for all the
doubled CO2 simulations show that had the sea surface
temperatures been allowed to adjust, the runs would have [19] The EP flux divergence changes for the different
simulations are shown in Figure 4. Associated with the
relative equatorward EP flux refraction change, increased
convergence occurs in the lower stratosphere in both hemi- Table 2. Annual Atmospheric Features in the Different Experiments
Unit
Control
Control (I)
2CO2
2CO2 (I)
2CO2 WT
2CO2 WT(I)
5-Year
Global surf temp
C
12.39
12.35
15.67
15.67
17.13
17.11
0.007
Sea ice coverage
%
4.4
4.4
2.6
2.6
2.3
2.3
0
Air temperature
C
25.33
25.30
22.62
22.55
20.87
20.87
0.013
Water vapor
mm
20.2
20.1
24.6
24.6
27.4
27.3
0.03
Cloud cover
%
68.8
68.7
66.3
66.2
65.3
65.1
0.03
Planetary albedo
%
32.33
32.27
30.28
30.39
30.18
30.30
0.08
Net radiation at model top
Wm2
0.1
0.4
1.1
1.5
3.5
3.9
0.016
Moist convective mass flux
109 Kgs1
1554
1568
1546
1550
1474
1483
1.5
Moist control mass flux at 195 mbar
109 Kgs1
141.3
145.5
222.8
232.0
278.7
290.0
0.6
Eddy kinetic energy
104 Jm2
64.9
65.0
63.5
64.2
67.2
67.5
0.27
EKE, wave numbers 1–4
1017 J
647
645
639
651
697
704
9.67
Peak D-F stream function
109 Kgs1
175
178
173
180
208
213
3
Peak J-A stream function
109 Kgs1
203
199
199
200
197
202
1.67 ACL
25 - 5 ACL
25 - 5 RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS Figure 2. Temperature changes in the different experiments, averaged over the last 10 years. Shown are
the results for the annual average and the two solstice seasons for (top) 2CO2, (second row) 2CO2(I),
(third row) 2CO2WT and (fourth row) 2CO2WT(I). Figure 2. Temperature changes in the different experiments, averaged over the last 10 years. 3.
Meteorological Effects of the Different Sea
Surface Temperature Patterns Shown are
the results for the annual average and the two solstice seasons for (top) 2CO2, (second row) 2CO2(I),
(third row) 2CO2WT and (fourth row) 2CO2WT(I). RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL
25 - 6 ACL
25 - 6 spheres from 45S to 45N, more so in the WT runs. In
addition, the wind changes in the upper troposphere/lower
stratosphere alter the wave refraction and lead to greater EP
poleward of 45N. In the extratropical troposphere, EP flux
convergences are associated with the east wind change a
the highest latitudes while divergences arise with the wes
Figure 3. Change in the zonal winds (shading/contours) and EP fluxes (arrows) for the two solstice
seasons in the different experiments (a) 2CO2 and 2CO2(I) and (b) 2CO2WT and 2CO2WT(I). urs) and EP fluxes (arrows) for the two solstice
O2(I) and (b) 2CO2WT and 2CO2WT(I). Figure 3. Change in the zonal winds (shading/contours) and EP fluxes (arrows) for the two solstice
seasons in the different experiments (a) 2CO2 and 2CO2(I) and (b) 2CO2WT and 2CO2WT(I). Figure 3. Change in the zonal winds (shading/contour
seasons in the different experiments (a) 2CO2 and 2CO2 Figure 3. Change in the zonal winds (shading/contours) and EP fluxes (arrows) for the two solstice
seasons in the different experiments (a) 2CO2 and 2CO2(I) and (b) 2CO2WT and 2CO2WT(I). poleward of 45N. In the extratropical troposphere, EP flux
convergences are associated with the east wind change at
the highest latitudes, while divergences arise with the west
wind change at upper midlatitudes. These effects are also spheres from 45S to 45N, more so in the WT runs. In
addition, the wind changes in the upper troposphere/lower
stratosphere alter the wave refraction, and lead to greater EP
flux convergences in the WT runs above 10 mbar primarily RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL
25 - 7 greatest in the WT runs, and also somewhat larger in the
interactive runs of each set. [20] Changes in the transformed Eulerian (residual) cir-
culation are presented in Figure 5. The intensified EP flux
convergences drive an increased residual circulation at low
latitudes in all the doubled CO2 simulations, more so in the
WT runs. 3.
Meteorological Effects of the Different Sea
Surface Temperature Patterns in the midstratosphere, while in 2CO2 there is greater
cyclonicity like the positive phase of the Arctic Oscillation. [22] However, from the perspective of the sea level
pressure (Figure 6, right) the results are very different. Now it appears qualitatively as if all the runs are producing
the positive phase of the AO and North Atlantic Oscillation,
the latter with lower pressure in the vicinity of the Icelandic
Low, and relatively higher pressure near Portugal. This
result illustrates that in a climate change situation one
cannot necessarily expect the recent barotropic nature of
AO response [Thompson et al., 2000] to prevail. We return
to this point in the discussion section. in the midstratosphere, while in 2CO2 there is greater
cyclonicity like the positive phase of the Arctic Oscillation. [21] These differences in high latitude response provide
for a different pattern of geopotential response. The WT
runs have increased 10 mbar heights around the pole in the
winter hemisphere (Figure 6, left), while the 2CO2 runs
have smaller changes or decreases. These results are con-
sistent not only with the amount of high latitude warming,
but also the EP flux convergence changes at high latitudes
above 10 mbar with their generation of the residual circu-
lation, i.e., EP flux convergence, a more direct circulation,
and subsidence warming in the WT runs. From this per-
spective, the WT runs have a change toward the negative
phase of the Arctic Oscillation (AO) (weaker polar vortex) 3.
Meteorological Effects of the Different Sea
Surface Temperature Patterns At higher latitudes during Northern Hemisphere
winter, the intensified EP flux convergences shown in
Figure 4 above 10 mbar help the 2CO2WT runs drive a
more direct circulation from 10 to 100 mbar via the down-
Figure 3. (continued) Figure 3. (c greatest in the WT runs, and also somewhat larger in the
interactive runs of each set. latitudes in all the doubled CO2 simulations, more so in the
WT runs. At higher latitudes during Northern Hemisphere
Figure 3. (continued) Figure 3. (continued) latitudes in all the doubled CO2 simulations, more so in the
WT runs. At higher latitudes during Northern Hemisphere
winter, the intensified EP flux convergences shown in
Figure 4 above 10 mbar help the 2CO2WT runs drive a
more direct circulation from 10 to 100 mbar via the down- greatest in the WT runs, and also somewhat larger in the
interactive runs of each set. greatest in the WT runs, and also somewhat larger in the
interactive runs of each set. [20] Changes in the transformed Eulerian (residual) cir-
culation are presented in Figure 5. The intensified EP flux
convergences drive an increased residual circulation at low ACL
25 - 8 25 - 8 ACL RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS Figure 4. Change in the EP flux divergence in the different experiments. Figure 4. Change in the EP flux divergence in the different experiments. in the midstratosphere, while in 2CO2 there is greater
cyclonicity like the positive phase of the Arctic Oscillation. [22] However, from the perspective of the sea level
pressure (Figure 6, right) the results are very different. Now it appears qualitatively as if all the runs are producing
the positive phase of the AO and North Atlantic Oscillation,
the latter with lower pressure in the vicinity of the Icelandic
Low, and relatively higher pressure near Portugal. This
result illustrates that in a climate change situation one
cannot necessarily expect the recent barotropic nature of
AO response [Thompson et al., 2000] to prevail. We return
to this point in the discussion section. ward control principle [Haynes et al., 1991]. In contrast, the
circulation is somewhat more indirect in the same region in
2CO2. In the troposphere, the more direct circulation at the
highest latitudes follows the EP flux convergences, both
being greatest in the WT simulations. Figure 6. Change in the (left) 10-mbar heights and (right) sea level pressure for December–January–
February. Figure 6. Change in the (left) 10-mbar heights and (right) sea level pressure for December–January–
February. 4.
Changes in Transports [23] In this section, we present changes in the transports
as discussed by Rind et al. [2001]. While 2CO2 uses the RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL
25 - 9 ACL
25 - 9 Figure 5. Change in the transformed Eulerian (residual) stream function in the different experiments. A
positive value indicates a relative counterclockwise change, to a more indirect (direct) circulation in the
Northern Hemisphere (Southern Hemisphere). Figure 5. Change in the transformed Eulerian (residual) stream function in the different experiments. A
positive value indicates a relative counterclockwise change, to a more indirect (direct) circulation in the
Northern Hemisphere (Southern Hemisphere). RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL
25 - 10 ACL ACL
25 - 11 ACL
25 - 11 ACL
25 - 11 Table 3. Interhemispheric Exchange Times (Years) in the
Different Simulations
SF6
CFC-11
CH4
Control
1.20
1.10
1.01
Control(I)
1.20
1.10
1.00
2CO2
1.21
1.15
1.00
2CO2(I)
1.20
1.13
1.00
2CO2WT
1.07
0.97
0.89
2CO2WT(I)
1.07
0.96
0.87
5-Year
0.04
0.007
0.007 Table 3. Interhemispheric Exchange Times (Years) in the
Different Simulations simulations, while the increased downward transport in the
subtropics is also evident. The global change is thus much
less than the tropical upwelling component. 4.6.
Transport Within the Stratosphere [30] Evident in both Figures 8a and 8b is a two-cell
distribution for the annual average change in the Northern
Hemisphere stratosphere, with relative upwelling at the pole
and equator, and downwelling around 45N. Quantitatively
the extratropical downwelling is greater in the WT runs as a
result of the greater EP flux convergences above 10 mbar. The transport change is somewhat similar in the Southern
Hemisphere except that the downwelling extends below 10
mbar only in the WT runs. 4.2.
Intrahemispheric Transport [25] For the same species shown in Table 3, there is
greater southward transport within the Northern Hemisphere
by a few percent (relative to the hemispheric tracer mass) in
2CO2WT, due both to the intensified Hadley Cell in North-
ern Hemisphere winter and the somewhat greater planetary
longwave energy. There is little change in 2CO2. 4.5.
Vertical Transport From the Stratosphere
to the Troposphere [29] The change in the residual stream function shown in
Figure 5 can be interpreted to imply that in all the doubled
CO2 simulations, there is increased downward transport
through the Northern Hemisphere lowermost stratosphere
at high latitudes and at midlatitudes (around 45N) relative
to the control run values (seen to some extent in Figure 7). To illustrate this more directly, given in Figure 8a is the
change in SF6 transport due to the large-scale dynamics
(hence not including convection) for 2CO2; results for
2CO2WT given in Figure 8b show qualitatively similar
features in the extratropics. The circulation change is
especially effective for bomb 14C which has its peak initial
source around 50 mbar. Globally, downward transport
increases by some 3–4% through 100 mbar, and by up to
10% through 240 mbar in the 2CO2WT experiments. This
results in a shorter residence time for bomb 14C in the
stratosphere, with the effects greatest in the 2CO2WT runs;
averaged over 6 years, there is 5.5% less
14C in the
atmosphere, with a decreased atmospheric residence time
of 15–20%. Changes are smaller, but in the same direction,
in the 2CO2 runs. As noted in the meteorology discussion,
this in turn results from the greater relative EP flux
convergences in the extratropics in driving a more direct
circulation, especially in winter (Figure 5). fied Hadley Cell during Northern Hemisphere winter (Table
2) in the WT experiments. 4.3.
Vertical Transport Within the Troposphere [26] With respect to the distribution of short-lived species
such as Rn222, increased convection diminishes tropical
gradients between low levels and the upper troposphere in
all of the doubled CO2 runs, and diminishes the gradients
between the surface and midtroposphere in 2CO2WT (despite
the overall decrease in convective mass flux indicated in
Table 2). At high latitudes, the increased warmth provides for
an additional radon source, from previously frozen ground,
and results in increased vertical gradients away from the
surface, more so in the warmer (WT) simulations. 4.1.
Interhemispheric Transport same sea surface temperatures and sea ice changes as in that
paper, this model has 53 layers (as compared to 31), and
several of the physics routines (boundary layer, momentum
advection, radiation) have been changed. The degree to
which the boundary conditions can enforce the same
response in these two related, but somewhat different
models, will be addressed in the discussion section. [24] Given in Table 3 are the interhemispheric exchange
times for three of the species in the different simulations, as
well as the differences between the two five year time
periods. There is little change in the 2CO2 simulation, but
interhemispheric exchange times decrease by 10–15% in
2CO2WT. This is qualitatively consistent with the intensi- [24] Given in Table 3 are the interhemispheric exchange
times for three of the species in the different simulations, as
well as the differences between the two five year time
periods. There is little change in the 2CO2 simulation, but
interhemispheric exchange times decrease by 10–15% in
2CO2WT. This is qualitatively consistent with the intensi- RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS 5.
Changes in Ozone Distribution [32] The results presented in the previous section can be
combined with the changes in ozone photochemistry to
understand how ozone changed in these doubled CO2
simulations. Shown in Figure 10 are the percentage changes
in ozone on the annual average in the different experiments. Increases in the tropical upper stratosphere are associated
with the colder temperatures due to doubled CO2 and they
do not depend on the different SST patterns, being similar in
2CO2 and 2CO2WT. The interactive runs have less ozone
increase there, because as the ozone increases, the temper-
ature does too, relatively, reducing the photochemical
source. Therefore, in the upper stratosphere, while the ozone
increase maximizes in the tropics in the noninteractive runs,
the changes are of comparable value at low and high
latitudes in the interactive simulations. [35] In a related development, below about 30 mbar, and
extending down through the middle troposphere, the inter-
active runs have greater EP flux convergences at high
latitudes, greater relative east winds, and warmer temper-
atures during Northern Hemisphere winter (Figures 2–4). This effect is the result of their weaker subtropical west
wind increase in the upper stratosphere diverting less wave
energy upward, allowing for greater convergences below. This greater high latitude warming results in a greater
increase in 10 mbar height at the pole (more negative phase
of the AO) in the interactive runs (most obvious in Figure 6
for the 2CO2 runs), an effect which extends down into the
upper troposphere (see also Table 5, discussed below). [33] Elsewhere, the SSTs do affect the ozone distribution. Ozone decreases in the tropical lower stratosphere, partly
due to reduced UV penetration and chemistry, partly the
result of increased tropical upwelling bringing up low
tropospheric ozone concentrations (note that the only tropo-
spheric ozone in these simulations is that which arises from
stratospheric transport). This latter effect is greater with the
warmer tropical SSTs. 2CO2WT also features greater ozone
increases at high latitudes and in the troposphere, due to its
increased poleward and downward transport, as occurred for
other species (e.g., methane, in Figure 9). The result is an
increase in midtropospheric ozone from the stratospheric
source in the 2CO2WT simulations, and a decrease in the
2CO2 runs (and for methane, with a tropospheric source, the
result is just the opposite). 4.4.
Vertical Transport From the Troposphere to the
Stratosphere 4.4. Vertical Transport From the Troposphere to the
Stratosphere [27] The ratios of upward transport from 20N to 20S
between experiment and control for levels in the upper
troposphere and lower stratosphere are given in Tables 4a
and 4b. In the 2CO2 runs, upward transport increases by
10–30% for all the species at all the levels, associated with
the intensified residual circulation at these altitudes (Figure
5). The transport increases by even larger percentages in the
2CO2WT simulations. [31] The change in CH4 distribution in the different runs
is used to illustrate the effect of the altered circulation in
Figure 9. Where air upwells, methane increases; regions of
decrease in the Northern Hemisphere lower stratosphere and
Southern Hemisphere middle stratosphere are associated
with the relative downwelling. The stratospheric methane
increase with greater flux from the troposphere is larger in
the 2CO2WT experiments, which had the greater residual
circulation change. Note that the increased interhemispheric
transport in the troposphere (reduced Northern Hemisphere [28] Also shown is the ratio of the global transports (in
brackets), and the values are much lower than the tropical
component. This is because the intensified tropical circula-
tion cells lead to increased downward transport as well. Shown in Figure 7 is the change in vertical transport of CH4
through 100 mbar for the four experiments; the other species
show similar characteristics. The increased upward transport
near the equator is apparent in all runs, greater with the WT Table 4a. Ratio of Vertical Transport Between 2CO2 and Control in the Tropics (20N to 20S) and Global (in Brackets)
Pressure, mbar
Run
CO2
N2O
CFC-11
CH4
5-Year
84
2CO2
1.19 [1.03]
1.19 [1.11]
1.18 [1.12]
1.19 [1.10]
0.032 [0.04]
2CO2(I)
1.16 [1.01]
1.17 [1.07]
1.16 [1.10]
1.16 [1.06]
0.005 [0.05]
100
2CO2
1.25 [1.03]
1.25 [1.11]
1.24 [1.11]
1.25 [1.09]
0.033 [0.04]
2CO2(I)
1.28 [1.02]
1.28 [1.07]
1.27 [1.09]
1.28 [1.05]
0.004 [0.05]
117
2CO2
1.20 [1.02]
1.20 [1.11]
1.18 [1.10]
1.19 [1.08]
0.005 [0.13]
2CO2(I)
1.27 [0.99]
1.27 [1.07]
1.26 [1.08]
1.27 [1.03]
0.029 [0.05]
135
2CO2
1.11 [0.97]
1.11 [1.12]
1.10 [1.08]
1.11 [1.06]
0.045 [0.04]
2CO2(I)
1.19 [0.95]
1.19 [1.08]
1.18 [1.06]
1.18 [1.00]
0.027 [0.06] RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS values relative to Southern Hemisphere) is also visible in
the figure for the WT experiment. 4.4.
Vertical Transport From the Troposphere to the
Stratosphere gences) at low latitudes in the middle and upper stratosphere
(Figure 4), and thus affects the tropical residual circulation
Table 4b. Ratio of Vertical Transport Between 2CO2WT Experiments and Control in the Tropics (20N to 20S) and Global (in
Brackets)
Pressure, mbar
Run
CO2
N2O
CFC-11
CH4
5-Year
84
2CO2WT
1.55 [1.05]
1.55 [1.26]
1.52 [1.26]
1.54 [1.22]
0.060 [0.09]
2CO2WT(I)
1.56 [1.04]
1.56 [1.17]
1.55 [1.21]
1.56 [1.15]
0.035 [0.08]
100
2CO2WT
1.74 [1.04]
1.74 [1.26]
1.69 [1.24]
1.73 [1.20]
0.036 [0.08]
2CO2WT(I)
1.82 [1.01]
1.83 [1.17]
1.79 1.19]
1.82 [1.12]
0.066 [0.04]
117
2CO2WT
1.64 [0.96]
1.64 [1.26]
1.60 [1.22]
1.63 [1.15]
0.011 [0.08]
2CO2WT(I)
1.74 [0.92]
1.74 [1.17]
1.70 [1.16]
1.73 [1.07]
0.066 [0.07]
135
2CO2WT
1.47 [0.87]
1.47 [1.26]
1.43 [1.19]
1.45 [1.05]
0.020 [0.06]
2CO2WT(I)
1.54 [0.83]
1.54 [1.17]
1.51 [1.14]
1.53 [0.97]
0.063 [0.07] Table 4b. Ratio of Vertical Transport Between 2CO2WT Experiments and Control in the Tropics (20N to 20S) and Global (in
Brackets) ansport Between 2CO2WT Experiments and Control in the Tropics (20N to 20S) and Global (in values relative to Southern Hemisphere) is also visible in
the figure for the WT experiment. gences) at low latitudes in the middle and upper stratosphere
(Figure 4), and thus affects the tropical residual circulation
in the lower stratosphere and upper troposphere. Hence the
increase in this circulation is slightly weaker in the non-
interactive runs below 100 mbar, as can be seen from the
tracer transports in Tables 4a and 4b. However, the down-
ward transport is also weaker (Figure 7), and so the global
transport upward is actually greater in the noninteractive
runs (Tables 4a and 4b). 5.
Changes in Ozone Distribution [36] We can summarize the results presented above by
showing how the age of air changed in the various experi-
ments (Figure 11), as deduced from the SF6 tracer (as given
by Rind et al. [2001]). The decrease in age of air throughout
the stratosphere is greatest in the 2CO2WT runs with their
intensified residual circulation, and the decrease is greater in
the noninteractive runs associated with their greater net
global upward transport (Table 4, again not due to greater
tropical upwelling). The noticeable increase in interhemi-
spheric transport in the 2CO2WT runs is evident in the
troposphere as younger ages in the Southern Hemisphere, Figure 7. Change in annual vertical transport of CH4
through 100 mbar in the different experiments. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS and to a lesser extent this appears to be true for portions of
the troposphere in the 2CO2 runs as well, although that was
not obvious from the hemispheric results given in Table 3. The difference relates to the increased age in the Southern
Hemisphere tropical upper troposphere for the 2CO2 runs
which, as can be seen from Figure 8a, is associated with an
upper tropospheric vortex spanning the equator, with
decreased age of air in the same region in the Northern
Hemisphere. The effect is entirely missing in the 2CO2WT
runs which have a coherent upward mass transport from low
levels just south of the equator (Figure 8b). This distinction
is related to the SST peak change south of the equator in
the WT runs (Figure 1a). Greater downward transport from
the stratosphere at extratropical northern latitudes in the
2CO2WT runs is indicated by the relative increase in age
of air, especially relative to the strong decreases seen in
the 2CO2 simulations. [39] The SST pattern, in particular the degree of tropical
warmth, affects the heating in the upper troposphere and its
latitudinal temperature gradient, the winds in the upper
troposphere and lower stratosphere, wave energy refraction
and convergence in the stratosphere, and hence the circu-
lation and winds throughout the middle atmosphere. Clearly,
to know what will happen in the Middle Atmosphere we
need to know the pattern of prospective SST change. [40] The ozone response affects the magnitude of temper-
ature change in the upper stratosphere, and the resulting
latitudinal temperature gradient; this in turn affects wave
energy propagation from the lower stratosphere, and wave
energy convergences and circulation in the lower strato-
sphere, with effects which extend down into the upper
troposphere. These dynamical responses illustrate how cli-
mate perturbations affecting the stratosphere can influence
tropospheric processes due to wave-mean flow interactions. With respect to both the meteorology and the change in
transports, the SST changes generally dominate the ozone
influence, although the effects are comparable in some
stratospheric regions. 6.
Dynamical Effect of Interactive Ozone [34] The net effect of the greater tropical ozone increase
in the noninteractive runs is to provide for warmer temper-
atures in the tropical upper stratosphere, by about 2C. The
warmer temperatures are then associated with a greater west
wind increase in the subtropical stratosphere (e.g., see the 1
mbar wind change in Figures 3a and 3b); this affects the
wave refraction and results in greater EP flux convergences
(or smaller divergences) in the extratropical middle and
upper stratosphere (e.g., between 10 and 1 mbar during
Northern Hemisphere winter in Figure 4), and a more direct
extratropical residual circulation in both winter hemispheres
(Figure 5). This alteration in wave refraction pattern also
produces greater EP flux divergences (or smaller conver- Figure 7. Change in annual vertical transport of CH4
through 100 mbar in the different experiments. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS 7.
Discussion [37] In this section we discuss a number of issues raised
by these results as well as some popular issues associated
with the response to increasing CO2 levels. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL
25 - 13 ACL
25 - 13 Figure 8. Schematic of the annual change in transport of SF6 by the large-scale dynamics (not including
convection) for (a) 2CO2 and (b) 2CO2WT. Shaded region indicates the lowermost stratosphere (potential
temperature less than 380 K, and potential vorticity greater than 2 105 k mbar1s1 outside the
tropics and above 100 mbar in the tropics). To illustrate the altered pathways of tracer transport, all
vectors are of unit length except in the tropical troposphere where the exceptionally large changes are
shown as exaggerated arrows. Separate log pressure scales are used above and below 100 mbar for
presentation purposes. Figure 8. Schematic of the annual change in transport of SF6 by the large-scale dynamics (not including
convection) for (a) 2CO2 and (b) 2CO2WT. Shaded region indicates the lowermost stratosphere (potential
temperature less than 380 K, and potential vorticity greater than 2 105 k mbar1s1 outside the
tropics and above 100 mbar in the tropics). To illustrate the altered pathways of tracer transport, all
vectors are of unit length except in the tropical troposphere where the exceptionally large changes are
shown as exaggerated arrows. Separate log pressure scales are used above and below 100 mbar for
presentation purposes. and to a lesser extent this appears to be true for portions of
the troposphere in the 2CO2 runs as well, although that was
not obvious from the hemispheric results given in Table 3. The difference relates to the increased age in the Southern
Hemisphere tropical upper troposphere for the 2CO2 runs
which, as can be seen from Figure 8a, is associated with an
upper tropospheric vortex spanning the equator, with
decreased age of air in the same region in the Northern
Hemisphere. The effect is entirely missing in the 2CO2WT
runs which have a coherent upward mass transport from low
levels just south of the equator (Figure 8b). This distinction
is related to the SST peak change south of the equator in
the WT runs (Figure 1a). Greater downward transport from
the stratosphere at extratropical northern latitudes in the
2CO2WT runs is indicated by the relative increase in age
of air, especially relative to the strong decreases seen in
the 2CO2 simulations. the 2CO2 and 2CO2WT runs), and what is influenced by the
ozone changes (the differences between the interactive and
noninteractive runs). 7.2.
Intermodel Differences [38] The range of experiments allows us to address the
issue of what in the troposphere/middle atmosphere system
is influenced by the SST patterns (the differences between [41] A number of these results are consistent with
expectations [Rind et al., 2001, 2002]. The upper tropo- RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL Figure 9. Change in annual average methane in the different experiments. Figure 9. Change in annual average methane in the different experiments. sphere/stratosphere tropical residual circulation did
increase in both climate simulations, and the magnitude
of increase was greater with warmer sea surface temper-
atures. These results were expected due to the influence of
the tropical sea surface temperature warming on temper-
atures in the tropical upper troposphere, and the subse-
quent effect on west winds and planetary wave refraction
at those altitudes. They support the assumption that increased transport from the troposphere to the strato-
sphere would be a robust result with different amounts
of tropical warming. However, the tracer transport changes
showed that the tropical upwelling is only part of the story,
for the global increase in transport into the stratosphere is
much less than the tropical change. The difference is
associated with increased downwelling in the subtropics
that accompanied the intensified tropical residual circula- RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS
ACL
25 - 15 RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL
25 - 15 Figure 10. Change in annual average ozone (in percent) in the different experiments. Note that the
tropospheric ozone and its change arises from stratospheric transport. Figure 10. Change in annual average ozone (in percent) in the different experiments. Note that the
tropospheric ozone and its change arises from stratospheric transport. tion. This recycling of air within the 30N to 30S regime
may be sensitive to the greater ‘‘leakiness’’ of the tropical
pipe in the model compared to the real world. faster. The warmer tropical sea surface temperatures also
led to a greater increase in penetrative convection and the
corresponding increased transport of tracers to the upper
troposphere, though that effect occurred in all the doubled
CO2 runs. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS Table 5. AO-Type Responses During December–Februarya
UNIT
2CO2
2CO2 (I)
2CO2WT
2CO2WT(I)
SLP
32N–52N minus 60N–80N
mbar
2.28 (1.14)
1.98
4.91b [3.88]
5.43b
500 mbar Ht
32N–52N minus 60N–80N
m
14 (41)
15
8 [7]
9
100 mbar Ht
32N–52N minus 60N–80N
m
76c (19)
12
103b [142]
45d
10 mbar Ht
32N–52N minus 60N–80N
m
196 (58)
57
96 [162]
220d
aResults from Rind et al. [1998, 2001] are shown (without significance testing) in brackets or parentheses, respectively, for comparison. bSignificant at 99% level. cSignificant at 95% level. dSignificant at 90% level. potential energy associated with hemispheric scales [Rind et
al., 1998]. of the model is somewhat less sensitive to SST patterns,
with weaker surface winds, surface fluxes and resulting
precipitation; hence it responded more weakly to the altered
SST fields it was given (e.g., no Hadley Cell change). The
newer model also had greater penetrative convection rela-
tive to convection at other levels, so when climate warmed,
and the penetrative convection increased, it was easier to
stabilize the atmosphere and reduce convection in general. Furthermore with the greater increase in penetrating con-
vection in the newer model, warming of the upper tropo-
sphere was greater; combined with the unchanged Hadley
circulation this resulted in a greater latitudinal temperature
gradient, stronger west wind increases and a more positive
AO phase (discussed further below). The tendency of
climate change simulations to mimic characteristics of the
control run has been noted previously [Mitchell et al., 1987;
Rind, 1988; Senior, 1995]. [43] However, a number of the results were unexpected,
relating primarily to changes associated with different
versions of the GISS GCMAM. Rind et al. [2001] use the
same marine boundary conditions as used in 2CO2 but with
an earlier version of the model II0 GCMAM. In that
simulation the Hadley Cell decreased in intensity, as did
interhemispheric transport, by 5%; in contrast, in this 2CO2
experiment there were negligible changes in both quantities. Also, in the earlier simulation, total convective mass flux
increased, as shown in Table 2, while this model produced a
small decrease. [44] The differing Hadley Cell response in the two
models also likely influenced the high latitude response,
by altering the magnitude of dynamical heat convergence in
the midlatitude troposphere [Rind et al., 2002]. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS Heating at
this latitude, compared with heating at high latitudes, affects
the latitudinal temperature gradient and zonal wind change
in the upper troposphere, and hence planetary wave refrac-
tion. Rind et al. [2001, their Figure 3] show that the
reduction in Hadley Cell intensity had minimized the
midlatitude heating leading to decreased west winds in
the upper troposphere at higher latitudes, relative poleward
planetary wave refraction and higher sea level pressures at
higher latitudes. In contrast, in the 2CO2 run here, during
winter west winds increase at the higher latitudes in the
upper troposphere, EP flux divergences occur and tropo-
spheric subpolar lows are intensified. Why were these
changes model dependent? [48] The 2CO2WT experiment should have produced
simulations that resembled the results discussed by Rind
et al. [1998] with the coarser resolution model, given that
the sea surface temperature/sea ice changes of that run (from
model II) were used in this one. The GCMAM II0 model has
weaker convective fluxes due to reduced mass associated
with each convective event, and hence the warming in the
tropical upper troposphere is about 25% less than it was in
the previous model. Consequently, the zonal wind increase
in the upper troposphere is not as strong, and the magnitude
of the wave refraction, EP flux divergence and residual
circulation changes are somewhat reduced, but the overall
pattern is similar. However, the AO phase changes differ
with altitude, as discussed below. g
p
[45] We can address that question by comparing the
current model to the version used by Rind et al. [2001]. The intermodel differences include the greater vertical
resolution in the current model, both in the boundary layer
(6 layers to 4 in the first 200 mbar) and overall (53 layers to
31 layers), and alterations in the model code, primarily in
the boundary layer. To determine which component of the
model changes affected each of the different results, we ran
a control run version with reduced boundary layer and
vertical resolution but with the newer 53-layer model code. [46] The results showed that increasing the vertical reso-
lution, including that in the boundary layer, reduced the
latent heat and total surface fluxes, and with it the precip-
itation. Changes in the model code resulted in the newer
model having weaker surface winds and often weaker eddy
mixing in the boundary layer under unstable conditions. 7.2.
Intermodel Differences And with greater tropical warming there was
increased generation of planetary wave energy at the longest
wavelengths, consistent with the increase in available [42] The Hadley Circulation response also matched
expectations, at least in the sense that with the warmer
tropical sea surface temperatures, the circulation would
increase, and interhemispheric transport would become RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS The newer version ACL
25 - 17 RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS T simulations, the values are larger than that, with
tratopause peak values of 15–20%, perhaps due to
temperature structure of these ozone differences produ
some additional cooling in the tropical lower stratosp
Figure 11. Percentage change in age of air in the various experiments as calculated from the SF6
surface concentrations. Figure 11. Percentage change in age of air in the various experiments as calculated from the SF6
surface concentrations. 2CO2WT simulations, the values are larger than that, with
tropical stratopause peak values of 15–20%, perhaps due to
the different ozone photochemical code. The ozone loss in
the tropical lower stratosphere is 5–10 times larger in the
new run, undoubtedly influenced by the ozone advection,
with the intensified upwelling exaggerating the lower strato-
sphere loss. High latitude ozone gains are also larger when
ozone advection is included. The radiative impact on the temperature structure of these ozone differences produces
some additional cooling in the tropical lower stratosphere
and slightly greater warming at high latitudes. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS Increased vertical resolution resulted in greater amounts of
penetrating convection to high levels and decreased fluxes
to low levels. [45] We can address that question by comparing the
current model to the version used by Rind et al. [2001]. The intermodel differences include the greater vertical
resolution in the current model, both in the boundary layer
(6 layers to 4 in the first 200 mbar) and overall (53 layers to
31 layers), and alterations in the model code, primarily in
the boundary layer. To determine which component of the
model changes affected each of the different results, we ran
a control run version with reduced boundary layer and
vertical resolution but with the newer 53-layer model code. ,
[49] Given these intermodel differences for their current
climate simulation, it would be reasonable to have the most
confidence in those simulations that produce the most
realistic current depictions. The reduced convective fluxes
in GCMAM II0 are likely to be more realistic than the
excessive values of the coarse grid model as deduced from
radon distributions (at least in comparison to other models’
radon results [Rind and Lerner, 1996]). Hence in that
respect (although not with respect to sensitivity to SSTs)
2CO2WT should be given preference over the results from
Rind et al. [1998]. The weaker surface response of 2CO2 is
not necessarily an improvement, for example, the surface
winds in the model were already too weak (as deduced from
modeled soil dust [Tegen and Miller, 1998]). Hence the
conclusions from 2CO2 are not necessarily to be preferred
over those given by Rind et al. [2001] where they differ. [46] The results showed that increasing the vertical reso-
lution, including that in the boundary layer, reduced the
latent heat and total surface fluxes, and with it the precip-
itation. Changes in the model code resulted in the newer
model having weaker surface winds and often weaker eddy
mixing in the boundary layer under unstable conditions. Increased vertical resolution resulted in greater amounts of
penetrating convection to high levels and decreased fluxes
to low levels. [50] The interactive ozone changes calculated by Rind et
al. [1998], with the ozone anomaly scheme described by
Shindell et al. [1998] that did not include ozone advection,
peaked at 12% in the tropical stratopause region. In the [47] The control run differences provide explanations for
the intermodel differences noted above. 7.3.
AO Changes [51] Many, but not all model simulations have shown an
increased positive AO phase with increasing atmospheric
CO2 [Shindell et al., 1999; Paeth et al., 1999; Zorita and RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL
25 - 18 ACL increased by 3–4C in the 2CO2 experiments, and 4–6C
in the 2CO2WT experiments. At 164 mbar in the tropics
specific humidity increased by 9%, in the 2CO2 runs and
increased by 45% in 2CO2WT. Nevertheless, colder temper-
atures just above the averaged tropopause associated with
the increased vertical velocities and localized penetrating
convection enhanced condensation, removing all the excess
moisture and more. Specific humidity decreased throughout
the stratosphere, with reductions of greater than 20%
between 100 and 30 mbar. Gonzalez-Rouco, 2000; Gillett et al., 2002a]. All the sim-
ulations shown here have decreased high latitude sea level
pressures as well, as is evident in Figure 6. To assess
whether these changes correspond to an altered phase of
the Arctic Oscillation, we show in Table 5 the differences in
sea level pressure or geopotential heights between approx-
imately 30N–50N and 60N–80N (characteristic of the
AO sea level pressure gradient), along with the significance
of the results using a two-tailed T-test on the interannual
variation for 10 modeled years. With the warmer tropical
sea surface temperatures, at sea level a change to the
positive phase of the AO-type oscillation is significant,
and this is also true at 100 mbar, but there is no significance
in the middle troposphere, and the sign is different in the
middle stratosphere. 8.
Conclusions These effects are weaker or missing entirely
in the other simulations. In all of the doubled CO2 simulations, there is a more
direct circulation at lower latitudes, a more indirect
circulation at midlatitudes in the troposphere and higher
latitudes in the stratosphere, and a more direct circulation at
high latitudes in the troposphere and lower stratosphere. All
of these circulation changes are greater in the WT
experiments. Ozone increases in the upper stratosphere and
decreases in the lower stratosphere in all of the doubled
CO2 simulations. In the WT experiments ozone increases at
high latitudes as well, an effect which extends down into the
troposphere, while the other simulations show decreases in
high latitude ozone in the midtroposphere. [54] We can also compare the results from the earlier
version of the current model (shown in parentheses in
Table 5) [Rind et al., 2001] to the 2CO2 results. The earlier
model featured a relative negative AO phase in the sea level
pressure field and throughout the troposphere, in contrast to
the newer run with the same SST field. This model difference
is also influenced by the different Hadley Cell convergences
at midlatitudes, as discussed earlier in this section. g
p
p
The interactive runs show the smallest ozone increase
in the tropics, as warmer temperatures reduce photochemi-
cal production. By altering the ozone gradients, the
interactive runs alter the temperature gradients, winds and
refractive properties of planetary waves, with some effect on
the circulation extending down into the troposphere. At sea level there is a change to the positive phase of
the AO-type oscillation and this is also true at 100 mbar, but
there is no significance in the middle troposphere, and the
sign is different in the middle stratosphere. 8.
Conclusions [56] Model simulations with doubled atmospheric CO2
and two sets of sea surface temperatures (2CO2 and
2CO2WT) were used in GCM simulations with eight online
tracers. Also included was a linearized ozone photochem-
istry run both interactively (I) and noninteractively with the
radiation. The primary results from these experiments are
summarized as follows: [52] The pattern of the EP flux change in all the experi-
ments features significant tropospheric divergences at upper
middle latitudes (providing a west wind acceleration), with
convergences at high latitudes (for an east wind acceler-
ation), as in Figures 3 and 4. This wind change is realized
with lower pressure circa 70N. The simulations with the greater increase in tropical
sea surface temperatures (2CO2WT, 2CO2WT (I)) caused an
amplified tropical residual circulation response, including
increased interhemispheric transport and an exaggerated
increase in transport out of the tropical troposphere into the
stratosphere. In the other simulations (2CO2, 2CO2(I))
interhemispheric transport did not change, and the increase
in transport out of the tropical troposphere was smaller. The simulations with the greater increase in tropical
sea surface temperatures (2CO2WT, 2CO2WT (I)) caused an
amplified tropical residual circulation response, including
increased interhemispheric transport and an exaggerated
increase in transport out of the tropical troposphere into the
stratosphere. In the other simulations (2CO2, 2CO2(I))
interhemispheric transport did not change, and the increase
in transport out of the tropical troposphere was smaller. p
[53] The increased positive phase alters sign by the
middle stratosphere in 2CO2WT, which then features a
weaker polar vortex (see also Figure 6), a result also found
by Butchart et al. [2000] and Gillett et al. [2002b]. We also
show in Table 5 (in brackets) the results from the previous
simulation with these SSTs in the coarse grid model, in
which the phase remained positive in the middle strato-
sphere; that is, the polar vortex was stronger. As can be seen
in Figure 2, the temperature change in 2CO2WT at high
northern latitudes in the lower stratosphere exceeds that in
the middle troposphere, and also exceeds that at midlati-
tudes, helping to change the phase of the AO. In contrast,
the older model did not have such amplified warming in the
polar lower stratosphere, and the positive AO phase change
was maintained into the stratosphere (i.e., a more barotropic
response). 8.
Conclusions This temperature difference is consistent with the
zonal wind change, which in the current simulations fea-
tures weaker west winds at high latitudes, with relative
poleward wave refraction, while the older model had
stronger west winds and relative equatorward wave refrac-
tion at most high latitudes [Rind et al., 1998, Figure 5]. The
greater convective fluxes and tropical response in the older
model led to greater energy convergence from the Hadley
Cell change at lower midlatitudes in the upper troposphere,
helping to produce these differences. [53] The increased positive phase alters sign by the
middle stratosphere in 2CO2WT, which then features a
weaker polar vortex (see also Figure 6), a result also found
by Butchart et al. [2000] and Gillett et al. [2002b]. We also
show in Table 5 (in brackets) the results from the previous
simulation with these SSTs in the coarse grid model, in
which the phase remained positive in the middle strato-
sphere; that is, the polar vortex was stronger. As can be seen
in Figure 2, the temperature change in 2CO2WT at high
northern latitudes in the lower stratosphere exceeds that in
the middle troposphere, and also exceeds that at midlati-
tudes, helping to change the phase of the AO. In contrast,
the older model did not have such amplified warming in the
polar lower stratosphere, and the positive AO phase change
was maintained into the stratosphere (i.e., a more barotropic
response). This temperature difference is consistent with the
zonal wind change, which in the current simulations fea-
tures weaker west winds at high latitudes, with relative
poleward wave refraction, while the older model had
stronger west winds and relative equatorward wave refrac-
tion at most high latitudes [Rind et al., 1998, Figure 5]. The
greater convective fluxes and tropical response in the older
model led to greater energy convergence from the Hadley
Cell change at lower midlatitudes in the upper troposphere,
helping to produce these differences. At high latitudes, zonal winds become more easterly in
the WT runs due to a combination of greater surface
warming and increased EP flux convergences in the
stratosphere. These effects are weaker or missing entirely
in the other simulations. At high latitudes, zonal winds become more easterly in
the WT runs due to a combination of greater surface
warming and increased EP flux convergences in the
stratosphere. References Andrews, A. E., et al., Mean ages of stratospheric air derived from in situ
observations of CO2, CH4, and N2O, J. Geophys. Res., 106, 32,295–
32,314, 2001. Rind, D., J. Lerner, K. Shah, and R. Suozzo, Use of on-line tracers as a
diagnostic tool in general circulation model development, 2, Transport
between the troposphere and stratosphere, J. Geophys. Res., 104, 9151–
9167, 1999. Butchart, N., J. Austin, J. R. Knight, A. A. Scaife, and M. L. Gallani, The
response of the stratospheric climate to projected changes in the concen-
trations of well-mixed greenhouse gases from 1992 to 2051, J. Clim., 13,
2142–2159, 2000. Rind, D., J. Lerner, and C. McLinden, Changes of tracer distributions in the
doubled CO2 climate, J. Geophys. Res., 106, 28,061–28,079, 2001. Rind, D., P. Lonergan, N. K. Balachandran, and D. Shindell, 2xCO2 and
solar variability influences on the troposphere through wave-mean flow
interactions, J. Meteorol. Soc. Jpn., 80, 863–876, 2002. Dai, A., T. M. L. Wigley, B. A. Boville, J. T. Kiehl, and L. E. Buja,
Climates of the twentieth and twenty-first centuries simulated by the
NCAR climate system model, J. Clim., 14, 485–519, 2001. p
Senior, C. A., The dependence of climate sensitivity on the horizontal
resolution of a GCM, J. Clim., 8, 2860–2880, 1995. Douglass, A. R., M. J. Prather, T. M. Hall, S. E. Strahan, P. J. Rasch, L. C. Sparling, L. Coy, and J. M. Rodriguez, Choosing meteorological input for
the global modeling initiative assessment of high-speed aircraft, J. Geo-
phys. Res., 104, 27,545–27,564, 1999. Shindell, D., D. Rind, and P. Lonergan, Climate change and the middle
atmosphere, IV:1, Ozone photochemical response to doubled CO2, J. Clim., 11, 895–918, 1998. p y
Fyfe, J. C., G. J. Boer, and G. M. Flato, The Arctic and Antarctic oscilla-
tions and their projected changes under global warming, Geophys. Res. Lett., 26, 1601–1604, 1999. Shindell, D., R. Miller, G. Schmidt, and L. Pandolfo, Simulation of recent
northern winter climate trends by greenhouse-gas forcing, Nature, 399,
452–455, 1999. Gillett, N. P., M. R. Allen, R. E. McDonald, C. A. Senior, D. T. Shindell,
and G. A. Schmidt, How linear is the Arctic Oscillation response to
greenhouse gases?, J. Geophys. Res., 107, 4022, doi:10.1029/
2001JD000589, 2002a. Tegen, I., and R. Miller, A general circulation model study on the inter-
annual variability of soil dust aerosol, J. Geophys. Res., 103, 25,975–
25,995, 1998. Thompson, D. W., J. M. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS - 19 face data sets applied to the GCMAM were also made
available to the GRIPS (GCM Reality Intercomparison
Project) [Pawson et al., 2000] so that different strato-
spheric modeling groups could run doubled CO2 experi-
ments with the same climate forcing. The goal of the
project is, in effect, to compare how important intermodel
differences are relative to the sea surface temperature
forcings. The results presented in this paper, when com-
pared with those from our earlier simulations, imply that
intermodel differences outweigh the sea surface temper-
ature forcings for a number of the features, and this is
from a comparison of old and new GISS GCMs. We might
expect the differences to be equivalent or larger among the
modeling groups in general. Pawson, S., et al., The GCM-Reality Intercomparison Project for SPARC
(GRIPS): Scientific issues and initial results, Bull. Am. Meteorol. Soc.,
81, 781–796, 2000. ,
,
Perlwitz, J., H. F. Graf, and R. Voss, The leading variability mode of the
coupled troposphere-stratosphere winter circulation in different climate
regimes, J. Geophys. Res., 105, 6915–6926, 2000. g
p y
Prather, M., Numerical advection by conservation of second-order mo-
ments, J. Geophys. Res., 91, 6671–6681, 1986. Ramstein, G., Y. Serafini-Le Treut, H. Le Treut, M. Forichon, and S. Joussaume, Cloud processes associated with past and future climate
changes, Clim. Dyn., 14, 233–247, 1998. Rind, D., Dependence of warm and cold climate depiction on climate model
resolution, J. Clim., 1, 965–997, 1988. Rind, D., and J. Lerner, The use of on-line tracers as a diagnostic tool in
GCM model development, J. Geophys. Res., 101, 12,667–12,683,
1996. Rind, D., R. Suozzo, N. K. Balachandran, A. Lacis, and G. L. Russell, The
GISS Global Climate/Middle Atmosphere Model, I, Model structure and
climatology, J. Atmos. Sci., 45, 329–370, 1988. Rind, D., R. Suozzo, N. K. Balachandran, and M. Prather, Climate change
and the middle atmosphere, 1, The doubled CO2 climate, J. Atmos. Sci.,
47, 475–494, 1990. [58]
Acknowledgments. This work was funded by the NASA
ACMAP and EOS programs. ,
,
Rind, D., D. Shindell, P. Lonergan, and N. K. Balachandran, Climate
change and the middle atmosphere, III, The doubled CO2 climate revis-
ited, J. Clim., 11, 876–894, 1998. J. Lerner, J. Perlwitz, and D. Rind, Goddard Institute for Space Studies at
Columbia University, 2880 Broadway, New York, N.Y. 10025, USA.
(jlerner@giss.nasa.gov;judith@giss.nasa.gov;drind@giss.nasa.gov) C. McLinden, Air Quality Research Branch, Meteorological Service of
Canada, 4905 Dufferin Street, Toronto, ON M3H 5T4, Canada.
(chris.mclinden@ec.gc.ca) M. J. Prather, Earth System Science Department, University of
California, Irvine, Irvine, CA 92697-3100, USA. (mprather@uci.edu) 7.4.
Stratospheric Water Vapor Change [55] Given the increase in fluxes of tropospheric species
into the stratosphere, as symbolized by the methane changes
shown in Figure 8, one might have expected that (non-
methane) stratospheric water vapor values would have
increased as well. In addition to the transport change,
tropical zonal and time-averaged tropopause temperatures [55] Given the increase in fluxes of tropospheric species
into the stratosphere, as symbolized by the methane changes
shown in Figure 8, one might have expected that (non-
methane) stratospheric water vapor values would have
increased as well. In addition to the transport change,
tropical zonal and time-averaged tropopause temperatures Many of these results differ from those generated in
older versions of the GISS GCMAM despite the same SST
forcing, due to differences in control run characteristics. [57] This last conclusion has implications concerning the
use of altered sea surface temperatures in different GCMs
as part of intercomparison projects. The two marine sur- ACL
25 - 19 [58]
Acknowledgments.
This work was funded by the NASA
ACMAP and EOS programs. References Wallace, and G. C. Hegerl, Annular modes in the
extratropical circulation, II, Trends, J. Clim., 13, 1018–1036, 2000. Gillett, N. P., M. R. Allen, and K. D. Williams, The role of stratospheric
resolution in simulating the Arctic Oscillation response to greenhouse
gases, Geophys. Res. Lett., 29, 1500, doi:10.1029/2001GL014444,
2002b. Yao, M.-S., and A. D. Del Genio, Effects of cloud parameterization on the
simulation of climate changes in the GISS GCM, J. Clim., 12, 761–779,
1999. Haynes, P. H., C. J. Marks, M. E. McIntyre, T. G. Shepherd, and K. P. Shine, On the ‘‘downward control’’ of extratropical diabatic circulations
by eddy-induced mean zonal forces, J. Atmos. Sci., 48, 651–678, 1991. Zorita, E., and F. Gonzalez-Rouco, Disagreement between predictions of
the future behavior of the Arctic Oscillation as simulated in two different
climate models: Implications for global warming, Geophys. Res. Lett., 27,
1755–1758, 2000. McLinden, C. A., S. C. Olsen, B. Hannegan, O. Wild, M. J. Prather, and J. Sundet, Stratospheric ozone in 3-D models: A simple chemistry and the
cross-tropopause flux, J. Geophys. Res., 105, 14,653–14,665, 2000. Mitchell, J. F. B., C. A. Wilson, and W. M. Cunnington, On CO2 climate
sensitivity and model dependence of results, Q. J. R. Meteorol. Soc., 113,
293–322, 1987. Monahan, A. H., J. C. Fyfe, and G. M. Flato, A regime view of Northern
Hemisphere atmospheric variability and change under global warming,
Geophys. Res. Lett., 27, 1139–1142, 2000. p y
Paeth, H., A. Hense, R. Glowienka-Hense, S. Voss, and U. Cubasch, The
North Atlantic Oscillation as an indicator for greenhouse-gas induced
regional climate change, Clim. Dyn., 15, 953–960, 1999.
|
https://openalex.org/W4381251060
|
http://www.thieme-connect.de/products/ejournals/pdf/10.1055/s-0043-1768472.pdf
|
English
| null |
The Effect of Dental Implant Drills Materials on Heat Generation in Osteotomy Sites: A Systematic Review
|
The european journal of dentistry/The european journal of dentistry
| 2,023
|
cc-by
| 6,386
|
© 2023. The Author(s).
This is an open access article published by Thieme under the terms of the
Creative Commons Attribution License, permitting unrestricted use,
distribution, and reproduction so long as the original work is properly cited.
(https://creativecommons.org/licenses/by/4.0/)
Thieme Medical and Scientific Publishers Pvt. Ltd., A-12, 2nd Floor,
Sector 2, Noida-201301 UP, India Article published online: 2023-06-19 Article published online: 2023-06-19 Article published online: 2023-06-19 Article published online: 2023-06-19 Review Article 65 The Effect of Dental Implant Drills Materials on
Heat Generation in Osteotomy Sites: A
Systematic Review Shreyasi Chakraborty1
Mohammad-Adel Moufti2,3
Waad Kheder2,3 1Department of Implant Dentistry, BPP University, Birmingham,
United Kingdom
2Department of Preventive and Restorative Dentistry, College of
Dental Medicine University of Sharjah, Sharjah, United Arab Emirates
3Research Institute for Medical and Health Sciences, University of
Sharjah, Sharjah, United Arab Emirates Address for correspondence Mohammad-Adel Moufti, BDS, Dip
(OMS), MFDS (RCSEd), PhD (Ncl), College of Dental Medicine,
University of Sharjah, Sharjah, P.O. Box 27272, United Arab Emirates
(e-mail: mamoufti@sharjah.ac.ae). 1Department of Implant Dentistry, BPP University, Birmingham,
United Kingdom
2Department of Preventive and Restorative Dentistry, College of
Dental Medicine University of Sharjah, Sharjah, United Arab Emirates
3Research Institute for Medical and Health Sciences, University of
Sharjah, Sharjah, United Arab Emirates Eur J Dent 2024;18:65–72. DOI https://doi.org/
10.1055/s-0043-1768472.
ISSN 1305-7456. 1Department of Implant Dentistry, BPP University, Birmingham,
United Kingdom
2Department of Preventive and Restorative Dentistry, College of
Dental Medicine University of Sharjah, Sharjah, United Arab Emirates
3Research Institute for Medical and Health Sciences, University of
Sharjah, Sharjah, United Arab Emirates article published online
June 19, 2023 Address for correspondence Mohammad-Adel Moufti, BDS, Dip
(OMS), MFDS (RCSEd), PhD (Ncl), College of Dental Medicine,
University of Sharjah, Sharjah, P.O. Box 27272, United Arab Emirates
(e-mail: mamoufti@sharjah.ac.ae). Address for correspondence Mohammad-Adel Moufti, BDS, Dip
(OMS), MFDS (RCSEd), PhD (Ncl), College of Dental Medicine,
University of Sharjah, Sharjah, P.O. Box 27272, United Arab Emirates
(e-mail: mamoufti@sharjah.ac.ae). article published online
June 19, 2023
DOI https://doi.org/
10.1055/s-0043-1768472.
ISSN 1305-7456. Introduction preparation?” was formulated based on predefined patient,
intervention, comparison, and outcome (PICO) elements that
are
explained
below. The
search
was
undertaken
in
Medline/PubMed Central, Science Direct, and Google Scholar
using the following keywords and Boolean operators: “im-
plant site preparation” OR “osteotomy” OR “bone drill” AND
“ceramic drill” OR “zirconia drill” OR “zirconium drill” OR
“zirconia oxide drill” OR “Zircon based drill” OR “Diamond
Like Carbon coated drill” OR “DLC coated drills” OR “black
diamond drill” OR “titanium nitride coated metal drills” OR
“tungsten carbide coated metal drill” OR “tin coated drill” OR
“stainless steel drill” OR “stainless steel drill” OR “titanium
drill” AND “heat generation” OR “thermal change” OR “ther-
mal osteonecrosis” OR “thermal variation” OR “intrabony
temperature” OR “temperature change.” The selection of
articles/studies followed the standardized staged process
of the Preferred Reporting Items for Systematic Reviews
and Meta-Analyses (PRISMA) as illustrated in ►Fig. 1. First,
the abstracts of the initially selected articles were screened
by the authors SC and WK independently based on the
inclusion and exclusion criteria listed below to confirm the
articles’ relevance to our study. Any disagreement was dis-
cussed among the three authors and resolved. The full text of
the selected articles was obtained and read in full, also by
both reviewers SC and WK independently, to confirm the
selection of those articles. The bibliographies of the studies
were manually searched to identify any article that was not
captured by the electronic search. Four articles were found
relevant and selected for this review. One of the most important challenges in bone drilling during
osteotomy site preparation is heat generation and thermal
damages to the alveolar bone. This could be due to several
biomechanical factors such as the drilling protocol (technique,
force, speed, drill geometry, and irrigation),1 properties of the
drills,2 and properties of the bone.3,4 Investigations have
reported that temperatures of over 44°C lead to irreversible
damage. Abstract The aim of this review was to examine the impact of dental implant drill materials and
wear profiles on heat generation in the osteotomy sites as reported in experimental
studies and to critically appraise these studies. The research question was formulated
based on predefined patient, intervention, comparison, and outcome (PICO) elements. A comprehensive electronic search was undertaken in Medline/PubMed Central,
Science Direct, and Google Scholar, using predetermined keywords, followed by a
manual search of the bibliography of the selected articles. The selection of the studies
for the critical appraisal part of our study was based on the criteria used to assess the
study designs such as study aims, outcome measure, clarity of method, sample
selection, randomization, allocation concealment, sample attrition, bias, method of
data analysis, and external validity. Increased heat generation was observed with both
ceramic and metal drills; the heat generation was proportional to drills’ wear. The
literature was inconclusive regarding the association between drill material and heat
generation. However, drill materials had a significant influence on the overall temper-
ature increase during osteotomy. The noncoated drills showed a higher wear resis-
tance, and it has been observed that using worn drills leads to more friction contact,
reduced drill cutting efficiency, and increased heat generation. Eleven in vitro studies
met the inclusion criteria, and showed a considerable methodological heterogeneity
and confounding factors, including drill geometry, speed and load, depth and
diameter, number of uses, irrigation protocol, study specimens, and the heat measur-
ing device. Besides, most of the studies have a potential operator and assessor bias, and
some have sponsorship bias. It is possible to conclude that the literature is not
conclusive on the effect of drill materials on heat generation during osteotomy. Lack of standardization and uniformity in the study design, along with potential bias
in the study methodology can be the reason for the heterogeneity of the results. Keywords
►ceramics
►dental implant
►drills
►friction
►heat
►osteotomy Heat Generation and Implant Drill Materials
Chakraborty et al. 66 Critical Appraisal Due to lack of standardized critical appraisal tools for in vitro
studies, our study selected appraisal criteria based on the
aspects that should be assessed in this type of study designs. The aspects appraised were study aims, outcome measure,
method clarity, sample selection, randomization, allocation
concealment, sample attrition, bias, method of data analysis,
and external validity as presented in ►Fig. 2. Results The initial electronic search identified eight articles. One
article was excluded after abstract screening because it did
not report the measurement of heat generation in relation to
the different drill materials (►Table 1). The remaining seven
articles met the inclusion criteria and were selected. An
additional four articles were identified from the manual Inclusion Criteria Included in this review were studies published on the influ-
ence of drill material on heat generation in osteotomy sites,
studies that answer the research question, and studies
including human, animal, or synthetic bone. Exclusion Criteria Case reports, case series studies, reviews, articles published
earlier than the year 2000, and publications in language
other than English were excluded from our review. The effect of the drill material on heat generation during
osteotomy has been debated. Although some reports con-
firmed this, other research claims that the tool material does
not have a significant effect on heat generation during
osteotomy.11 Despite the advances in the manufacturing of
ceramic burs, only a limited number of studies have tried to
address such an important effect, and there is still a lack of
consensus when it comes to its dental applications.12 The
aim of this study was to explore the experimental studies in
the literature regarding the impact of drill materials and
their wear profile on heat generation in osteotomy sites, and
to critically appraise these studies. Introduction Drill wear is one of the important causes of the rise in
temperature during osteotomy, which needs to be controlled
to avoid thermal osteonecrosis.5 The elevated bone tempera-
ture constitutes a major cause of early dental implant failure
since it may lead to hyperemia, osteocyte degeneration, in-
creased osteoclastic activity, and necrosis, which ultimately
affects the regenerative capacity of the bone and consequently
ends with osseointegration failure.4 Delayed healing, failure of
osseointegration, and necrosis of the bone were reported
during the osteotomy when the temperature exceeded 47°C
for more than 1 minute.6 Therefore, for successful osseointe-
gration, the osteotomy preparation in low temperature is of
utmost importance. For many decades, dental implant drills were made of
stainless steel because of their high cutting efficiency and
durability. Ceramic drills, which are mainly composed of 80%
zirconia oxide and 20% alumina oxide, were lately introduced
in the market. Combining zirconia with magnesium or
alumina stabilizes zirconia, resulting in better biomechanical
properties.7,8 Ceramic drills have the advantage of being
biocompatible,
which
reduces
the
chances
of
allergic
responses to metal during osteotomy preparation. They
show good resistance against high temperature, corrosion,
and wear/abrasion.9 Thus, they are expected to generate less
heat during implant site preparation.10 However, literature
on the use of ceramic drills in implantology is scarce and is
not conclusive on their advantages in lowering heat genera-
tion during drilling. Understanding whether drill material
can affect heat generation during osteotomy preparation is of
high clinical relevance as it can influence the implant success
and survival rate. European Journal of Dentistry
Vol. 18
No. 1/2024
© 2023. The Author(s). Articles Search Strategy The research question “Is there a difference in the amount of
heat generation using different drill materials for osteotomy Heat Generation and Implant Drill Materials
Chakraborty et al. 67 Fig. 1
Flowchart of the articles search strategy. Fig. 1
Flowchart of the articles search strategy. the implant site has a significant impact on the success of
osseointegration,3 because heat-induced bone injuries re-
duce the implant primary stability, leading to implant fail-
ure.17 Furthermore, if the bone is exposed during osteotomy
to a temperature exceeding 47°C for more than 1 minute,
irreversible cellular damage will happen, and the bone will
be replaced with fibrous tissue.18 Drill materials can influ-
ence the heat generation during osteotomy. Therefore, the
selection of drills is of high clinical relevance as it can help in
implant osseointegration and increase their success and
survival rate. These studies have been performed in vitro
on a variety of bone models using different temperature
measurement systems (various thermocouples or infrared
thermography devices). search of the bibliography of the 7 selected articles, resulting
in a total of 11 articles being selected for our review
(►Table 2). The article search strategy was designed to
capture articles that go back to the year 2000; however,
relevant studies were found only after 2010. All the included articles were in vitro studies, of which 10
were performed on bovine bone specimens except 1 study
that was performed on artificially manufactured bone speci-
mens. Drills used in the studies were fabricated from differ-
ent materials such as stainless steel, ceramic, tungsten
carbide, black diamond, and diamond like carbon-coated
drills. Ten of the studies used thermocouples for tempera-
ture measurement placed close to the osteotomy at different
depths to measure the real-time temperature. In study
number 11, the temperature was measured using an infrared
thermometer. The studies evaluated other variables such as
drill wear, effect of depth, and irrigation. The rotational speed
and load used for osteotomy preparation was constant
during the experiments and was reported in all studies. All
selected articles observed an increase in the bone tempera-
ture while preparing the osteotomy sites. The overall tem-
perature increase varied distinctly among the studies, which
could be explained by the confounding factors in each study
(►Table 3). Composition of the Drills Most of the included studies compared stainless steel and
ceramic materials along with some less common drill types,
such as tungsten carbide carbon, titanium nitride, and other
coated drills. In comparison between metal and ceramic
drills, the studies have reported contradictory findings. Some found that stainless steel drills generate more heat
than the ceramic drills9,11,15,19; others showed no differ-
ence,12,14,20 while one study reported higher mean temper-
ature with ceramic drills than metal drills.16 The effect of drill coating was also examined. One study by
Allsobrook et al13 found that tungsten carbide-coated stain-
less steel generated less heat compared with the stainless
steel drills, while Hochscheidt et al reported that steel
diamondlike carbon coating showed highest temperature
than other drills fabricated from surface-treated steel, and
aluminum-toughened zirconium ceramic.21 A direct com-
parison between the study results is neither possible nor Articles Search Strategy Most studies reported the bone baseline temper-
ature, while a few studies did not report the bone baseline
temperature and only compared the maximum temperature
reached by the different drill types.13–16 European Journal of Dentistry
Vol. 18
No. 1/2024
© 2023. The Author(s). Discussion Osseointegration is the direct functional and structural
connection between the surface of the dental implant and
the living bone. Heat generation during drilling to prepare Heat Generation and Implant Drill Materials
Chakraborty et al. 68 g. 2
Traffic light system summarizing critical appraisal of the included articles. g. 2
Traffic light system summarizing critical appraisal of the included articles. Fig. 2
Traffic light system summarizing critical appraisal of the included articles. Fig. 2
Traffic light system summarizing critical appraisal of the included articles. Table 1 List of excluded articles
SN
Study
Database
Article title
1
Batista
Mendes
et al10
PubMed
Influence of implant drill
materials on wear,
deformation, and roughness
after repeated drilling and
sterilization temperature increase during osteotomy, which may be re-
lated to a higher wear resistance of the noncoated drills, as
explored below. Table 1 List of excluded articles European Journal of Dentistry
Vol. 18
No. 1/2024
© 2023. The Author(s). Drill Wear and Number of Uses Heat Generation and Implant Drill Materials
Chakraborty et al. 69 Table 2 List of included articles in a chronological order
SN
Author/year
Database
Article title
1
Sumer et al/2011
PubMed
Comparison of heat generation during implant drilling using stainless steel
and ceramic drills
2
Allsobrook et al/2011
Manual search
Descriptive study of the longevity of dental implant surgery drills
3
Oliveira et al/2012
PubMed
Thermal changes and drill wear in bovine bone during implant site
preparation. A comparative in vitro study: twisted stainless steel and
ceramic drills
4
Harder et al/2013
Manual search
Influence of the drill material and method of cooling on the development
of intrabony temperature during preparation of the site of an implant
5
Moshiri et al/2013
Manual search
Evaluation the effect of drill type on heat generation in implant drilling site
6
Koo et al/2015
PubMed
Effects of implant drill wear, irrigation, and drill materials on heat
generation in osteotomy sites
7
Hochscheidt et al/2017
Manual search
Thermal variation during osteotomy with different dental implant drills: a
standardized study in bovine ribs
8
Er et al/2018
PubMed
Improved dental implant drill durability and performance using heat and
wear resistant protective coatings
9
Koopaie et al/2019
Science Direct
Comparison of wear and temperature of zirconia and tungsten carbide
tools in drilling bone: in vitro and finite element analysis
10
Scarano et al/2020
PubMed
Infrared thermographic evaluation of temperature modifications induced
during implant site preparation with steel vs. zirconia implant drill
11
Tur et al/2020
PubMed
Thermal effects of various drill materials during implant site preparation—
ceramic vs. stainless steel drills: a comparative in vitro study in a
standardised bovine bone model Table 2 List of included articles in a chronological order Table 3 Overall temperature changes during osteotomy
Author/Year
Results
Sumer et al9
The mean temperature in the water bath was 30.1°C. The mean maximum temperature with stainless
steel drills was 35.05°C, whereas that with ceramic drills was 35.9°C
Allsobrook et al13
The mean temperature measured over the experiment was 20.0°C. The maximum temperature reached
was 27.7°C, and the temperature seldom varied 2°C from the initial temperature
Oliveira et al11
Mean baseline bone temperature for the stainless steel and ceramic drills were 21.59 0.1 and
21.6 0.1°C, respectively. The mean maximum increase in temperature was 1.64 1.11°C
Harder et al12
The baseline temperature was 23°C. Drill Wear and Number of Uses The mean maximum temperature recorded was 64.7°C
Hochscheidt et al21
Baseline temperature was 20.32°C and the peak temperatures reached were as follows, in sequence:
steel with a diamondlike carbon coating, 67.6°C at 5-mm drilling depth; aluminum-toughened
zirconium ceramic, 52.1°C at 13-mm depth; and experimentally surface-treated steel, 32.0°C at 13-mm
depth
Er et al20
The baseline temperature was 23–24°C, with the mean maximum bone temperature at 35.5°C
Koopaie et al22
Baseline temperature was 20°C. The mean temperature was 38.3°C
Scarano et al15
The steel drills showed a bone temperature of 42.45 1.70°C, compared with the zirconia drills, which
reported average values of 40.80 0.85°C
Tur et al16
The maximum mean temperature increases for 16-mm drilling without irrigation was 27.20°C
Table 2 List of included articles in a chronological order
SN
Author/year
Database
Article title
1
Sumer et al/2011
PubMed
Comparison of heat generation during implant drilling using stainless steel
and ceramic drills
2
Allsobrook et al/2011
Manual search
Descriptive study of the longevity of dental implant surgery drills
3
Oliveira et al/2012
PubMed
Thermal changes and drill wear in bovine bone during implant site
preparation. A comparative in vitro study: twisted stainless steel and
ceramic drills
4
Harder et al/2013
Manual search
Influence of the drill material and method of cooling on the development
of intrabony temperature during preparation of the site of an implant
5
Moshiri et al/2013
Manual search
Evaluation the effect of drill type on heat generation in implant drilling site
6
Koo et al/2015
PubMed
Effects of implant drill wear, irrigation, and drill materials on heat
generation in osteotomy sites
7
Hochscheidt et al/2017
Manual search
Thermal variation during osteotomy with different dental implant drills: a
standardized study in bovine ribs
8
Er et al/2018
PubMed
Improved dental implant drill durability and performance using heat and
wear resistant protective coatings
9
Koopaie et al/2019
Science Direct
Comparison of wear and temperature of zirconia and tungsten carbide
tools in drilling bone: in vitro and finite element analysis
10
Scarano et al/2020
PubMed
Infrared thermographic evaluation of temperature modifications induced
during implant site preparation with steel vs. zirconia implant drill
11
Tur et al/2020
PubMed
Thermal effects of various drill materials during implant site preparation—
ceramic vs. stainless steel drills: a comparative in vitro study in a
standardised bovine bone model
Heat Generation and Implant Drill Materials
Chakraborty et al. Drill Wear and Number of Uses Ithasbeenobservedthat using worndrillsleadstomorefriction
contact, reduced drill cutting efficiency, and increased heat
generation. This may increase the possibility of bone necrosis,
irreversible cellular damage, and replacement of bone tissue
with fat tissue.20 Therefore, the number of drill reuse is an
important factor in heat generation during osteotomy. valid, as the wear resistance of pure zirconia, for instance, is
different to that of zirconium oxide coating. It is apparent
that drill materials had a significant influence on the overall valid, as the wear resistance of pure zirconia, for instance, is
different to that of zirconium oxide coating. It is apparent
that drill materials had a significant influence on the overall The number of drill uses has varied among the reviewed
studies, but most of them have used the drills for 50 times, European Journal of Dentistry
Vol. 18
No. 1/2024
© 2023. The Author(s). and different conclusions were reached. Oliveira et al com-
pared scanning electron microscope (SEM) images of both
stainless steel and zirconia drills before and following 50
uses, and identified different patterns of wear.11 The ceramic
tip, contrasting with the stainless steel drill, which showed a
higher tip wear in both edges. However, none of the drills
appeared to present severe deformation or blunting after 50
uses. This is not consistent with Koopaie et al in which the
Table 3 Overall temperature changes during osteotomy
Author/Year
Results
Sumer et al9
The mean temperature in the water bath was 30.1°C. The mean maximum temperature with stainless
steel drills was 35.05°C, whereas that with ceramic drills was 35.9°C
Allsobrook et al13
The mean temperature measured over the experiment was 20.0°C. The maximum temperature reached
was 27.7°C, and the temperature seldom varied 2°C from the initial temperature
Oliveira et al11
Mean baseline bone temperature for the stainless steel and ceramic drills were 21.59 0.1 and
21.6 0.1°C, respectively. The mean maximum increase in temperature was 1.64 1.11°C
Harder et al12
The baseline temperature was 23°C. The mean maximum intrabony temperature increased was 3.9°C
Moshiri et al19
Baseline temperature not mentioned. The mean maximum temperature was 33°C
Koo et al14
The mean internal temperature is 36.5°C and the surface temperature of 28°C (baseline temperature). European Journal of Dentistry
Vol. 18
No. 1/2024
© 2023. The Author(s). Drill Wear and Number of Uses The mean maximum intrabony temperature increased was 3.9°C
Moshiri et al19
Baseline temperature not mentioned. The mean maximum temperature was 33°C
Koo et al14
The mean internal temperature is 36.5°C and the surface temperature of 28°C (baseline temperature). The mean maximum temperature recorded was 64.7°C
Hochscheidt et al21
Baseline temperature was 20.32°C and the peak temperatures reached were as follows, in sequence:
steel with a diamondlike carbon coating, 67.6°C at 5-mm drilling depth; aluminum-toughened
zirconium ceramic, 52.1°C at 13-mm depth; and experimentally surface-treated steel, 32.0°C at 13-mm
depth
Er et al20
The baseline temperature was 23–24°C, with the mean maximum bone temperature at 35.5°C
Koopaie et al22
Baseline temperature was 20°C. The mean temperature was 38.3°C
Scarano et al15
The steel drills showed a bone temperature of 42.45 1.70°C, compared with the zirconia drills, which
reported average values of 40.80 0.85°C
Tur et al16
The maximum mean temperature increases for 16-mm drilling without irrigation was 27.20°C Table 3 Overall temperature changes during osteotomy and different conclusions were reached. Oliveira et al com-
pared scanning electron microscope (SEM) images of both
stainless steel and zirconia drills before and following 50
uses, and identified different patterns of wear.11 The ceramic
drills had slightly visible alterations on one edge of the drill tip, contrasting with the stainless steel drill, which showed a
higher tip wear in both edges. However, none of the drills
appeared to present severe deformation or blunting after 50
uses. This is not consistent with Koopaie et al in which the
overall wear of the ceramic drills after 45 uses was about half European Journal of Dentistry
Vol. 18
No. 1/2024
© 2023. The Author(s). Heat Generation and Implant Drill Materials
Chakraborty et al. 70 that of the tungsten carbide drills. However, the comparator
in both studies was different.22 The SEM images in the third
study by Allsobrook et al13 found that tungsten carbide drills
display least corrosion and wear and lowest mean drilling
temperatures after 20 osteotomies when compared with
stainless steel drills. Interestingly Scarano et al15 found
that in case of using zirconia drills, the outline of the implant
bed was well defined even after 120 osteotomies. Another
noteworthy finding is that by Koo et al14 who found that only
the initial/pilot drills generate more heat after 50 uses. Drill Wear and Number of Uses subject, which reported higher temperature while preparing
the deeper parts of the osteotomies due to ineffective cooling
with irrigation. These contradictory results again raise con-
cerns regarding the validity of the study methods. Study Models/Specimens The drill geometry is of critical importance to heat genera-
tion, and has been investigated in many studies. Therefore, to
make a valid comparison between the studies, the geometry
of the drills used in the studies should be similar. Koo et al14
used drills of different shapes, which could affect the inter-
pretation of the data. The other studies either have used
similar drills or have not reported the design of the drills
used in their experiments. No human-based articles were identified, which may be due
to ethical reasons, since these experiments require an inva-
sive insertion of special equipment to detect the temperature
change. Most of the included articles have used bovine bone
for the experiment, which has been used successfully in
many studies to investigate heat generation in response to
implant site preparation. However, there is no standard
study model for conducting experiments on heat generation
during osteotomy.6 Thus, the studies have used different
parts of bovine bone like ribs, femur, and scapular bone to
conduct the experiment, which was considered to have a
similar density as the human bone. However, none of the
studies reported the bone density type, despite the signifi-
cant importance of bone density in heat generation and
thermal conductivity. The
included studies have only
claimed that the density is comparable to that of the human
bone. Irrigation during Osteotomy The use of irrigation has been proven to decrease the amount
of heat generation during osteotomy. Most of the studies
have used external irrigation with saline solution, but the
flow rate and solution temperature are either variant or not
reported, which can affect the rigor of the studies. Drill Speed and Applied Load The speed and load used during osteotomy were also not
consistent among the studies. It has been established that the
increase or decrease in speed and load can affect the tem-
perature change during implant site preparation. Hence, the
results obtained in different studies are not comparable. Furthermore, not all studies reported these parameters. For instance, Koopaie et al22 and Er et al20 did not mention
the used load in the experiments, while Tur et al16 did not
mention both the speed and load. There are other concerns regarding the generalizability of
results obtained from dead bone. First, in addition to bone
density, thermal conductivity varies significantly between
live and dead bone. This can be due to the difference in
cellularity, water content, and fluid movement.23 Second,
dead bone can be fresh or frozen and thawed. It is of
paramount importance to know the baseline temperature
of the bone block prior to experiment to appreciate the exact
increase in the temperature during drilling. Ten of the
included articles have used bovine bone specimens, which
were frozen and then thawed in a thermostat-controlled
water bath or immersed in saline solution to minimize the
thermophysical and mechanical changes of the proteins in
the dead bones. This also maintained the internal tempera-
ture of the bone specimen before the start of the experiment. Only one study by Moshiri et al19 failed to explain the
procedure used to maintain the bovine bones and the
baseline temperature in the experiment. European Journal of Dentistry
Vol. 18
No. 1/2024
© 2023. The Author(s). Depth and Diameter of the Osteotomy The temperature was measured in the included studies at
different depths and for different diameters of the final drill. Koo et al14 suggested that the initial (pilot) drill should be
changed after 50 uses, as this drill leads to more heat
generation because it is the first drill to cut the bone in
the osteotomy compared with the following drills, but most
of the studies have measured temperature increase with the
final drill as it can provide accurate temperature change
because the thermocouple placement needs to be closer to
the prepared osteotomy wall. Similarly, the temperature was measured for one particu-
lar depth in all the studies instead of measuring throughout
the osteotomy and the level of depth varies in each study. Again, this raises similar doubts in the validity of comparing
the studies. Few of the studies have found that the superficial
part of the osteotomy cavity generates more heat compared
with the deeper parts.12,15 The study by Moshiri et al19 found
that the temperature was higher at the depth of 6 mm when
compared with that at 3 and 9 mm. The authors observed
that this result is similar to other studies stating that there
will be more heat generation during the preparation of the
superficial part of the osteotomy site. The studies explained
this finding by stating that friction is higher in the superficial
part due to the higher content of cortical bone. This conclu-
sion contradicts with many previous publications on the References 1 Chauhan CJ, Shah DN, Sutaria FB. Various bio-mechanical factors
affecting heat generation during osteotomy preparation: a sys-
tematic review. Indian J Dent Res 2018;29(01):81–92 2 Hein C, Inceoglu S, Juma D, Zuckerman L. Heat generation during
bone drilling: a comparison between industrial and orthopaedic
drill bits. J Orthop Trauma 2017;31(02):e55–e59 2 Hein C, Inceoglu S, Juma D, Zuckerman L. Heat generation during
bone drilling: a comparison between industrial and orthopaedic
drill bits. J Orthop Trauma 2017;31(02):e55–e59 3 Mishra SK, Chowdhary R. Heat generated by dental implant drills
during osteotomy-a review: heat generated by dental implant
drills. J Indian Prosthodont Soc 2014;14(02):131–143 3 Mishra SK, Chowdhary R. Heat generated by dental implant drills
during osteotomy-a review: heat generated by dental implant
drills. J Indian Prosthodont Soc 2014;14(02):131–143 There are many confounding factors in studies evaluating
the heat generation during the osteotomy preparation. There-
fore, standardization and uniformity in the study design is
required to comparetheresultsand to make avalidconclusion. The present review attempted to standardize the processes of
identifyingandappraising therelevantarticlesintheliterature
despite the lack of standardized appraisal tools for in vitro
studies. Finally, a comprehensive evaluation of all the relevant
confounding factors within all the studies was conducted,
which helped identify the missing gap of knowledge in the
literature. The results of this study can be generalized to
clinical practice with caution as the included articles were in
vitro studies, with a significant dissimilarity between live
human and dead animal bone. This, besides the remarkable
heterogeneity and potential bias in the study methods. 4 Trisi P, Berardini M, Falco A, Vulpiani MP. Effect of temperature on the
dental implant osseointegration development in low-density bone:
an in vivo histological evaluation. Implant Dent 2015;24(01):96–100 4 Trisi P, Berardini M, Falco A, Vulpiani MP. Effect of temperature on the
dental implant osseointegration development in low-density bone:
an in vivo histological evaluation. Implant Dent 2015;24(01):96–100 5 Augustin G, Zigman T, Davila S, et al. Cortical bone drilling and
thermal osteonecrosis. Clin Biomech (Bristol, Avon) 2012;27(04):
313–325 5 Augustin G, Zigman T, Davila S, et al. Cortical bone drilling and
thermal osteonecrosis. Clin Biomech (Bristol, Avon) 2012;27(04):
313–325 6 Möhlhenrich SC, Modabber A, Steiner T, Mitchell DA, Hölzle F. Heat generation and drill wear during dental implant site prepa-
ration: systematic review. Br J Oral Maxillofac Surg 2015;53(08):
679–689 7 Bayerlein T, Proff P, Richter G, Dietze S, Fanghänel J, Gedrange T. Temperature Changes Measurement Temperature Changes Measurement
Thermocouples can detect only spot temperatures and not
the overall thermal profile. Therefore, factors like depth of
recording, distance of the sensor from the osteotomy site,
and material of sensor element can affect the overall results. Nine of the included articles have used thermocouples for
measuring the changes in the temperature during implant
bed site preparation. But their use is not uniform, especially
in respect to distance of the sensor from the osteotomy, Heat Generation and Implant Drill Materials
Chakraborty et al. 71 which ranges from 0.2 to 1.5 mm. Sumer et al9 and Moshiri
et al19 have not even mentioned the distance. However,
Koopaie et al22 have used infrared thermometer for heat
measurement, which is considered a better option as it
provides the overall thermal profile with less degree of error. On the other hand, Scarano et al15 used infrared thermogra-
phy for evaluating the change in temperature during implant
bed preparation. There is no study in the literature that
compares the accuracy of heat generation using both tech-
niques. Hence, determining a preference is still difficult. Furthermore, despite the importance of calibrating the in-
strument before the experiment to verify the precision of
temperature recording (device reliability), only few of the
reviewed studies have reported undertaking this using a
proper method for calibration. osteotomy. It is important for clinicians to be aware of these
findings, as they underscore the need to carefully inspect
wear patterns and consider the appropriate drill material
when performing osteotomy procedures. By taking these
factors into account, clinicians can help minimize the risk of
excessive heat generation, which can have a negative impact
on implant success rates and treatment outcomes. Study Bias Blinding the operator and assessor is difficult in these studies
as the operator needs to see the drills while performing the
experiment and the assessor has to measure the heat instan-
taneously. Having said that, these types of bias can be reduced
by assigning different operators for each group along with an
independent assessor. None of the studies have performed
this, which makes them all proneto considerablebias. Another
important source of bias observed in these studies is sponsor-
ship bias. Some articles failed to acknowledge the source of
funding andtoprovide a clear statementonconflictof interest. There are many confounding factors in studies evaluating
the heat generation during the osteotomy preparation. There-
fore, standardization and uniformity in the study design is
required to comparetheresultsand to make avalidconclusion. The present review attempted to standardize the processes of
identifyingandappraising therelevantarticlesintheliterature
despite the lack of standardized appraisal tools for in vitro
studies. Finally, a comprehensive evaluation of all the relevant
confounding factors within all the studies was conducted,
which helped identify the missing gap of knowledge in the
literature. The results of this study can be generalized to
clinical practice with caution as the included articles were in
vitro studies, with a significant dissimilarity between live
human and dead animal bone. This, besides the remarkable
heterogeneity and potential bias in the study methods. Blinding the operator and assessor is difficult in these studies
as the operator needs to see the drills while performing the
experiment and the assessor has to measure the heat instan-
taneously. Having said that, these types of bias can be reduced
by assigning different operators for each group along with an
independent assessor. None of the studies have performed
this, which makes them all proneto considerablebias. Another
important source of bias observed in these studies is sponsor-
ship bias. Some articles failed to acknowledge the source of
funding andtoprovide a clear statementonconflictof interest. Conflict of Interest
None declared. Conflict of Interest
None declared. Conclusion 10 Batista Mendes GC, Padovan LEM, Ribeiro-Júnior PD, Sartori EM,
Valgas L, Claudino M. Influence of implant drill materials on wear,
deformation, and roughness after repeated drilling and steriliza-
tion. Implant Dent 2014;23(02):188–194 It is possible to conclude that the literature about the effect of
drill materials on heat generation during osteotomy is not
conclusive. The lack of standardization and uniformity in the
study design, along with potential bias in the study meth-
odology, makes it challenging to draw concrete conclusions
based on the findings. However, the findings of this study can
still serve as a helpful guide for clinicians seeking to optimize
their approach to implant procedures. 11 Oliveira N, Alaejos-Algarra F, Mareque-Bueno J, Ferrés-Padró E,
Hernández-Alfaro F. Thermal changes and drill wear in bovine
bone during implant site preparation. A comparative in vitro
study: twisted stainless steel and ceramic drills. Clin Oral
Implants Res 2012;23(08):963–969 12 Harder S, Egert C, Wenz HJ, Jochens A, Kern M. Influence of the
drill material and method of cooling on the development of
intrabony temperature during preparation of the site of an
implant. Br J Oral Maxillofac Surg 2013;51(01):74–78 References The use of ceramic drills on a zirconium oxide basis in bone
preparation. Folia Morphol (Warsz) 2006;65(01):72–74 8 Scarano A, Carinci F, Quaranta A, Di Iorio D, Assenza B, Piattelli A. Effects of bur wear during implant site preparation: an in vitro
study. Int J Immunopathol Pharmacol 2007;20(1, Suppl 1):23–26 9 Sumer M, Misir AF, Telcioglu NT, Guler AU, Yenisey M. Comparison
of heat generation during implant drilling using stainless steel
and ceramic drills. J Oral Maxillofac Surg 2011;69(05):1350–1354 Limitations of the Study To minimize the effect of clinical heterogeneity and con-
founding factors on the study outcomes, this review focused
solely on benchtop research related to the subject. Therefore,
the generalizability of the findings to clinical practice may be
limited. European Journal of Dentistry
Vol. 18
No. 1/2024
© 2023. The Author(s). Clinical Implications 13 Allsobrook OFL, Leichter J, Holborrow D, Swain M. Descriptive
study of the longevity of dental implant surgery drills. Clin
Implant Dent Relat Res 2011;13(03):244–254 13 Allsobrook OFL, Leichter J, Holborrow D, Swain M. Descriptive
study of the longevity of dental implant surgery drills. Clin
Implant Dent Relat Res 2011;13(03):244–254 The findings of this review emphasize the significant impact
of drill material and wear behavior on heat generation during 72 Heat Generation and Implant Drill Materials
Chakraborty et al. 14 Koo KT, Kim MH, Kim HY, Wikesjö UME, Yang JH, Yeo IS. Effects of
implant drill wear, irrigation, and drill materials on heat genera-
tion in osteotomy sites. J Oral Implantol 2015;41(02):e19–e23 19 Moshiri Z, Roshanaei G, Vafaei F, Kadkhodazadeh M. Evaluation
the effect of drill type on heat generation in implant drilling site. Res J Med Sci 2013;7(5–6):118–122 15 Scarano A, Lorusso F, Noumbissi S. Infrared thermographic evalu-
ation of temperature modifications induced during implant site
preparation with steel vs. zirconia implant drill. J Clin Med 2020;9
(01):148 20 Er N, Alkan A, Ilday S, Bengu E. Improved dental implant drill
durability and performance using heat and wear resistant protec-
tive coatings. J Oral Implantol 2018;44(03):168–175 21 Hochscheidt CJ, Shimizu RH, Andrighetto AR, Moura LM, Golin AL,
Hochscheidt RC. Thermal variation during osteotomy with differ-
ent dental implant drills: a standardized study in bovine ribs. Implant Dent 2017;26(01):73–79 16 Tur D, Giannis K, Unger E, Mittlböck M, Rausch-Fan X, Strbac GD. Thermal effects of various drill materials during implant site
preparation—ceramic vs. stainless steel drills: a comparative in
vitro study in a standardised bovine bone model. Clin Oral
Implants Res 2021;32(02):154–166 22 Koopaie M, Kolahdouz S, Kolahdouz EM. Comparison of wear and
temperature of zirconia and tungsten carbide tools in drilling
bone: in vitro and finite element analysis. Br J Oral Maxillofac Surg
2019;57(06):557–565 17 Yoshida K, Uoshima K, Oda K, Maeda T. Influence of heat stress to
matrix on bone formation. Clin Oral Implants Res 2009;20(08):
782–790 23 Tehemar SH. Factors affecting heat generation during implant
site preparation: a review of biologic observations and future
considerations. Int J Oral Maxillofac Implants 1999;14(01):
127–136 23 Tehemar SH. Factors affecting heat generation during implant
site preparation: a review of biologic observations and future
considerations. Int J Oral Maxillofac Implants 1999;14(01):
127–136 18 Eriksson RA, Adell R. Temperatures during drilling for the place-
ment of implants using the osseointegration technique. European Journal of Dentistry
Vol. 18
No. 1/2024
© 2023. The Author(s). Clinical Implications J Oral
Maxillofac Surg 1986;44(01):4–7
|
https://openalex.org/W3190795812
|
https://www.biorxiv.org/content/biorxiv/early/2021/08/04/2021.08.04.455019.full.pdf
|
English
| null |
Probing the stability of the SpCas9-DNA complex after cleavage
|
bioRxiv (Cold Spring Harbor Laboratory)
| 2,021
|
cc-by
| 11,875
|
Probing the stability of the SpCas9-DNA complex after cleavage Pierre Aldag1, Fabian Welzel1, Leonhard Jakob2, Andreas Schmidbauer2, Marius Rutkauskas1, Fergus
Fettes1, Dina Grohmann2,3, and Ralf Seidel1,*
1 Peter Debye Institute for Soft Matter Physics, University of Leipzig, Leipzig, 04103, Germany
2 Institute of Microbiology & Archaea Centre, Single-Molecule Biochemistry Lab, University of
Regensburg, Regensburg, 93053, Germany
3 Regensburg Center of Biochemistry (RCB), University of Regensburg, 93053 Regensburg, Germany
* To whom correspondence should be addressed. Tel: +49 341 9732501; Email: ralf.seidel@physik.uni-
leipzig.de
Present Address: Leonhard Jakob, Department of Pharmacology and Toxicology, Institute of Pharmacy,
University of Regensburg, Regensburg, 03053, Germany Pierre Aldag1, Fabian Welzel1, Leonhard Jakob2, Andreas Schmidbauer2, Marius Rutkauskas1, Fergus
Fettes1, Dina Grohmann2,3, and Ralf Seidel1,* 1 Peter Debye Institute for Soft Matter Physics, University of Leipzig, Leipzig, 04103, Germany
2 Institute of Microbiology & Archaea Centre, Single-Molecule Biochemistry Lab, University of
Regensburg, Regensburg, 93053, Germany g
g
y (
)
y
g
g
g
g
y
* To whom correspondence should be addressed. Tel: +49 341 9732501; Email: ralf.seidel@physik.uni-
leipzig.de * To whom correspondence should be addressed. Tel: +49 341 9732501; Email: ralf.seidel@physik.uni-
leipzig.de * To whom correspondence should be addressed. Tel: +49 341 9732501; Email: ralf.seidel@physik.uni-
leipzig.de Present Address: Leonhard Jakob, Department of Pharmacology and Toxicology, Institute of Pharmacy,
University of Regensburg, Regensburg, 03053, Germany Present Address: Leonhard Jakob, Department of Pharmacology and Toxicology, Institute of Pharmacy,
University of Regensburg, Regensburg, 03053, Germany Present Address: Leonhard Jakob, Department of Pharmacology and Toxicology, Institute of Pharmacy,
University of Regensburg, Regensburg, 03053, Germany Present Address: Leonhard Jakob, Department of Pharmacology and Toxicology, Institute of Pharmacy,
University of Regensburg, Regensburg, 03053, Germany .
CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprin
this version posted August 4, 2021.
;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprin
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint ABSTRACT CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-
stranded DNA. Wildtype Cas9 as well as its nickase and cleavage-incompetent mutants have been
used in various biological techniques due to their versatility and programmable specificity. Cas9 has
been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting
further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in
genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the
binding stability of different S. pyogenes Cas9 variants after cleavage by challenging them with
supercoiling. We find that different release mechanisms occur depending on which DNA strand is
cleaved. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling
of the non-target strand around the DNA with friction. Consequently, Cas9 and its non-target strand
nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress. After
target-strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified
several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage
state of Cas9 exhibits a higher stability compared to the pre-cleavage state. This suggests that Cas9
has evolved to remain tightly bound to its cut target. . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint INTRODUCTION The TS and NTS are cleaved by the HNH and the RuvC
domains, respectively, followed by a further conformational change into a post-cleavage product state
(16,12). In this state, the HNH domain undocks from the cleavage site and is thought to become
completely disordered. After inducing a double-strand break, an extremely slow release of the DNA
products essentially limits SpCas9 to undergo further turnovers (17). This results in an inhibited and
slow in-vivo DNA repair process (18,19). However, even for target bound SpCas9, the 3’ end of the
cleaved NTS can be released and targeted by complementary ssDNA or 3’ to 5’ ssDNA exonucleases
(16,19). CRISPR (Clustered Regularly Interspaced Palindromic Repeats)-Cas (CRISPR associated) systems
constitute adaptive immune systems against nucleic acid-containing invaders in prokaryotic cells (1). CRISPR-Cas9 from the bacterial species Streptococcus pyogenes (SpCas9) is a 160 kD protein that
forms a ribonucleoprotein (RNP) complex with CRISPR-RNA (crRNA) and trans-activating RNA
(tracrRNA). This complex can site-specifically target double-stranded DNA (dsDNA) and induces a
double-strand break using its RuvC and HNH nuclease domains (2–4). Since its discovery, SpCas9
and other Cas9 variants have been widely used in rapidly emerging genome engineering applications
in prokaryotic, plant and animal cells (5–7). The target specificity is mainly encoded by the 20 nt spacer
region at the 5’ end of the crRNA of the SpCas9 RNP complex. The tracrRNA and crRNA can be
replaced by a single-guide RNA chimera (sgRNA) (4) which simplifies RNP formation. A prerequisite
for site-specific DNA targeting is a three-nucleotide PAM sequence (protospacer-adjacent motif, NGG
for SpSpas9) next to the target site. SpCas9 weakly interacts with suitable PAM sites via facilitated
diffusion (8). PAM recognition triggers an initial melting of the DNA double strand and primes base
pairing between the crRNA spacer and the DNA target strand (TS) while displacing the non-target DNA
strand (NTS) such that a so-called R-loop structure is formed (9). R-loop expansion is initiated from the
PAM proximal nucleotides over the entire spacer region and constitutes the actual recognition of
matching targets. In case of strongly mismatched targets, R-loop expansion is impeded and stops or
the R-loop collapses (10). Successful base pairing between TS and crRNA spacer is verified by two
conformational changes of the SpCas9 RNP. Formation of an R-loop intermediate of 9 bp length triggers
the transition into a “checkpoint” state, opening up a channel to further accommodate the TS-crRNA
hybrid. INTRODUCTION CRISPR (Clustered Regularly Interspaced Palindromic Repeats)-Cas (CRISPR associated) systems
constitute adaptive immune systems against nucleic acid-containing invaders in prokaryotic cells (1). CRISPR-Cas9 from the bacterial species Streptococcus pyogenes (SpCas9) is a 160 kD protein that
forms a ribonucleoprotein (RNP) complex with CRISPR-RNA (crRNA) and trans-activating RNA
(tracrRNA). This complex can site-specifically target double-stranded DNA (dsDNA) and induces a
double-strand break using its RuvC and HNH nuclease domains (2–4). Since its discovery, SpCas9
and other Cas9 variants have been widely used in rapidly emerging genome engineering applications
in prokaryotic, plant and animal cells (5–7). The target specificity is mainly encoded by the 20 nt spacer
region at the 5’ end of the crRNA of the SpCas9 RNP complex. The tracrRNA and crRNA can be
replaced by a single-guide RNA chimera (sgRNA) (4) which simplifies RNP formation. A prerequisite
for site-specific DNA targeting is a three-nucleotide PAM sequence (protospacer-adjacent motif, NGG
for SpSpas9) next to the target site. SpCas9 weakly interacts with suitable PAM sites via facilitated
diffusion (8). PAM recognition triggers an initial melting of the DNA double strand and primes base
pairing between the crRNA spacer and the DNA target strand (TS) while displacing the non-target DNA
strand (NTS) such that a so-called R-loop structure is formed (9). R-loop expansion is initiated from the
PAM proximal nucleotides over the entire spacer region and constitutes the actual recognition of
matching targets. In case of strongly mismatched targets, R-loop expansion is impeded and stops or
the R-loop collapses (10). Successful base pairing between TS and crRNA spacer is verified by two
conformational changes of the SpCas9 RNP. Formation of an R-loop intermediate of 9 bp length triggers
the transition into a “checkpoint” state, opening up a channel to further accommodate the TS-crRNA
hybrid. This channel is formed by a conformational change of the REC2 and REC3 domains (11,12). In
this state, the HNH domain is still positioned more than 30 Å away from the cleavage site in the TS. The second transition into the “docked” state is induced once the R-loop reaches the PAM-distal end,
causing a large movement of the HNH domain to reach its site of catalytic activity (13–15). Mg2+ ions
as cofactor and a maximum of three terminal mismatches are prerequisites for the transition into the
docked state that allows DNA cleavage. INTRODUCTION CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint Cas9-mediated functionalities (22,23,21). While a large body of research has investigated the structure
of the conformational states of SpCas9 during R-loop formation and after DNA cleavage, the stability
of the SpCas9-DNA complex in these states and their dependence on supercoiling remains elusive. Developing a thorough understanding of the post-cleavage stability of SpCas9 and the respective
mutants can be helpful to improve the efficiencies of genome editing techniques. Here, we investigated the post-cleavage state stability of SpCas9 and its respective nickase mutants
by challenging it with supercoiling using single-molecule magnetic tweezers experiments (9,24,25). We
show that SpCas9 bound to a target with a dsDNA break or a nicked NTS can easily relax superhelical
tension. It can thus not be dissociated in processes that generate this kind of mechanical stress, such
as transcription and replication (26–28). In contrast, SpCas9 bound to an intact target or a target with a
nicked TS cannot release superhelical tension and can thus be dissociated from the DNA when elevated
tension is induced. In both cases, protein states with different stabilities are found indicating dynamic
structural transitions in the post-celavage state. We show that dissociation from a target DNA with a
nicked TS requires considerably higher levels of supercoiling compared to an intact target, suggesting
an increased stability of the post-cleavage conformational state. DNA substrates for magnetic tweezers measurements Double-stranded DNA constructs for magnetic tweezers experiments with lengths of 1500 bp and
2700 bp were prepared as previously described (25). A single copy of a given protospacer matching
the spacer of the SpCas9 sgRNA and including a 5’-AGG-3’PAM on the non-target strand (see Table
S1) was cloned into the SmaI site of a pUC19 plasmid. From the plasmid, a 1.5 kbp or 2.7 kbp fragment
including the SpCas9 target site was amplified by PCR, using primers in which either a NotI or a SpeI
restriction enzyme site was introduced (see Table S1). After digestion with NotI and SpeI, the fragment
was ligated at either end to ~600-bp-PCR fragments containing multiple biotin (SpeI site) or digoxigenin
(NotI site) modifications (29). INTRODUCTION This channel is formed by a conformational change of the REC2 and REC3 domains (11,12). In
this state, the HNH domain is still positioned more than 30 Å away from the cleavage site in the TS. The second transition into the “docked” state is induced once the R-loop reaches the PAM-distal end,
causing a large movement of the HNH domain to reach its site of catalytic activity (13–15). Mg2+ ions
as cofactor and a maximum of three terminal mismatches are prerequisites for the transition into the
docked state that allows DNA cleavage. The TS and NTS are cleaved by the HNH and the RuvC
domains, respectively, followed by a further conformational change into a post-cleavage product state
(16,12). In this state, the HNH domain undocks from the cleavage site and is thought to become
completely disordered. After inducing a double-strand break, an extremely slow release of the DNA
products essentially limits SpCas9 to undergo further turnovers (17). This results in an inhibited and
slow in-vivo DNA repair process (18,19). However, even for target bound SpCas9, the 3’ end of the
cleaved NTS can be released and targeted by complementary ssDNA or 3’ to 5’ ssDNA exonucleases
(16,19). Introducing inactivating point mutations at positions D10A (RuvC) or H840A (HNH) turns SpCas9 into
TS or NTS nickases (4). Cas9 nickase mutants and cleavage-incompetent Cas9, which carries both
mutations, have been used in various genome editing applications to increase specificity or to avoid
double-strand breaks (20,21). Fusion of Cas9 variants with other enzymes, such as the repair enzyme
Rad51, the endonuclease FokI, or an engineered reverse transcriptase, expanded the repertoire of Introducing inactivating point mutations at positions D10A (RuvC) or H840A (HNH) turns SpCas9 into
TS or NTS nickases (4). Cas9 nickase mutants and cleavage-incompetent Cas9, which carries both
mutations, have been used in various genome editing applications to increase specificity or to avoid
double-strand breaks (20,21). Fusion of Cas9 variants with other enzymes, such as the repair enzyme
Rad51, the endonuclease FokI, or an engineered reverse transcriptase, expanded the repertoire of . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . Preparation of SpCas9 Plasmids for the expression of wildtype SpCas9 and the catalytically inactive SpCas9 variants carrying
the mutations D10A/H840A were obtained from Addgene (plasmid ID: 39312 and 39318). SpCas9
variants that carried a single mutation of the catalytic site (D10A or H840A) were generated by site-
directed mutagenesis using the wildtype SpCas9 expression plasmid as template. Expression was
performed in E. coli BL21 (DE3) cells. Expression and cell lysis conditions were chosen as described
by Jinek et al. (4). The cleared cell lysate was applied to a HisTrap FF Ni-NTA affinity column (GE
healthcare) equilibrated with HisA buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl2, 5% (v/v)
glycerol, 20 mM imidazole). The column was washed with 2 column volumes (cv) of HisA buffer and
10 cv of HisA buffer containing 1 M NaCl to liberate unspecifically bound nucleic acids from SpCas9. Subsequently, the column was equilibrated with 2 cv of HisA buffer and pre-eluted with 2 cv buffer HisA
with increased imidazole concentration of 40 mM. In the final elution step, the column was washed with
5 cv elution buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl2, 5% (v/v) glycerol, 200 mM
imidazole). Elution fractions were pooled and digested with 50 U AcTEV protease (Thermo Fisher
Scientific) over night at 4 °C. The cleaved His6-MBP-tag was removed by gel filtration using a HiLoad
16/60 Superdex 200 column (GE healthcare) equilibrated in size exclusion buffer (20 mM Tris-HCl,
150 mM NaCl, 5 mM MgCl2, 5% (v/v) glycerol, 1 mM DTT). Elution fractions contained nucleic acid free
SpCas9 or SpCas9 variants (A260/280 < 0.6). Fractions were flash frozen in liquid nitrogen and stored
at -80 °C. sgRNA synthesis for magnetic tweezers experiments The template for T7 transcription of the SpCas9 single sgRNA was generated by PCR, using the High
Fidelity Phusion Polymerase (Thermo Fisher Scientific) and overlapping oligonucleotides. RNAs were
produced by in vitro transcription using the TranscriptAid T7 High Yield Transcription Kit (Thermo
Fisher Scientific) and purified using the GeneJet RNA Cleanup and Concentration Kit (Thermo Fisher
Scientific). The sequences of the oligonucleotides and sgRNA used in this study are available in
Table S1. . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint Reconstitution of guideRNA-SpCas9 complexes For characterizing DNA cleavage, SpCas9 was pre-loaded with either a sgRNA or a pre-annealed
crRNA:tracrRNA complex (Table S1 and S2). To reconstitute SpCas9-RNA complexes, a stock solution
containing 1 µM SpCas9 and 1 µM guide RNA in 1x Cas Buffer (20 mM Tris–HCl pH 7.5, 100 mM
NaCl, 10 mM MgCl2, 2% (v/v) glycerol, 1 mM DTT, 0.05% (v/v) Tween20) was incubated for 10 minutes
at room temperature followed by a centrifugation step (20.000 g, 5 min, room temperature). Plasmid-DNA cleavage assays As substrate for plasmid-DNA cleavage assays, we cloned target plasmid (pM53.1; 6290 bp) containing
the sequence of the S. cerevisiae PHO5 gene with its native genomic context 500 bp upstream and
downstream of the gene in a pBluescript vector backbone. The PHO5 locus was amplified from yeast
genomic
DNA
using
primers
5’-GGCCGCAGATCAAGTCAGAG-3’
and
5’-
GCAAGTCACGAGAAATACCA-3’. The gene is flanked by a Z. rouxii sequence-specific recombinase
R recognition site (RS) upstream, and a triple E. coli LexA binding site downstream of the gene followed
by another RS element. The plasmid was generated making use of pM49.2 containing the upstream
RS element and the downstream triple LexA binding site and RS element (for details of the cloning
procedure see (30)). Reconstituted SpCas9-sgRNA complexes were added in 10-fold excess to 5 nM
pM53.1 plasmid DNA containing the target sequence and a 5’-AGG-3’ PAM motif in 1x Cas buffer. For
kinetic analysis, 15 µl samples were taken at different time points and the reactions were stopped by
addition of EDTA (83 mM final concentration). Subsequently, SpCas9 was digested by the addition of . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint 0.36 U Proteinase K (Thermo Fisher Scientific) followed by an incubation at 55 °C for one hour. After
Proteinase K digestion, 6x DNA loading dye (10 mM Tris-HCl pH 7.5, 0.03% bromophenol blue (w/v),
60% glycerol (v/v), 6 mM EDTA) was added to the samples and fragments were resolved on a 0.9%
agarose gel. Cleavage products were visualized using a ChemiDoc Imaging System (Bio-Rad) after
ethidium bromide staining of the agarose gel. Short target DNA cleavage assay for determination of SpCas9 strand-specific cleavage
Fluorescently labeled double-stranded target DNA was formed annealing two single-stranded
oligonucleotides. Single strands were purchased from Microsynth Seqlab (Göttingen, Germany) with a
fluorescent label coupled to the 5‘-end. Plasmid-DNA cleavage assays The target strand carried a Cy3 label and the non-target strand
a Cy5 label (Table S2). Annealing was performed by incubation of equimolar amounts of TS and NTS
in annealing buffer (final concentration: 3 mM HEPES pH 7.4, 10 mM CH3COOK, 0.2 mM
Mg(CH3COO)2 at 95 °C for 3 min and passive cool-down to room temperature. The reconstituted
SpCas9-crRNA:tracrRNA complex (final concentration of 250 nM) was added to 5 nM of the double-
stranded target DNA in a total volume of 10 µl in 1x Cas buffer. Cleavage reactions were incubated at
37 °C for 1 h and the reaction was stopped by the addition of 0.36 U Proteinase K (Thermo Fisher
Scientific), followed by incubation at 55 °C for 45 min. The samples were mixed with loading dye (final
concentration: 47.5% (v/v) formamide, 0.01% (w/v) SDS, 0.01% (w/v) bromophenol blue, 0.005% (w/v)
xylene cyanol, 0.5 mM EDTA). Immediately prior to loading of the samples, the samples were denatured
at 95 °C for 5 min. Denatured samples were separated for 30 min on a pre-heated 15% PAA, 6 M urea,
1× TBE gel (300 V, 45 min). Cleavage products were visualized using a ChemiDoc Imaging System
(Bio-Rad). Magnetic Tweezers experiments The measurements were performed in a custom-built magnetic tweezers setup allowing the GPU-
assisted real-time tracking of the DNA length for up to 100 DNA molecules in parallel (31). DNA
constructs for the tweezers experiments were bound to magnetic beads of 1 µm-diameter (MyOne;
Invitrogen) and flushed into the glass flowcell of the setup allowing the anchoring of the digoxigen-
modified end to the anti-digoxigenin coated surface of the cell (29,32). After removing unbound beads
by flushing, force was applied by lowering the magnets towards the flow cell and DNA tethered beads
were selected. Using bead tracking with a camera (Mikrotron EoSens), the length of the individual DNA
molecules was determined and the applied forces were calibrated (33). During experiments, desired
forces on the DNA construct could be set by placing the magnets at a particular distance from the flow
cell according to the calibration results. Supercoiling of DNA was achieved by turning the magnets. The
resulting torque that depended mainly on the stretching force was calculated based on previous
theoretical work (34,35). Time trajectories of the DNA length were recorded at 120 Hz and smoothed
with a sliding average to 6 Hz for analysis. For measurements with SpCas9, reconstituted SpCas9-
sgRNA was added in tweezers buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM MgCl2, and 1
mg/ml BSA) at a concentration of 0.25 nM or 0.5 nM. After adding SpCas9, DNA length changes were . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint monitored in real-time. For experiments where we exclusively challenged the DNA-bound SpCas9
complex, SpCas9 was allowed to prebind to negatively supercoiled DNA in the flowcell for 20 minutes
to ensure efficient R-loop formation on most molecules before the experiment was started. The
successful formation of an R-loop was confirmed by the occurrence of a pronounced change in DNA
length. Data Analysis Data analysis for the tweezers experiments was performed with Origin (OriginLab) and custom-built
Matlab (The Mathworks Inc.) scripts. Supercoil release velocities Supercoil release velocities For Cas9dHNH experiments, a slow supercoil release was observed. The velocities of the release were
determined in Matlab by performing linear fits to the individual release events (see Fig. 2). The velocities
at each particular torque were then plotted in a histogram and fitted with a Gamma distribution. The
Gamma distribution describes the total waiting time 𝑡 of 𝑛 successive events with exponentially
distributed dwell times with mean value 𝜆. The Gamma distribution is given by: 𝑓(𝑡) = 𝑒−𝜆𝑡⋅𝜆𝑛⋅𝑡𝑛−1
(𝑛−1)! . 𝑓(𝑡) = 𝑒−𝜆𝑡⋅𝜆𝑛⋅𝑡𝑛−1
(𝑛−1)! . 𝑓(𝑡) = 𝑒−𝜆𝑡⋅𝜆𝑛⋅𝑡𝑛−1
(𝑛−1)! . By using this distribution, we assume that SpCas9 releases the non-target strand one turn at a time,
with each turn released after an exponentially distributed time with the same mean waiting time 1/𝜆. R-loop collapse times To determine transition times, R-loop transitions into the intermediate state or full collapse were fitted
by step functions in Matlab. Survival plots of the collapse times were then fitted with bi- or tri-exponential
functions using Origin. Investigating the post-cleavage behavior of SpCas9 in single-molecule multiplex magnetic
tweezers measurements To investigate the post-cleavage behavior of different SpCas9 variants, we employed a single-molecule
DNA twisting assay on a high-throughput magnetic tweezers platform (31). Numerous 2.7 kbp dsDNA
molecules containing a single SpCas9 target site matching the crRNA spacer and multiple biotin or
digoxigenin modifications at either end were attached on the respective DNA end to 1 µm streptavidin-
coated superparamagnetic beads and to the antidigoxigenin-coated surface of the flowcell of the setup. The three-dimensional positions of multiple DNA-tethered beads were tracked in parallel in a home-
built microscope (36) allowing the real-time observation of the DNA length of the molecules (Fig. 1A). The field gradient from a pair of magnets enabled the application of force onto the DNA molecule. The . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
oRxiv preprint . CC-BY 4.0 International license
made available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
oRxiv preprint force could be controlled by adjusting the distance between the magnets and the flow cell. Turning the
magnets allowed supercoiling of the DNA molecules in parallel. To monitor R-loop formation by SpCas9, we applied negative supercoiling which reduces the DNA
length due to writhe formation (Fig 1B, top). R-loop formation results in untwisting of the DNA by about
two turns, which absorbs part of the applied supercoiling such that it becomes visible as a sudden
increase in the DNA length (Fig. 1B bottom). Figure 1. (A) Schematic representation of the magnetic tweezers assay used to twist single double-
stranded DNA molecules that are tethered at either end to the surface of the fluidic cell and to a
magnetic microbead. Investigating the post-cleavage behavior of SpCas9 in single-molecule multiplex magnetic
tweezers measurements CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint As was previously shown for cleavage-incompetent dCas9 and other CRISPR-Cas effector complexes,
the application of positive twist on DNA can promote the collapse of the R-loop and dissociation of the
complex (9,25,10). In a simplified view, the R-loop is wrung out with the opposing superhelicity. At
positive supercoiling, R-loop collapse on intact target DNA is also observable as a sudden DNA length
change that, however, occurs in two steps (Fig. 1D, first trajectory). The first step corresponds to the
transition into the checkpoint state, corresponding to the formation of the partial R-loop, while the
second step corresponds to the total collapse of the R-loop and typically the dissociation of the protein. We started to test the stability of the post-cleavage product state by challenging R-loops formed by
wildtype SpCas9 (Cas9wt thereafter) (Fig. 1C), SpCas9 with a mutated HNH domain (Cas9dHNH
thereafter), and SpCas9 with a mutated RuvC domain (Cas9dRuvC thereafter) with positive supercoiling. Even though Cas9wt induced double-strand breaks (DSBs) within ~2 min after enzyme addition (Fig. S1), the DNA tethers in the tweezers stayed in place (Fig. S2) for several hours, indicating that Cas9wt
remained stably bound to both DNA ends as previously reported (13,16). We observed different
behaviors of post-cleavage SpCas9-DNA complexes depending on which strand was cleaved (Fig. 1D). For Cas9wt we did not observe the characteristic R-loop collapse with a 2-step supercoil release limited
to ~2 turns. Instead, an immediately starting but slow release of all applied supercoiling was observed
(Fig. 1D, second trajectory). This behavior resembled that of eukaryotic topoisomerase IB, which nicks
DNA and releases twist by a free swivel mechanism in which the release velocity is reduced by protein
friction (37). Cas9dHNH, which solely nicks the NTS of the DNA, showed a similar behavior as Cas9wt
indicating that also in this case a swivel mechanism causes the slow release of supercoiling (Fig. 1D,
third trajectory). In contrast, for Cas9dRuvC, which induces a nick on the TS, no immediately starting
supercoil release was observed. Instead, the supercoiling level remained constant for prolonged times
and was completely released in a fast, sudden step (Fig. Investigating the post-cleavage behavior of SpCas9 in single-molecule multiplex magnetic
tweezers measurements 1D, fourth trajectory). Such fast supercoil
release is indicative of a DNA nick in absence of bound protein (38) suggesting that Cas9dRuvC suddenly
dissociated leaving a free nick behind. This idea was supported by the observation that the fast release
of all supercoils was frequently preceeded by a short substep indicative of the first step of R-loop
collapse as seen for dCas9 (see small step in Fig. 1D, fourth trajectory). Investigating the post-cleavage behavior of SpCas9 in single-molecule multiplex magnetic
tweezers measurements A pair of permanent magnets allows the application of a stretching force and
molecule twisting. Up to one hundred beads were tracked in parallel (right: full microscope image with
enlarged view of a single bead in the inset). (B) Top: Representative DNA supercoiling curve taken at
0.3 pN showing the characteristic DNA length reduction due to writhe formation upon twisting. Bottom:
Representative time trajectory of the DNA length of a negatively supercoiled molecule (- 6 turns, 0.3
pN) exhibiting a DNA length jump due to R-loop formation by Cas9wt. (C) Schematic representation of
the SpCas9-sgRNA ribonucleoprotein complex forming an R-loop on double-stranded DNA. The
scissors indicate the active sites of the RuvC and HNH nuclease domains. The orange section on the
NTS represents the PAM. (D) Representative time trajectories when challenging R-loops formed by
dCas9, Cas9wt, Cas9dHNH, and Cas9dRuvC with positive supercoiling. Figure 1. (A) Schematic representation of the magnetic tweezers assay used to twist single double-
stranded DNA molecules that are tethered at either end to the surface of the fluidic cell and to a
magnetic microbead. A pair of permanent magnets allows the application of a stretching force and
molecule twisting. Up to one hundred beads were tracked in parallel (right: full microscope image with
enlarged view of a single bead in the inset). (B) Top: Representative DNA supercoiling curve taken at
0.3 pN showing the characteristic DNA length reduction due to writhe formation upon twisting. Bottom:
Representative time trajectory of the DNA length of a negatively supercoiled molecule (- 6 turns, 0.3
pN) exhibiting a DNA length jump due to R-loop formation by Cas9wt. (C) Schematic representation of
the SpCas9-sgRNA ribonucleoprotein complex forming an R-loop on double-stranded DNA. The
scissors indicate the active sites of the RuvC and HNH nuclease domains. The orange section on the
NTS represents the PAM. (D) Representative time trajectories when challenging R-loops formed by
dCas9, Cas9wt, Cas9dHNH, and Cas9dRuvC with positive supercoiling. . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . Bound Cas9dHNH releases supercoiling through detachment of the NTS in a swivel-like manner
governed by torque and friction Next, we investigated the different responses of the SpCas9 variants to supercoiling after DNA cleavage
in more detail. Both, Cas9wt and Cas9dHNH released supercoiling slowly by a swivel mechanism (Fig. 2A). The slow supercoil release was observed repeatably, indicating that the protein remained stably
bound to the target. Otherwise, supercoils would be very rapidly released by a protein-free nick (38)
and the introduction of additional supercoils in the DNA would be inhibited (as observed for Cas9dRuvC,
see below). Hence, we observed a similar behavior for the Cas9wt and Cas9dHNH variants, which both
nick the NTS, suggesting that occasional detachment of the NTS from the protein enables the
swiveling/supercoil release (Fig. 2B). Despite the presence of a TS nick for Cas9wt, the DNA tethers did
not rupture during supercoil release, indicating that the TS is tightly bound. . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint For Cas9dHNH, we carefully characterized the supercoil release velocity as function of the applied
positive and negative supercoiling levels. For both supercoiling directions, a similar release behavior
was recorded. Application of different forces allowed varying the acting torque during supercoil release. We measured the velocity of individual release events by applying a linear fit through each event. At
each respective torque, we determined the linear slopes of the supercoiling curve which allowed us to
convert the velocity from nanometers per second to turns per second. The distributions of the release
velocities had a pronounced maximum and were well described by a Gamma distribution (Fig. 2C). The
Gamma distribution describes a process containing 𝑛 substeps with exponentially distributed dwell
times (see Methods). Figure 2. (A) Representative time trajectories of repeated supercoil release measured with magnetic
tweezers (smoothed to 6Hz). Upon repeated step-wise DNA supercoiling, the DNA length is shortened. Bound Cas9dHNH releases supercoiling through detachment of the NTS in a swivel-like manner
governed by torque and friction It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint For the parameter which describes the number of substeps in the process, we obtained on average
𝑛 = 6.6 ± 1.3 and 𝑛 = 4.3 ± 0.3 for positive and negative supercoiling, respectively. These numbers
agree well with the number of turns within the linear regime of the supercoiling curve, indicating the
release of supercoils one turn at a time. The mean release velocities approximately showed an
exponential increase with the applied torque for positive and negative supercoiling (Fig. 2D). Following
Arrhenius transition-state rate theory, we modeled the supercoil release as a step-wise process in which
supercoils can be released (or introduced) as single full turns. For this process, a periodic energy
landscape can be drawn with energy minima at full turns (corresponding to the NTS docked onto
Cas9dHNH) and transition state maxima in between (corresponding to a detached NTS and partially
turned DNA) (Fig. 2E). Applied torque tilts the energy landscape to favor supercoil release. The torque
dependence can be described using an exponential Arrhenius-term in which the activation energy
required to reach the transition state is changed by the work to reach the transition state in presence of
the torque. To consider supercoil release in positive and negative direction and to ensure a zero net
release at zero torque, two torque-dependent Arrhenius-terms were used: 𝑣(𝛤) = 𝑣0 ⋅(exp (𝛤⋅ 2𝜋⋅Δ𝑁+
𝑘𝑏𝑇
) −exp (−𝛤⋅2𝜋⋅Δ𝑁−
𝑘𝑏𝑇
)),
(1) (1) where 𝑣0 is the spontaneous swiveling rate in either direction at zero torque, 𝛤 is the applied torque,
and Δ𝑁+/− are the angular distances of the transition state from the energy minima for positive and
negative supercoil release, respectively. Fitting this model to the data in Figure 2C resulted in transition
state distances of Δ𝑁+= 0.4 ± 0.1 turns and Δ𝑁−= 0.3 ± 0.1, indicating that the transition state is located
approximately half a turn from the energy minima. The swiveling rate at zero torque was 𝑣0= 0.04 ± 0.01
turns/s, i.e. Cas9dHNH would spontaneously introduce or deplete a single supercoil every ~25s even for
relaxed DNA. Overall, this data supports a model in which Cas9dHNH releases the NTS by a swiveling mechanism
governed by torque and friction. Due to the slight difference in the transition state distance, the release
occurred slightly faster at positive than negative torque. Bound Cas9dHNH releases supercoiling through detachment of the NTS in a swivel-like manner
governed by torque and friction Supercoil release can be detected as a slow increase in DNA length, which is limited by friction due to
the presence of Cas9dHNH. (B) Cartoon illustrating the supercoil release for DNA nicked by Cas9dHNH. When the NTS of a supercoiled dsDNA is cleaved, the applied torque is released by swivelling of the
NTS around the TS. The swivelling is sterically obstructed by the protein. (C) Histogram of the measured
supercoil release velocities given in turns per second (blue bars). The red line shows a fit with a Gamma
distribution. (D) Torque-dependence of the supercoil release velocity (blue). The red line shows a fit of
the data with Eq. (1) (E) Hypothetic scheme of the energy landscape for NTS release. The energy
landscape is tilted downhill if positive or negative torque is applied (red line) in contrast to the absence
of torque (blue line). Figure 2. (A) Representative time trajectories of repeated supercoil release measured with magnetic
tweezers (smoothed to 6Hz). Upon repeated step-wise DNA supercoiling, the DNA length is shortened. Supercoil release can be detected as a slow increase in DNA length, which is limited by friction due to
the presence of Cas9dHNH. (B) Cartoon illustrating the supercoil release for DNA nicked by Cas9dHNH. When the NTS of a supercoiled dsDNA is cleaved, the applied torque is released by swivelling of the
NTS around the TS. The swivelling is sterically obstructed by the protein. (C) Histogram of the measured
supercoil release velocities given in turns per second (blue bars). The red line shows a fit with a Gamma
distribution. (D) Torque-dependence of the supercoil release velocity (blue). The red line shows a fit of
the data with Eq. (1) (E) Hypothetic scheme of the energy landscape for NTS release. The energy
landscape is tilted downhill if positive or negative torque is applied (red line) in contrast to the absence
of torque (blue line). . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. TS cleavage causes two differently stable SpCas9 states In a next step, we explored the stability of Cas9dRuvC in the post-cleavage state on DNA when subjected
to supercoiling. Cas9dRuvC only cleaves the TS, such that supercoils cannot be released by NTS
detachment and swiveling around the protein (Fig. 3A). Experimentally, this is seen as a constant
supercoiling level over extended time periods (Fig. 3B). Full supercoil release can be observed as a
sudden rapid step similar to the free nick generated by a nicking enzyme (38). This suggests that the
twist is only released after R-loop collapse. Notably, the sudden supercoil release due to R-loop
collapse is preceeded by a substep corresponding to a partial R-loop collapse i.e. the transition into the
intermediate R-loop state. Subsequent repeated introduction of additional supercoiling failed, indicating
that the enzyme dissociated upon R-loop collapse. After a waiting time of several minutes, the
introduction of supercoils became possible again, indicating that a new Cas9dRuvC molecule had bound
to the nicked target site (Fig S3). . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint We carefully investigated the stability of Cas9dRuvC bound to the nicked target site. To this end, we
incubated the protein and DNA in the flowcell at negative supercoiling to allow R-loop formation. We
then induced positive twist on the DNA to enforce the collapse of the R-loop and characterized the time
between introduction of positive supercoiling and the collapse of the full R-loop. The collapse times
were best described by a bi-exponential distribution (Fig. 3C). We, therefore, propose the existence of
two distinct states distinguishable by their collapse times which were on the order of minutes for the
unstable state and hours for the stable state. Repeating the experiment at different applied torques, we
observed an exponential dependence of the R-loop collapse time on the torque for both states (Fig. 3D). The post-cleavage product state stabilizes the R-loop in a torque-dependent manner To reveal the relative stability of the post-cleavage state, we finally investigated the stability of SpCas9
in the pre-cleavage state. To this end, we challenged intact R-loops formed by a catalytically fully
inactive dCas9 variant with positive supercoiling (Fig. 4A). The measurements were performed in
presence of magnesium, allowing dCas9 to adopt the “docked” conformation of the HNH domain. As
described before, R-loops formed by dCas9 collapsed in two steps at positive torque (Fig. 4B). The first
step can be attributed to the transition from the docked to the intermediate state as observed for
Cas9dRuvC . The second step, which was limited only to partial supercoiling release due to the lack of a
DNA nick, can be attributed to the collapse of the intermediate state and dissociation of dCas9. Allowing
a new R-loop formation at negative turns, the collapse process could be repeated multiple times (Fig. S5). The R-loop collapse times were best described by an exponential distribution with three distinct
components, therafter called stable, semi-stable, and unstable state (Fig. 4C). As for Cas9dRuvC, R-loop
collapse occurred in two distinct steps for all three components. The respective collapse times fell into
the range of hours, minutes, and seconds with each component showing a slight torque dependence
(Fig. 4D). Compared to Cas9dRuvC, the collapse time for the stable state was slightly decreased, while
the collapse time of the unstable state did not significantly change. The occurrence of a torque-
dependent semi-stable state was not observed for Cas9dRuvC. This may indicate flexibility of the docked
state of dCas9. Similar to Cas9dRuvC, the fraction of molecules in the unstable state did not change with
torque (Fig. 4E). However, the fraction of molecules in the semi-stable state increased, while the fraction
of molecules in the stable state decreased with increasing torque. Due to the torque dependence of the
fraction of molecules found in the different populations, the mean time of the R-loop collapse was
strongly dependent on the torque applied (Fig. 4F). When comparing these results to Cas9dRuvC (Fig. 4F), we found that the mean time of the R-loop collapse was generally higher for Cas9dRuvC compared
to dCas9. This indicates a further stabilization of SpCas9 after TS cleavage. The stabilization was found
to be torque-dependent with pronounced stabilization at high torques. TS cleavage causes two differently stable SpCas9 states CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint This corresponds to a DNA rewinding (most likely at the PAM-distal end) of about 2 and 1 bp. Furthermore, the fitting allowed to extrapolate the collapse times to zero torque for which we obtained
408 ± 58 min and 3.2 ± 1.5 min for the stable and the unstable state, respectively. On average, 72 ± 3%
of the R-loops were in the stable state and 28% ± 3% in the unstable state with no obvious torque-
dependence (Fig. 3E). We did not observe any dsDNA release by Cas9wt until many hours after
cleavage. This suggests that the occurrence of the unstable state after TS cleavage is unique to
Cas9dRuvC and does not occur in case of Cas9wt, since stable TS capturing would only be possible for a
formed R-loop. Overall, the results support that Cas9dRuvC stays tightly bound to the target after TS cleavage until the
full R-loop collapses. During this time, the Cas9dRuvC-sgRNA-DNA complex can adopt different
conformational states, possibly in a dynamic manner. TS cleavage causes two differently stable SpCas9 states This indicated the presence of a transition state for R-loop collapse (similar to the supercoil release
above) as observed previously for various CRISPR-Cas proteins (25,10,39,9). Fitting the torque
dependent data with a single Arrhenius function provided angular distances to the transition states of
0.18 ± 0.03 turns and 0.07 ± 0.01 turns for the unstable and stable state, respectively. Figure 3. (A) Cartoon illustrating the nicking of the TS by Cas9dRuvC. The TS is nicked but remains tightly
bound to Cas9dRuvC such that supercoils are not released in presence of the protein. (B) Representative
magnetic tweezers trajectory (smoothed to 6 Hz) showing data for an experiment in which Cas9dRuvC-
nicked the DNA and the complex is challenged by strong positive supercoiling (high positive torque). No supercoils are released until part of the R-loop collapses (see orange arrow). This is followed by a
rapid release of all supercoiling as observed for nicked DNA in absence of protein (red arrow). (C)
Survival plot of the introduced R-loops (black squares) fitted with a bi-exponential decay function
(orange/blue line). (D) Characteristic time constants for the R-loop collapse as function of torque
obtained from the bi-exponential fits revealing an unstable (orange) and a stable (blue) population of R-
loops. (E) Torque-dependence of the fractions of unstable (orange) and stable (blue) R-loops. Figure 3. (A) Cartoon illustrating the nicking of the TS by Cas9dRuvC. The TS is nicked but remains tightly
bound to Cas9dRuvC such that supercoils are not released in presence of the protein. (B) Representative
magnetic tweezers trajectory (smoothed to 6 Hz) showing data for an experiment in which Cas9dRuvC-
nicked the DNA and the complex is challenged by strong positive supercoiling (high positive torque). No supercoils are released until part of the R-loop collapses (see orange arrow). This is followed by a
rapid release of all supercoiling as observed for nicked DNA in absence of protein (red arrow). (C)
Survival plot of the introduced R-loops (black squares) fitted with a bi-exponential decay function
(orange/blue line). (D) Characteristic time constants for the R-loop collapse as function of torque
obtained from the bi-exponential fits revealing an unstable (orange) and a stable (blue) population of R-
loops. (E) Torque-dependence of the fractions of unstable (orange) and stable (blue) R-loops. . The post-cleavage product state stabilizes the R-loop in a torque-dependent manner To correlate the two states with
different stabilities to known conformations of SpCas9, we carried out experiments under Mg2+-free
conditions at which the docked state of SpCas9 is suppressed (14). Even though the docked state is . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint suppressed, a full R-loop is formed (Fig. S6A). We obtained an average collapse time that matches the
collapse time of the previously observed unstable state (Fig. S6B). This suggests that the stable and
semi-stable states represent the docked conformation of SpCas9. suppressed, a full R-loop is formed (Fig. S6A). We obtained an average collapse time that matches the
collapse time of the previously observed unstable state (Fig. S6B). This suggests that the stable and
semi-stable states represent the docked conformation of SpCas9. In conclusion, we showed that dCas9 adopts three different conformations when a full R-loop has been
formed. Additionally, we observed a torque-dependent stabilization of the R-loop after cleavage of the
TS. Figure 4. (A) Cartoon illustrating the pre-cleavage state of SpCas9, a state produced by the cleavage-
incompetent dCas9 variant. (B) Representative magnetic tweezers trajectory (smoothed to 6 Hz) when
challenging an R-loop formed by dCas9 with positive supercoiling. The R-loop spontaneously collapses
in two characteristic steps. Absence of full supercoil release indicates that the DNA was not nicked by
dCas9. (C) Survival plot of R-loops formed by dCas9 (black triangles) fitted with a tri-exponential decay
function (orange/purple/blue line). (D) Characteristic time constants for the R-loop collapse as function
of torque obtained from the tri-exponential fits revealing an unstable (orange), a semi-stable (purple),
and a stable (blue) population of R-loops. (E) Torque-dependence of the fractions of unstable (orange),
semi-stable (purple), and stable (blue) R-loops. (F) Mean R-loop collapse time averaged over all
populations for dCas9 (green) and Cas9dRuvC (red). Cleavage of the TS appears to stabilize SpCas9 on
the DNA. Figure 4. DISCUSSION Several studies showed that SpCas9 stays tightly bound to its target DNA after cleavage of double-
stranded DNA is completed. In this study, we elucidate the mechanisms by which SpCas9 achieves
this high stability even under unfavorable conditions of positive supercoiling, a situation encountered
during transcription and replication of genomic DNA. Using single-molecule magnetic tweezers
measurements, we show that the post-cleavage behavior of SpCas9 strongly depends on which strand
of the target site is cleaved. NTS cleavage allows the NTS to detach from the protein and to swivel around the TS under friction. By
this mechanism, even high supercoiling levels are quickly relaxed such that R-loop collapse and
SpCas9 dissociation are circumvented. We determined a spontaneous swivel rate of 0.04 ± 0.01 turns/s
at zero torque. This provides evidence for a spontaneous release of the NTS even in absence of
supercoiling such that it becomes accessible for complementary ssDNA or exonucleases (16). Characterizing the torque dependence of this supercoil release allowed us to determine the transition
state distances, indicating that the transition state lies at approximately half a turn. A different behavior was observed for a SpCas9 variant that can bind the DNA target site but can only
cleave the TS. Here, a facilitated supercoil release was impeded. As observed in the cryo-EM structure
of the post-cleavage state of SpCas9 (12), the cleaved TS stays hybridized to the sgRNA in the post-
cleavage state which can explain the absence of an immediate release of supercoiling. Applying
elevated positive torque resulted in the collapse of the R-loop and dissociation of Cas9dRuvC. By carefully
analyzing the collapse times, we observed two states of different stabilities with collapse times in the
order of a few minutes and several hours, respectively. After TS cleavage, the HNH domain has been
reported to be highly flexible (16,12). The different stability states observed in this work might stem from
this flexibility. Additionally, there has been some debate on the dynamics of the pre-cleavage
conformational states. While some studies have shown that SpCas9 reaches a stable, cleavage-
competent ‘docking’ conformation in the presence of Mg2+ ions (14,40), other studies have observed
only brief visits of the docked state with the majority of time spent in the ‘checkpoint’ conformation
(15,41,42). The post-cleavage product state stabilizes the R-loop in a torque-dependent manner (A) Cartoon illustrating the pre-cleavage state of SpCas9, a state produced by the cleavage-
incompetent dCas9 variant. (B) Representative magnetic tweezers trajectory (smoothed to 6 Hz) when
challenging an R-loop formed by dCas9 with positive supercoiling. The R-loop spontaneously collapses
in two characteristic steps. Absence of full supercoil release indicates that the DNA was not nicked by
dCas9. (C) Survival plot of R-loops formed by dCas9 (black triangles) fitted with a tri-exponential decay
function (orange/purple/blue line). (D) Characteristic time constants for the R-loop collapse as function
of torque obtained from the tri-exponential fits revealing an unstable (orange), a semi-stable (purple),
and a stable (blue) population of R-loops. (E) Torque-dependence of the fractions of unstable (orange),
semi-stable (purple), and stable (blue) R-loops. (F) Mean R-loop collapse time averaged over all
populations for dCas9 (green) and Cas9dRuvC (red). Cleavage of the TS appears to stabilize SpCas9 on
the DNA. . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint DISCUSSION Our results suggest that even though a complete R-loop is formed and cleavage is carried
out, Cas9dRuvC exhibits conformational flexibility, transitioning between an undocked state and a
stabilized post-cleavage state. It is possible that the undocked state corresponds to the previously
reported ‘checkpoint’ state. However, we note that even in the undocked state a full R-loop is formed,
as confirmed by the extent of the length jump and the presence of the intermediate step during R-loop
collapse (Figure S7). We show that the lifetime of the undocked state resembles that of a Mg2+-free
control for which the “docked” state is not formed. For wildtype SpCas9, we did not observe DNA release
after a cleavage of both DNA strands even after several hours of measurement indicating that it does
not exhibit the undocked state after cleavage which was observed for Cas9dRuvC. Cleavage-incompetent
dCas9 exhibits even three distinct states and, potentially, a higher conformational flexibility. Combining
the results of Cas9dRuvC and dCas9 we suggest that the stable state that is observed for both variants
corresponds to the post-cleavage product state. This state would appear to be transiently visited even
for the uncleaved target, albeit only at low levels of supercoiling. The semi-stable state is observed
when dCas9 is bound to the target DNA without DNA cleavage . This state dominates under conditions . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint of high supercoiling and most likely corresponds to the pre-cleavage state with a docked HNH domain. The unstable state is found for both dCas9 and Cas9dRuvC and most likely represents the undocked
state in agreement with the previously observed dynamic sampling between the different states
(15,41,42) (Fig. 5). Overall, the dominance of the post-cleavage product state accompanying TS
cleavage stabilizes the R-loop compared to the uncut target, especially at higher torques. DISCUSSION This suggests
that, under in-vivo conditions, SpCas9 has evolved to remain bound to its cut target. Possibly, this not
only inactivates the invader DNA but also blocks transcription and/or replication of the foreign DNA,
thereby fully blocking replication of a bacteriophage. However, this represents a challenge for efficient
genome engineering applications. Overall, the presented results expand the knowledge about the post-cleavage state(s) of SpCas9 and
the mechanisms by which SpCas9 avoids R-loop collapse and dissociation even under high twist that
can be generated in vivo by the genome processing machineries. Nickase mutants of Cas9 have been
used in various approaches for genome editing with strong differences in results depending on which
nickase was used (22,43,44). Our findings may help to understand the described differences and might
facilitate future genome editing applications with SpCas9 nickases. Figure 5. Schematic overview of the different states identified for SpCas9 bound to intact DNA and
DNA with a nicked TS. We assigned the unstable state to the intermediate state with undocked HNH
domain, the semi-stable state to the precleavage state with the HNH domain docked to the active site,
and the stable state to the post-cleavage product state with a disordered (or flexible) HNH domain
(12,16). Catalytically inactive dCas9 can occupy all states and most likely samples these states in a
dynamic manner. Cas9dRuvC was predominantly found in the stable (post-cleavage) state and rarely
occupied the unstable state. Figure 5. Schematic overview of the different states identified for SpCas9 bound to intact DNA and
DNA with a nicked TS. We assigned the unstable state to the intermediate state with undocked HNH
domain, the semi-stable state to the precleavage state with the HNH domain docked to the active site,
and the stable state to the post-cleavage product state with a disordered (or flexible) HNH domain
(12,16). Catalytically inactive dCas9 can occupy all states and most likely samples these states in a
dynamic manner. Cas9dRuvC was predominantly found in the stable (post-cleavage) state and rarely
occupied the unstable state. We thank Joachim Griesenbeck for providing the pM53.1 plasmid. Furthermore, we thank Virginijus
Siksnys, Tautvydas Karvelis, and Greta Bigelytė for providing the sgRNA. REFERENCES 1. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A. and Horvath,
P. (2007) CRISPR provides acquired resistance against viruses in prokaryotes, Science (New York, N.Y.), 315,
1709–1712. 2. Garneau, J.E., Dupuis, M.-È., Villion, M., Romero, D.A., Barrangou, R., Boyaval, P., Fremaux, C., Horvath, P.,
Magadán, A.H. and Moineau, S. (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage
and plasmid DNA, Nature, 468, 67–71. 2. Garneau, J.E., Dupuis, M.-È., Villion, M., Romero, D.A., Barrangou, R., Boyaval, P., Fremaux, C., Horvath, P.,
Magadán, A.H. and Moineau, S. (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage
and plasmid DNA, Nature, 468, 67–71. 3. Gasiunas, G., Barrangou, R., Horvath, P. and Siksnys, V. (2012) Cas9-crRNA ribonucleoprotein complex
mediates specific DNA cleavage for adaptive immunity in bacteria, Proc Natl Acad Sci U S A, 109, E2579-86. 4. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A. and Charpentier, E. (2012) A programmable
dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science (New York, N.Y.), 337, 816–
821. 5. Le Cong, Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W. and Marraffini, L.A. et
al. (2013) Multiplex genome engineering using CRISPR/Cas systems, Science (New York, N.Y.), 339, 819–
823. 6. Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E. and Church, G.M. (2013) RNA-
guided human genome engineering via Cas9, Science (New York, N.Y.), 339, 823–826. 7. Hsu, P.D., Lander, E.S. and Zhang, F. (2014) Development and applications of CRISPR-Cas9 for genome
engineering, Cell, 157, 1262–1278. 8. Globyte, V., Lee, S.H., Bae, T., Kim, J.-S. and Joo, C. (2019) CRISPR/Cas9 searches for a protospacer adjacent
motif by lateral diffusion, The EMBO journal, 38. 9. Szczelkun, M.D., Tikhomirova, M.S., Sinkunas, T., Gasiunas, G., Karvelis, T., Pschera, P., Siksnys, V. and
Seidel, R. (2014) Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector
complexes, Proc Natl Acad Sci U S A, 111, 9798–9803. 10. Ivanov, I.E., Wright, A.V., Cofsky, J.C., Aris, K.D.P., Doudna, J.A. and Bryant, Z. (2020) Cas9 interrogates DNA
in discrete steps modulated by mismatches and supercoiling, Proc Natl Acad Sci U S A, 117, 5853–5860. 11. Jiang, F., Taylor, D.W., Chen, J.S., Kornfeld, J.E., Zhou, K., Thompson, A.J., Nogales, E. and Doudna, J.A. (2016) Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage, Science (New York, N.Y.),
351, 867–871. 12. ACKNOWLEDGEMENT We thank Joachim Griesenbeck for providing the pM53.1 plasmid. Furthermore, we thank Virginijus
Siksnys, Tautvydas Karvelis, and Greta Bigelytė for providing the sgRNA. . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint REFERENCES Zhu, X., Clarke, R., Puppala, A.K., Chittori, S., Merk, A., Merrill, B.J., Simonović, M. and Subramaniam, S. (2019) Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9, Nature
structural & molecular biology, 26, 679–685. 13. Sternberg, S.H., LaFrance, B., Kaplan, M. and Doudna, J.A. (2015) Conformational control of DNA target
cleavage by CRISPR-Cas9, Nature, 527, 110–113. . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint 14. Dagdas, Y.S., Chen, J.S., Sternberg, S.H., Doudna, J.A. and Yildiz, A. (2017) A conformational checkpoint
between DNA binding and cleavage by CRISPR-Cas9, Science advances, 3, eaao0027. 15. Yang, M., Peng, S., Sun, R., Lin, J., Wang, N. and Chen, C. (2018) The Conformational Dynamics of Cas9
Governing DNA Cleavage Are Revealed by Single-Molecule FRET, Cell reports, 22, 372–382. 16. Wang, Y., Mallon, J., Wang, H., Singh, D., Hyun Jo, M., Hua, B., Bailey, S. and Ha, T. (2021) Real-time
observation of Cas9 postcatalytic domain motions, Proc Natl Acad Sci U S A, 118. 17. Raper, A.T., Stephenson, A.A. and Suo, Z. (2018) Functional Insights Revealed by the Kinetic Mechanism of
CRISPR/Cas9, J Am Chem Soc, 140, 2971–2984. 18. Brinkman, E.K., Chen, T., Haas, M. de, Holland, H.A., Akhtar, W. and van Steensel, B. (2018) Kinetics and
Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks, Molecular cell, 70, 801-813.e6. 19. Richardson, C.D., Ray, G.J., DeWitt, M.A., Curie, G.L. and Corn, J.E. (2016) Enhancing homology-directed
genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA, Nature
biotechnology, 34, 339–344. 20. Ran, F.A., Hsu, P.D., Lin, C.-Y., Gootenberg, J.S., Konermann, S., Trevino, A.E., Scott, D.A., Inoue, A.,
Matoba, S. and Zhang, Y. et al. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome
editing specificity, Cell, 154, 1380–1389. 21. Anzalone, A.V., Randolph, P.B., Davis, J.R., Sousa, A.A., Koblan, L.W., Levy, J.M., Chen, P.J., Wilson, C.,
Newby, G.A. REFERENCES and Raguram, A. et al. (2019) Search-and-replace genome editing without double-strand
breaks or donor DNA, Nature, 576, 149–157. 22. Rees, H.A., Yeh, W.-H. and Liu, D.R. (2019) Development of hRad51-Cas9 nickase fusions that mediate HDR
without double-stranded breaks, Nat Commun, 10, 2212. 23. Guilinger, J.P., Thompson, D.B. and Liu, D.R. (2014) Fusion of catalytically inactive Cas9 to FokI nuclease
improves the specificity of genome modification, Nat Biotechnol, 32, 577–582,
https://www.nature.com/articles/nbt.2909. 24. Brutzer, H., Luzzietti, N., Klaue, D. and Seidel, R. (2010) Energetics at the DNA supercoiling transition,
Biophys J, 98, 1267–1276. 25. Rutkauskas, M., Sinkunas, T., Songailiene, I., Tikhomirova, M.S., Siksnys, V. and Seidel, R. (2015) Directional
R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site
Rejection, Cell reports, 10, 1534–1543. 26. Kouzine, F., Sanford, S., Elisha-Feil, Z. and Levens, D. (2008) The functional response of upstream DNA to
dynamic supercoiling in vivo, Nature structural & molecular biology, 15, 146–154. 27. Ma, J., Bai, L. and Wang, M.D. (2013) Transcription under torsion, Science (New York, N.Y.), 340, 1580–
1583. . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint 28. Wu, H.-Y., Shyy, S., Wang, J.C. and Liu, L.F. (1988) Transcription generates positively and negatively
supercoiled domains in the template, Cell, 53, 433–440. 29. Luzzietti, N., Knappe, S., Richter, I. and Seidel, R. (2012) Nicking enzyme-based internal labeling of DNA at
multiple loci, Nature protocols, 7, 643–653. 30. Griesenbeck, J., Boeger, H., Strattan, J. and Kornberg, R.D. (2003) Purification of Defined Chromosomal
Domains, Methods in Enzymology : Chromatin and Chromatin Remodeling Enzymes, Part A. Academic
Press, pp. 170–178. 31. Huhle, A., Klaue, D., Brutzer, H., Daldrop, P., Joo, S., Otto, O., Keyser, U.F. and Seidel, R. (2015) Camera-
based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy, Nat Commun, 6,
5885, https://www.nature.com/articles/ncomms6885. 32. REFERENCES Schwarz, F.W., Tóth, J., van Aelst, K., Cui, G., Clausing, S., Szczelkun, M.D. and Seidel, R. (2013) The
helicase-like domains of type III restriction enzymes trigger long-range diffusion along DNA, Science (New
York, N.Y.), 340, 353–356. 33. Daldrop, P., Brutzer, H., Huhle, A., Kauert, D.J. and Seidel, R. (2015) Extending the range for force
calibration in magnetic tweezers, Biophys J, 108, 2550–2561. 34. Maffeo, C., Schöpflin, R., Brutzer, H., Stehr, R., Aksimentiev, A., Wedemann, G. and Seidel, R. (2010) DNA-
DNA interactions in tight supercoils are described by a small effective charge density, Physical review
letters, 105, 158101. 35. Schöpflin, R., Brutzer, H., Müller, O., Seidel, R. and Wedemann, G. (2012) Probing the elasticity of DNA on
short length scales by modeling supercoiling under tension, Biophys J, 103, 323–330. 36. Klaue, D. and Seidel, R. (2009) Torsional stiffness of single superparamagnetic microspheres in an external
magnetic field, Physical review letters, 102, 28302. 37. Koster, D.A., Croquette, V., Dekker, C., Shuman, S. and Dekker, N.H. (2005) Friction and torque govern the
relaxation of DNA supercoils by eukaryotic topoisomerase IB, Nature, 434, 671–674. 38. Crut, A., Koster, D.A., Seidel, R., Wiggins, C.H. and Dekker, N.H. (2007) Fast dynamics of supercoiled DNA
revealed by single-molecule experiments, Proc Natl Acad Sci U S A, 104, 11957–11962. 39. Singh, D., Wang, Y., Mallon, J., Yang, O., Fei, J., Poddar, A., Ceylan, D., Bailey, S. and Ha, T. (2018)
Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis,
Nature structural & molecular biology, 25, 347–354. 40. Chen, J.S., Dagdas, Y.S., Kleinstiver, B.P., Welch, M.M., Sousa, A.A., Harrington, L.B., Sternberg, S.H., Joung,
J.K., Yildiz, A. and Doudna, J.A. (2017) Enhanced proofreading governs CRISPR-Cas9 targeting accuracy,
Nature, 550, 407–410. 41. Osuka, S., Isomura, K., Kajimoto, S., Komori, T., Nishimasu, H., Shima, T., Nureki, O. and Uemura, S. (2018)
Real-time observation of flexible domain movements in CRISPR-Cas9, The EMBO journal, 37. . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. .
CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021.
;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint REFERENCES It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint 42. Shibata, M., Nishimasu, H., Kodera, N., Hirano, S., Ando, T., Uchihashi, T. and Nureki, O. (2017) Real-space
and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy, Nature
communications, 8, 1430. 43. Shen, B., Zhang, W., Zhang, J., Zhou, J., Wang, J., Chen, L., Wang, L., Hodgkins, A., Iyer, V. and Huang, X. et
al. (2014) Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects, Nature
methods, 11, 399–402. 43. Shen, B., Zhang, W., Zhang, J., Zhou, J., Wang, J., Chen, L., Wang, L., Hodgkins, A., Iyer, V. and Huang, X. et
al. (2014) Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects, Nature
methods, 11, 399–402. 44. Miyaoka, Y., Berman, J.R., Cooper, S.B., Mayerl, S.J., Chan, A.H., Zhang, B., Karlin-Neumann, G.A. and
Conklin, B.R. (2016) Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell
type on genome-editing, Scientific reports, 6, 23549. 44. Miyaoka, Y., Berman, J.R., Cooper, S.B., Mayerl, S.J., Chan, A.H., Zhang, B., Karlin-Neumann, G.A. and
Conklin, B.R. (2016) Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell
type on genome-editing, Scientific reports, 6, 23549. . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted August 4, 2021. ;
https://doi.org/10.1101/2021.08.04.455019
doi:
bioRxiv preprint
|
https://openalex.org/W3209619552
|
https://zenodo.org/record/5594474/files/UKavak_PhDThesis_Title_and_contents.pdf
|
English
| null |
Interaction of Massive Stars with Gas Clouds in the Milky Way
| null | 2,021
|
cc-by
| 3,766
|
Citation for published version (APA):
Kavak, U. (2021). Interaction of Massive Stars with Gas Clouds in the Milky Way: from shooting stars to
breaking bubbles. University of Groningen. https://doi.org/10.33612/diss.187660161 University of Groningen University of Groningen Kavak, Umit DOI:
10.33612/diss.187660161 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below. Document Version
Publisher's PDF, also known as Version of record Link to publication in University of Groningen/UMCG research database Citation for published version (APA):
Kavak, U. (2021). Interaction of Massive Stars with Gas Clouds in the Milky Way: from shooting stars to
breaking bubbles. University of Groningen. https://doi.org/10.33612/diss.187660161 Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-
amendment. Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim. Take-down policy
If
b li
h Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum. Download date: 23-10-2021 Download date: 23-10-2021 Download date: 23-10-2021 Cover design by: Yakup Kurt/Adgency Koeln. The background image on the
front cover is a star-forming region known as LH-95 in the Large Magellanic
Cloud taken from hubblesite.org. The interpretation of Starry Night on the
back-cover and figures at the end of each chapter were provided by Garip Ay.
The blue tulip created by Evren Kaan at page 220 was taken from kulturpor-
tali.gov.tr with ID of #8942.
Printed by: Gildeprint Contents 1
Introduction
1
1.1
Interstellar Medium
. . . . . . . . . . . . . . . . . . . . . 2
1.2
Low and Massive Star Formation . . . . . . . . . . . . . . 3
1.3
Massive Stars . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.3.1
Jets and Outflows
. . . . . . . . . . . . . . . . . . 9
1.3.2
Photodissociation Regions . . . . . . . . . . . . . . 10
1.3.3
Interstellar Bubbles . . . . . . . . . . . . . . . . . . 13
1.4
Feedback from Massive Stars
. . . . . . . . . . . . . . . . 15
1.4.1
Mechanical Feedback . . . . . . . . . . . . . . . . . 16
1.4.2
Radiative Feedback . . . . . . . . . . . . . . . . . . 16
1.5
Aims and Methods . . . . . . . . . . . . . . . . . . . . . . 18
1.5.1
Herschel Space Observatory . . . . . . . . . . . . . 18
1.5.2
Very Large Array (VLA) . . . . . . . . . . . . . . . 20
1.5.3
Stratospheric Observatory for Infrared Astronomy
(SOFIA) . . . . . . . . . . . . . . . . . . . . . . . . 21
1.5.4
Meudon PDR code . . . . . . . . . . . . . . . . . . 22
1.5.5
RADEX . . . . . . . . . . . . . . . . . . . . . . . . 23
1.5.6
GILDAS . . . . . . . . . . . . . . . . . . . . . . . . 23
1.5.7
CASA . . . . . . . . . . . . . . . . . Ümit Kavak Supervisors
Prof. F. F. S. van der Tak
Prof. A. G. G. M. Tielens Assessment committee
Prof. J. Tan
Prof. G. J. Stacy
Prof. I. E. E. Kamp Assessment committee
Prof. J. Tan
Prof. G. J. Stacy
Prof. I. E. E. Kamp To my family... Cover design by: Yakup Kurt/Adgency Koeln. The background image on the
front cover is a star-forming region known as LH-95 in the Large Magellanic
Cloud taken from hubblesite.org. The interpretation of Starry Night on the
back-cover and figures at the end of each chapter were provided by Garip Ay. The blue tulip created by Evren Kaan at page 220 was taken from kulturpor-
tali.gov.tr with ID of #8942. Printed by: Gildeprint Contents . . . . . . . . 23
1.5.8
HIPE
. . . . . . . . . . . . . . . . . . . . . . . . . 24
1.5.9
Python Libraries . . . . . . . . . . . . . . . . . . . 24
1.6
Goals of this thesis . . . . . . . . . . . . . . . . . . . . . . 24
1.7
Outline
. . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2
Search for radio jets from massive young stellar objects. Association of radio jets with H2O and CH3OH masers
29
2.1
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
i 1
Introduction
1
1.1
Interstellar Medium
. . . . . . . . . . . . . . . . . . . . . 2
1.2
Low and Massive Star Formation . . . . . . . . . . . . . . 3
1.3
Massive Stars . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.3.1
Jets and Outflows
. . . . . . . . . . . . . . . . . . 9
1.3.2
Photodissociation Regions . . . . . . . . . . . . . . 10
1.3.3
Interstellar Bubbles . . . . . . . . . . . . . . . . . . 13
1.4
Feedback from Massive Stars
. . . . . . . . . . . . . . . . 15
1.4.1
Mechanical Feedback . . . . . . . . . . . . . . . . . 16
1.4.2
Radiative Feedback . . . . . . . . . . . . . . . . . . 16
1.5
Aims and Methods . . . . . . . . . . . . . . . . . . . Contents . . . 18
1.5.1
Herschel Space Observatory . . . . . . . . . . . . . 18
1.5.2
Very Large Array (VLA) . . . . . . . . . . . . . . . 20
1.5.3
Stratospheric Observatory for Infrared Astronomy
(SOFIA) . . . . . . . . . . . . . . . . . . . . . . . . 21
1.5.4
Meudon PDR code . . . . . . . . . . . . . . . . . . 22
1.5.5
RADEX . . . . . . . . . . . . . . . . . . . . . . . . 23
1.5.6
GILDAS . . . . . . . . . . . . . . . . . . . . . . . . 23
1.5.7
CASA . . . . . . . . . . . . . . . . . . . . . . . . . 23
1.5.8
HIPE
. . . . . . . . . . . . . . . . . . . . . . . . . 24
1.5.9
Python Libraries . . . . . . . . . . . . . . . . . . . 24
1.6
Goals of this thesis . . . . . . . . . . . . . . . . . . . . . . 24
1.7
Outline
. . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2
Search for radio jets from massive young stellar objects. Association of radio jets with H2O and CH3OH masers
29
2.1
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 1
Introduction 2.2
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.3
Observations
. . . . . . . . . . . . . . . . . . . . . . . . . Contents 33
2.3.1
Selected sample . . . . . . . . . . . . . . . . . . . . 33
2.3.2
VLA observations . . . . . . . . . . . . . . . . . . . 35
2.4
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.4.1
Continuum emission . . . . . . . . . . . . . . . . . 36
2.4.2
Spectral index analysis . . . . . . . . . . . . . . . . 37
2.4.3
Maser emission . . . . . . . . . . . . . . . . . . . . 41
2.5
Analysis and discussion
. . . . . . . . . . . . . . . . . . . 41
2.5.1
Nature of the radio continuum emission
. . . . . . 42
2.5.2
Association with molecular outflows
. . . . . . . . 44
2.5.3
Association with EGOs
. . . . . . . . . . . . . . . 45
2.5.4
Association with masers . . . . . . . . . . . . . . . 48
2.5.5
Radio-jet candidates . . . . . . . . . . . . . . . . . 48
2.6
Implications for high-mass star formation
. . . . . . . . . 57
2.7
Summary
. . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.8
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 60
2.9
Comments on individual sources
. . . . . . . . . . . . . . 60
2.10 Catalog of the continuum sources . . . . . . . . . . . . . . 72
3
Origin of hydrogen fluoride emission in the Orion Bar. An excellent tracer for CO-dark H2 gas clouds
109
3.1
Abstract . . . . . . . . Contents . . . . . . . . . . . . . . . . . . . . 109
3.2
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 111
3.3
Observation and data reduction . . . . . . . . . . . . . . . 113
3.4
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
3.5
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
3.5.1
Column density . . . . . . . . . . . . . . . . . . . . 119
3.5.2
Spatial distribution of HF . . . . . . . . . . . . . . 122
3.6
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
3.6.1
Collisional excitation . . . . . . . . . . . . . . . . . 124
3.6.2
Infrared pumping . . . . . . . . . . . . . . . . . . . 129
3.6.3
Chemical Pumping . . . . . . . . . . . . . . . . . . 130
3.7
Summary
. . . . . . . . . . . . . . . . . . . . . . . . . . . 130
3.8
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 131
3.9
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
3.9.1
SEDs of Three Positions in the HF map . . . . . . 133 ii 4
Breaking Orion’s Veil bubble with fossil outflows
139
4.1
Abstract . . . . . . . . . . . . . Contents . . . . . . . . . . . . . . . 139
4.2
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 141
4.3
Observations
. . . . . . . . . . . . . . . . . . . . . . . . . 145
4.3.1
[C ii] Observations . . . . . . . . . . . . . . . . . . 145
4.3.2
Molecular Gas Observations . . . . . . . . . . . . . 147
4.3.3
Ionized Gas Observations
. . . . . . . . . . . . . . 148
4.3.4
Far-IR photometric observations
. . . . . . . . . . 148
4.3.5
Mid-IR Observations . . . . . . . . . . . . . . . . . 150
4.4
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
4.5
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
4.5.1
Expansion Velocity . . . . . . . . . . . . . . . . . . 152
4.5.2
Morphology of the protrusion . . . . . . . . . . . . 152
4.5.3
Expansion Timescale . . . . . . . . . . . . . . . . . 155
4.5.4
Line Profile Analysis . . . . . . . . . . . . . . . . . 155
4.5.5
Kinetic Energy and Momentum . . . . . . . . . . . 157
4.6
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
4.6.1
Persistence of fossil outflow cavity
. . . . . . . . . 165
4.6.2
Ionizing source . . . . . . . . . . . . . . . . . . . Contents . 166
4.6.3
Correlation of Intensities . . . . . . . . . . . . . . . 167
4.7
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
4.8
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 170
4.9
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
4.9.1
Geometric correction Factor . . . . . . . . . . . . . 170
4.10 Additional Maps
. . . . . . . . . . . . . . . . . . . . . . . 172
5
Unveiling the Veil: Protostellar feedback in Orion
183
5.1
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
5.2
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 185
5.3
Observations
. . . . . . . . . . . . . . . . . . . . . . . . . 187
5.3.1
[C ii] observations
. . . . . . . . . . . . . . . . . . 187
5.3.2
Molecular Gas observations . . . . . . . . . . . . . 189
5.3.3
Mid-IR observations . . . . . . . . . . . . . . . . . 189
5.3.4
Far-IR photometric observations
. . . . . . . . . . 189
5.4
Identification of Dents . . . . . . . . . . . . . . . . . . . . 190
5.4.1
Position-velocity (PV) Diagrams
. . . . . . . . . . 190
5.4.2
High-velocity [C ii] emission . . . . . . . . . . . . . 193 iii 5.4.3
Line profiles . . . . . . . . . . . Contents . . . . . . . . . . . 193
5.5
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
5.5.1
Characteristics of the dents . . . . . . . . . . . . . 194
5.5.2
Origin of the dents . . . . . . . . . . . . . . . . . . 196
5.5.3
Momentum of the dents . . . . . . . . . . . . . . . 196
5.5.4
Potential shock signature of the dents
. . . . . . . 199
5.5.5
Collimation factor and opening angle . . . . . . . . 201
5.6
Summary
. . . . . . . . . . . . . . . . . . . . . . . . . . . 202
5.7
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 203
5.8
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
5.8.1
Gaussian Fitting Results . . . . . . . . . . . . . . . 204
5.8.2
Massive Stars and Geometry
. . . . . . . . . . . . 204
5.8.3
PV diagram of the dents . . . . . . . . . . . . . . . 205
6
Conclusions and Outlook
215
6.1
Summary and conclusions . . . . . . . . . . . . . . . . . . 215
6.2
Future Outlook . . . . . . . . . . . . . . . . . . . . . . . . 218
7
Additional Sections
223
7.1
Contributed Publications
. . . . . . . . . . . . . . . . . . 223
7.2
Talks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
7.3
Posters . Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
7.4
Türkçe Özet . . . . . . . . . . . . . . . . . . . . . . . . . . 226
7.5
Nederlandse samenvatting . . . . . . . . . . . . . . . . . . 231
7.6
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 236 iv iv
|
https://openalex.org/W2911539515
|
https://www.jmir.org/2019/1/e11658/PDF
|
English
| null |
Digital Recruitment and Acceptance of a Stepwise Model to Prevent Chronic Disease in the Danish Primary Care Sector: Cross-Sectional Study
|
JMIR. Journal of medical internet research/Journal of medical internet research
| 2,019
|
cc-by
| 11,572
|
Digital Recruitment and Acceptance of a Stepwise Model to
Prevent Chronic Disease in the Danish Primary Care Sector:
Cross-Sectional Study Lars Bruun Larsen1, MPH; Jens Sondergaard1, PhD; Janus Laust Thomsen2, PhD; Anders Halling3, PhD; Anders
Larrabee Sønderlund1, PhD; Jeanette Reffstrup Christensen1, PhD; Trine Thilsing1, PhD
1Research Unit of General Practice, Institute of Public Health, University of Southern Denmark, Odense, Denmark
2Research Unit for General Practice, Department of Clinical Medicine, Aalborg University, Aalborg, Denmark
3Department of Clinical Sciences in Malmö, Centre for Primary Health Care Research, Lund University, Lund, Sweden Corresponding Author:
Lars Bruun Larsen, MPH
Research Unit of General Practice
Institute of Public Health
University of Southern Denmark
JB Winsløws Vej 9A
Odense, 5000
Denmark
Phone: 45 65503830
Email: lblarsen@health.sdu.dk Abstract Background: During recent years, stepwise approaches to health checks have been advanced as an alternative to general health
checks. In 2013, we set up the Early Detection and Prevention project (Tidlig Opsporing og Forebyggelse, TOF) to develop a
stepwise approach aimed at patients at high or moderate risk of a chronic disease. A novel feature was the use of a personal digital
mailbox for recruiting participants. A personal digital mailbox is a secure digital mailbox provided by the Danish public authorities. Apart from being both safe and secure, it is a low-cost, quick, and easy way to reach Danish residents. Objective: In this study we analyze the association between the rates of acceptance of 2 digital invitations sent to a personal
digital mailbox and the sociodemographic determinants, medical treatment, and health care usage in a stepwise primary care
model for the prevention of chronic diseases. Methods: We conducted a cross-sectional analysis of the rates of acceptance of 2 digital invitations sent to randomly selected
residents born between 1957 and 1986 and residing in 2 Danish municipalities. The outcome was acceptance of the 2 digital
invitations. Statistical associations were determined by Poisson regression. Data-driven chi-square automatic interaction detection
method was used to generate a decision tree analysis, predicting acceptance of the digital invitations. Results: A total of 8814 patients received an invitation in their digital mailbox from 47 general practitioners. A total of 40.22%
(3545/8814) accepted the first digital invitation, and 30.19 % (2661/8814) accepted both digital invitations. The rates of acceptance
of both digital invitations were higher among women, older patients, patients of higher socioeconomic status, and patients not
diagnosed with or being treated for diabetes mellitus, chronic obstructive pulmonary disease, or cardiovascular disease. Conclusions: To our knowledge, this is the first study to report on the rates of acceptance of digital invitations to participate in
a stepwise model for prevention of chronic diseases. More studies of digital invitations are needed to determine if the acceptance
rates seen in this study should be expected from future studies as well. Similarly, more research is needed to determine whether
a multimodal recruitment approach, including digital invitations to personal digital mailboxes will reach hard-to-reach
subpopulations more effectively than digital invitations only. Original Paper
Digital Recruitment and Acceptance of a Stepwise Model to
Prevent Chronic Disease in the Danish Primary Care Sector:
Cross-Sectional Study
Lars Bruun Larsen1, MPH; Jens Sondergaard1, PhD; Janus Laust Thomsen2, PhD; Anders Halling3, PhD; Anders
Larrabee Sønderlund1, PhD; Jeanette Reffstrup Christensen1, PhD; Trine Thilsing1, PhD
1Research Unit of General Practice, Institute of Public Health, University of Southern Denmark, Odense, Denmark
2Research Unit for General Practice, Department of Clinical Medicine, Aalborg University, Aalborg, Denmark
3Department of Clinical Sciences in Malmö, Centre for Primary Health Care Research, Lund University, Lund, Sweden
Larsen et al
JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Original Paper (J Med Internet Res 2019;21(1):e11658) doi: 10.2196/11658 (J Med Internet Res 2019;21(1):e11658) doi: 10.2196/11658 J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 1
(page number not for citation purposes) Background General health checks are seen as one way to mitigate the rising
prevalence of chronic diseases such as cardiovascular disease
(CVD), type 2 diabetes mellitus (T2DM), and chronic
obstructive pulmonary disease (COPD). Consequently,
periodical general health checks are provided to citizens by
various national health care systems around the world, including
those in the United States, South Korea, Australia, and Germany. However, general health checks have not only failed to show
population health effects on CVD and total mortality but may
have also widen health inequalities [1-6]. This is probably
because of the generally higher uptake of health check initiatives
in the populations who are likely to benefit the least—including
most notably women and patients of higher age, better health,
and higher socioeconomic status (SES). Population-level uptake
seems to be determined by an interrelationship between
individual and societal facilitators and barriers as well as
self-selection [7,8]. A novel feature of the pilot study was the use of a personal
digital mailbox for recruiting participants. A personal digital
mailbox is a secure digital mailbox provided by the Danish
public authorities. It is accessed either via a webpage or an app
developed for all major operating systems. The digital mailbox
is secured by a national 2-phased log-in system (NemID) and
is used by all public authorities as well as an increasing number
of private companies such as banks and insurance companies. Beyond being both safe and secure, it is a low-cost, quick, and
easy way to reach Danish residents [19]. Permanent residents
of Denmark are obliged by law to have a digital mailbox and
are expected to check it regularly. Short message service text
message and mail reminders are optional. Opting out is only
possible in special cases, mainly in the event of low information
and technology literacy (usually age-related) or cognitive
impairment. A total of 90% of the entire population in Denmark
and 95% of the target population have a digital mailbox (May
2016) [20]. During recent years, stepwise approaches to health checks have
been advanced as an alternative to general health checks [9,10]. Stepwise approaches usually comprise a risk assessment to
identify the at-risk population, followed by health checks
tailored to this population. If deemed necessary, behavior change
interventions, preventive medical treatment, or a combination
of the two may also be included. KEYWORDS promotion of health; clinical decision support systems; cross-sectional studies http://www.jmir.org/2019/1/e11658/ XSL•FO
RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al targeted intervention would be complemented by further
behavior change intervention or preventive medical treatment. Patients
already
diagnosed
with
hypertension,
hypercholesterolemia, T2DM, CVD, or COPD by the GP, or
who displayed no health risk behaviors were only offered the
joint intervention. In line with the Medical Research Council’s
recommendations for complex interventions, we tested the
acceptability, feasibility, and short-term effects of the
intervention in a pilot study in 2 municipalities between April
and December 2016 [18]. Objective This study reports on the association between the rates of
acceptance of 2 digital invitations sent to a personal digital
mailbox and sociodemographic determinants, medical treatment,
and health care usage in a stepwise primary care model for the
prevention of chronic diseases. Design We conducted a cross-sectional analysis of the rates of
acceptance of 2 digital invitations sent to residents from 2
Danish municipalities randomly selected to take part in a pilot
study of the TOF project (NCT02797392). Population The target population consisted of citizens born between 1957
and 1986 and residing in Haderslev or Varde, 2 rural
municipalities located in the southern part of Jutland, Denmark. The population of both municipalities totals 106,081 citizens
(2015). Background Various stepwise approaches
to health checks have been tested in research studies; however,
no long-term effects have been reported [11-15]. In the Danish
health care system, health checks are provided to the general
population on an opportunistic, nonperiodic, and nontargeted
basis. On the basis of a technical feasibility study from 2012
[16], we set up the Tidlig Opsporing og Forebyggelse (TOF;
early detection and prevention) project in a partnership with the
general practitioners’(GPs) organization and 10 municipalities
of the Region of Southern Denmark [17]. Over a period of 2
years, we developed a stepwise model for systematic and
targeted prevention of chronic diseases to be used in the Danish
primary care sector. The intervention consisted of a joint
intervention and a targeted intervention. The joint intervention
was applied to the entire study population in the form of a
personal digital health profile. The targeted intervention was
only applied to patients who were deemed to potentially benefit
from either a health check at their GP or lifestyle coaching
provided by the municipal health center. Patients at high risk
of a chronic disease were identified using validated risk
algorithms for COPD, T2DM, and CVD and were offered a
health check at their GP in the form of a medical examination
and a health dialogue. Patients with health risk behavior included
patients who were not at high risk as determined by the risk
algorithms but who engaged in one or more health-risk behaviors
such as smoking, high-risk alcohol consumption, poor dietary
habits, sedentary behavior, and/or a body mass index above 35. This cohort was offered a short 15-min telephone-based health
dialogue with a health professional from the municipal health
center. For patients with limited health capabilities, the initial
telephone-based health dialogue could be followed up by a
1-hour face-to-face health dialogue. If deemed necessary, the J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 2
(page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ Variables In January 2016, all 66 GPs residing in the municipalities of
Varde and Haderslev received a written invitation, with an
enclosed project agreement form and a prepaid return envelope. Nonresponse was followed up by a telephone call to the GP. Using the Regional Primary Care Administrative System (KMD
Sygesikring), the regional health authorities identified a source
population of 200 randomly selected patients extracted from
each of the participating GPs’ patient lists. From the source
population, we excluded patients having either no digital
mailbox or residing outside the municipalities of Varde or
Haderslev. Registry variables for the entire study population were retrieved
from the administrative registry and Statistics Denmark and
linked with the patients’Danish Personal Identification numbers. EPR information was retrieved directly from participating GPs’
EPR systems and related purely to consenting patients (Table
1). All participants were pseudonymized when linking project
data and national registers from Statistics Denmark. Age was categorized in 10-year age groups. Country of origin
was retrieved for the year 2016 and categorized as Danish,
Western, or non-Western origin. Cohabitational status was
retrieved for the year 2016 and categorized as cohabiting or
single. Highest attainable educational level was retrieved for
October 2015 and categorized as secondary school, high school,
vocational education, or higher education. Occupation was
retrieved for November 2014 and categorized according to the
Organisation for Economic Co-operation and Development
equivalence scales into 5 groups: employed, self-employed,
unemployed or on benefits, social welfare recipients, or other
[30]. The distinction between unemployment benefits and social
welfare is that unemployment benefits are accessible to citizens
who have been unemployed for less than 2 years and who are
members of a voluntary unemployment benefit fund. Social
welfare benefits are for all other unemployed persons who can
take up a job. Others represent, for example, nonworking
persons from a family that relies on 1 income only. Family
income was retrieved for 2013, 2014, and 2015, defined by the
mean annual net income of the household, and categorized into
quartiles. “Partner in project” describes whether your partner
(if cohabiting) participated as well. Partner in project is
categorized in a binary yes or no variable. Participants were recruited using 2 digital invitations sent to
their personal digital mailbox (Multimedia Appendix 1). a personal digital health profile. The results from the second
digital invitation are presented in Multimedia Appendices 2 and
3. a personal digital health profile. The results from the second
digital invitation are presented in Multimedia Appendices 2 and
3. secondary prevention, including treatment for hypertension,
hypercholesterolemia, and diabetes. secondary prevention, including treatment for hypertension,
hypercholesterolemia, and diabetes. Variables Both
digital invitations consisted of a 1-page PDF file in Danish and
included a highly visible hyperlink to a Web-based digital
support system, on which participants would provide both
consent and access to the personal digital health profile [22]. The first digital invitation was sent out in April 2016, with the
aim of obtaining consent to participate in the study and to access
specific information from their GP’s electronic patient record
(EPR) system, including International Classification of Primary
Care, 2nd edition) codes for diagnoses and anatomical
therapeutic chemical (ATC) codes for medical prescriptions. The second digital invitation was aimed at providing participants
with a digital health profile. This invitation was sent out in
September 2016 to participating patients who were registered
with the same GP as when they consented and who still resided
in the municipalities of Varde or Haderslev. A nonresponse
triggered up to 2 reminders 1 week apart. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 3
(page number not for citation purposes) Setting The Danish health care system comprises a strong, publicly
funded primary care sector, which includes municipal health
centers and GP clinics [21]. GPs operate a patient list system,
with an average of 1600 patients per GP. On average, 2 GPs
work in a given clinic. Municipal health centers serve the entire
population with primary prevention such as smoking cessation
and dietary advice, whereas GPs manage and coordinate http://www.jmir.org/2019/1/e11658/ XSL•FO
RenderX XSL•FO
RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Table 1. Analyses of associations between patient characteristics and acceptance. Second digital invitation
First digital invitation
Variable
Multimedia Appendix 3
Multimedia Appendix 2
Results section
Presentation of results
Consent to the first digital invitation
Study population
Study population
Denominator
Uptake or nonuptake of the second
digital invitation
Uptake or nonuptake of the second
digital invitation
Consent or nonconsent to the first
digital invitation
Outcome variable
Exposures
Agea,b
Agea,b
Agea,b
Sociodemographics
Sexa,b
Sexa,b
Sexa,b
Country of origin
Country of origin
Country of originb
Highest educational attainmentc
Highest educational attainmentc
Highest educational attainmentc
Occupational statusd
Occupational statusd
Occupational statusd
Family incomee
Family incomee
Family incomee
Cohabitational statusb
Cohabitational statusb
Cohabitational statusb
Partner consentf
Partner consentf
Partner consentf
Prescriptions from primary care or
hospitals (ATC codes)g
Prescriptions from primary care or
hospitals (ATC codes)g
Prescriptions from primary care or
hospitals (anatomical therapeutic
chemical [ATC] codes)g
Medical treatment
Hospital discharge diagnoses (ICD-
10 codes)h
Hospital discharge diagnoses (ICD-
10 codes)h
Hospital discharge diagnoses (Interna-
tional Classification of Diseases 10th
edition [ICD-10] codes)h
Administrative primary care codesi,j;
health checksi,j
Administrative primary care codesi,j
Administrative primary care codesi,j
Health care usage
aDanish National Administrative Primary Care System (Praksys). bDanish Civil Registration System [23]. cDanish Education Register [24]. dDanish Registers of Labour Market Affiliation [25]. eDanish Registers of Personal Income and Transfer Payments [26]. fQuestionnaire data. gDanish National Prescription Registry [27]. hDanish National Patient Registry [28]. iGeneral practitioners’ electronic patient record. jDanish National Health Service Register [29]. Table 2. International Classification of Diseases 10th edition codes and anatomical therapeutic chemical classification codes used to define when a
medical condition of chronic obstructive pulmonary disease, cardiovascular disease, or diabetes mellitus had been registered. ATCb therapeutic codes for prescribed medicine registered
from May 2014 to April 2016
ICD-10a codes registered from January 2013 to April 2016
Medical condition
R03AC18, R03AC19, R03AL03, R03AL04, R03AL05,
R03BB04, R03BB05, and R03BB06
J44
COPDc
C (except: C01CA and C05)
I1-I7 and E78 (except: I0, I16, I60, I73, and I78)
CVDd
A10
E10-E13
Diabetes mellitus
aICD-10: International Classification of Diseases 10th edition. bATC: anatomical therapeutic chemical. cCOPD
h
i
b
i
l
di
Larsen et al
JOURNAL OF MEDICAL INTERNET RESEARCH Table 1. Analyses of associations between patient characteristics and acceptance. Outcomes In this paper, we report on both the consent to take part in the
study and the uptake of the personal digital health profile. The
main outcome relates to the acceptance of the first digital
invitation and is operationalized in terms of consent or
nonconsent to take part in the study. Consent is defined as the
provision of informed consent to participate in the study;
nonconsent includes both nonresponse and active nonconsent. The second outcome relates to the acceptance of the second
digital invitation and is operationalized in terms of uptake or
nonuptake of the personal digital health profile. Uptake is
defined as patients who gave their active consent and received Information on prescriptions and diagnoses was combined as a
proxy for medical treatment (Table 2). Prescriptions were
retrieved for the period from May 2014 to April 2016 as ATC
codes. We chose a 2-year period, as prescriptions may be filed
up to 2 years after their date of issue. International Classification
of Diseases 10th edition (ICD-10) codes were retrieved for the
period from January 2013 to April 2016. Medical treatment was
defined as either registered with an ATC code, ICD-10 code,
or both during the periods specified above. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 3
(page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ http://www.jmir.org/2019/1/e11658/ XSL•FO
RenderX http://www.jmir.org/2019/1/e11658/ JOURNAL OF MEDICAL INTERNET RESEARCH Second digital invitation
First digital invitation
Variable
Multimedia Appendix 3
Multimedia Appendix 2
Results section
Presentation of results
Consent to the first digital invitation
Study population
Study population
Denominator
Uptake or nonuptake of the second
digital invitation
Uptake or nonuptake of the second
digital invitation
Consent or nonconsent to the first
digital invitation
Outcome variable
Exposures
Agea,b
Agea,b
Agea,b
Sociodemographics
Sexa,b
Sexa,b
Sexa,b
Country of origin
Country of origin
Country of originb
Highest educational attainmentc
Highest educational attainmentc
Highest educational attainmentc
Occupational statusd
Occupational statusd
Occupational statusd
Family incomee
Family incomee
Family incomee
Cohabitational statusb
Cohabitational statusb
Cohabitational statusb
Partner consentf
Partner consentf
Partner consentf
Prescriptions from primary care or
hospitals (ATC codes)g
Prescriptions from primary care or
hospitals (ATC codes)g
Prescriptions from primary care or
hospitals (anatomical therapeutic
chemical [ATC] codes)g
Medical treatment
Hospital discharge diagnoses (ICD-
10 codes)h
Hospital discharge diagnoses (ICD-
10 codes)h
Hospital discharge diagnoses (Interna-
tional Classification of Diseases 10th
edition [ICD-10] codes)h
Administrative primary care codesi,j;
health checksi,j
Administrative primary care codesi,j
Administrative primary care codesi,j
Health care usage
aDanish National Administrative Primary Care System (Praksys). Table 1. Analyses of associations between patient characteristics and acceptance. Exposures aDanish National Administrative Primary Care System (Praksys). bDanish Civil Registration System [23]. cDanish Education Register [24]. dDanish Registers of Labour Market Affiliation [25]. eDanish Registers of Personal Income and Transfer Payments [26]. fQuestionnaire data. gDanish National Prescription Registry [27]. hDanish National Patient Registry [28]. iGeneral practitioners’ electronic patient record. jDanish National Health Service Register [29]. Table 2. International Classification of Diseases 10th edition codes and anatomical therapeutic chemical classification codes used to define when a
medical condition of chronic obstructive pulmonary disease, cardiovascular disease, or diabetes mellitus had been registered. ATCb therapeutic codes for prescribed medicine registered
from May 2014 to April 2016
ICD-10a codes registered from January 2013 to April 2016
Medical condition
R03AC18, R03AC19, R03AL03, R03AL04, R03AL05,
R03BB04, R03BB05, and R03BB06
J44
COPDc
C (except: C01CA and C05)
I1-I7 and E78 (except: I0, I16, I60, I73, and I78)
CVDd
A10
E10-E13
Diabetes mellitus
aICD-10: International Classification of Diseases 10th edition. bATC
i
l h
i
h
i
l ATCb therapeutic codes for prescribed medicine registered
from May 2014 to April 2016 J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 4
(page number not for citation purposes) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 4
(page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Health care usage was determined from administrative codes
registered by the GP and retrieved for the period from May 2013
to April 2016. To this end, we also examined EPR information
on laboratory test results. Administrative codes were used to
extract frequent GP attenders, GP attenders, and usage of
specific administrative codes pertaining to laboratory tests and
preventive consultations. Frequent attenders referred to the top
10% of patients, who on average contacted the GP the
most—either in person or by phone—during the 3 years from
May 2013 to April 2016 [31]. GP attenders were defined as
patients having contacted their GP during the 2-year period
from May 2014 to April 2016. Laboratory tests comprised blood
samples (administrative codes 2101 and 2601), peak flow tests
(7183), spirometries (7113), and home blood pressure
monitoring (2146) during the period from May 2014 to April
2016. Furthermore, we retrieved information on specific
preventive consultations (0120)—that is, special consultations
for coaching patients with health risk behaviors and patients
diagnosed with a chronic disease. Ethics Approval and Consent to Participate The study was approved by the Danish Data Protection Agency
(J.Number 2015-57-0008) and registered at Clinical Trial Gov
(Unique Protocol ID: TOFpilot2016). According to Danish
regulations (Act on Research Ethics Review of Health Research
Projects [section 14,2]), this study does not need approval from
a health research ethics committee as no research on human
tissue or other biological material is performed. The study
complies with the Helsinki Declaration, with informed consent
to study participation and to disclosure of data from the GPs’
EPR obtained from all participants. Exposures To determine if a patient had
received a preventive health check within a period of 2 years
before consenting to the study (May 2014 to May 2016), we
retrieved blood pressure (systolic and diastolic blood pressure),
lung function (forced expiratory volume [FEV1], forced vital
capacity [FVC], and FEV1/FVC), glycated hemoglobin, and
lipids (total cholesterol, high-density lipoprotein, and
low-density lipoprotein) measurements from the GPs’ EPR
systems. A health check was defined as having had 2 or more
of the above-mentioned values measured in the same
consultation. tests of interdependence, a CHAID analysis clusters categories
within each predictor variable to determine what predictor
variables are associated with the outcome. Subsequently, the
predictor variables associated with the outcome are ordered
hierarchically. The specific variable order is determined by the
Bonferroni P value of each variable such that the predictor
variable with the smallest P value (strongest association) is
placed at the top of the hierarchy (parent node). The minimum
number of observations for each split in the decision tree was
set at 500 (child node) and 200 for each node (terminal node). In the analysis of the acceptance of the second digital invitation,
each split in the decision tree was set to 100 (child node) and
20 for each node (terminal node) because of the limited number
of observations. Statistical analysis was performed on secure servers at Statistics
Denmark using Stata 14 (Statacorp). J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 5
(page number not for citation purposes) Recruitment and Overall Uptake Statistical associations are presented as crude figures, age- and
sex-adjusted figures, and as models minimally adjusted for
known confounders. Poisson regression with robust variance
error was used rather than logistic regression to obtain incidence
rate ratios (IRR). The binary outcome variable of consent or
nonconsent was interpreted as a continuous variable with the
only counts being 0 or 1. The minimally adjusted model was
developed from a causal direct acyclic graph (DAG), built on
the current evidence of the determinants of attendance at health
checks [32]. Attendance at health checks was the outcome
variable of the DAG. Significance level was set at P<.05. Of the 68 GPs residing in the 2 municipalities, 47 GPs from 18
clinics agreed to participate in the study (Figure 1). This
provided us with a source population of 9400 patients. However,
a total of 586 patients did not meet the inclusion criteria, which
is why only 8814 received the first invitation. Initially, a total
of 3587 patients consented to participate, but among them, 30
patients moved from the municipality to a nonparticipating GP,
and 12 withdrew their consent after receiving the second
invitation. This resulted in 3545 active consenters from the first
round of invitations (Multimedia Appendix 2). Of the patients
who accepted the first digital invitation (n=3545), 75.06%
(2661/3545) also accepted the second digital invitation
(Multimedia Appendix 3; Table 3). A data-driven chi-square automatic interaction detection
(CHAID) method was used to generate a decision tree analysis
to identify interactions and a hierarchy of variables predicting
the chosen outcome variable (root node) [33]. Using chi-square J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 5
(page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ http://www.jmir.org/2019/1/e11658/ JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Figure 1. Flow diagram from source population to study population. GP: general practitioner. Larsen et
JOURNAL OF MEDICAL INTERNET RESEARCH J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 6
(page number not for citation purposes) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 6
(page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Table 3. Descriptive analysis of determinants of the acceptance of the first digital invitation. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 7
(page number not for citation purposes) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 7
(
b
f
i
i
) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 7
http://www.jmir.org/2019/1/e11658/
(page number not for citation purposes)
FO http://www.jmir.org/2019/1/e11658/ Recruitment and Overall Uptake Missing, n (%
Total (N=8814), n (%)
Nonconsenters (N=5269), n (%)
Consenters (N=3545), n (%)
Determinants
Demographya
Age (years)
0 (0.00)
2653 (30.09)
1921 (36.45)
732 (20.64)
29-39
0 (0.00)
3026 (34.33)
1875 (35.58)
1151 (32.46)
40-49
0 (0.00)
3135 (35.56)
1473 (27.95)
1662 (46.88)
50-60
Sex
0 (0.00)
4379 (49.68)
2845 (53.99)
1590 (44.85)
Male
0 (0.00)
4435 (50.31)
2424 (46.00)
1955 (55.14)
Female
Country of origin
18 (0.20)
7831 (88.44)
4446 (84.38)
3385 (95.48)
Denmark
18 (0.20)
549 (6.22)
458 (8.69)
91 (2.56)
Western
18 (0.20)
416 (4.71)
347 (6.58)
69 (1.94)
Non-Western
Cohabitation
18 (0.20)
2242 (25.43)
1516 (28.77)
726 (20.47)
Single
18 (0.20)
6554 (74.35)
3735 (70.88)
2819 (79.52)
Cohabiting
Partner in project
18 (0.20)
4752 (53.91)
2715 (51.52)
2037 (57.46)
Yes
18 (0.20)
4044 (45.88)
2536 (48.13)
1508 (42.53)
No
Socioeconomy
Educational attainment
583 (6.61)
1707 (19.36)
1194 (22.66)
513 (14.47)
Secondary school
583 (6.61)
356 (4.03)
213 (4.04)
143 (4.103)
High school
583 (6.61)
3803 (46.14)
2199 (41.73)
1604 (45.24)
Vocational education
583 (6.61)
2365 (26.83)
1149 (21.80)
1216 (34.30)
Higher education
Employment status
105 (1.19)
6610 (74.99)
3719 (70.58)
2891 (81.55)
Employed
105 (1.19)
430 (4.87)
260 (4.93)
170 (4.79)
Self-employed
105 (1.19)
272 (3.08)
184 (3.49)
88 (2.48)
Benefits
105 (1.19)
1146 (13.00)
806 (15.29)
340 (9.59)
Social welfare
105 (1.19)
251 (2.84)
200 (3.79)
51 (1.43)
Other
Family income
130 (1.47)
2047 (23.22)
1488 (28.24)
559 (15.76)
Low
130 (1.47)
2147 (24.35)
1341 (25.45)
806 (22.73)
Middle-low
130 (1.47)
2235 (25.35)
1246 (24.64)
989 (27.89)
Middle-high
130 (1.47)
2255 (25.58)
1072 (20.34)
1183 (33.4)
High
Medical treatmentb
Prescriptions and diagnoses
0 (0.00)
1736 (19.69)
973 (18.46)
763 (21.52)
Treatment
0 (0.00)
7078 (80.30)
4296 (81.53)
2782 (78.47)
No treatment
Health care usagec
Att
d
t
l
titi
(GP) Table 3. Descriptive analysis of determinants of the acceptance of the first digital invitation. Recruitment and Overall Uptake Missing, n (%)
Total (N=8814), n (%)
Nonconsenters (N=5269), n (%)
Consenters (N=3545), n (%)
Determinants http://www.jmir.org/2019/1/e11658/ XSL•FO
RenderX XSL•FO
RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Missing, n (%)
Total (N=8814), n (%)
Nonconsenters (N=5269), n (%)
Consenters (N=3545), n (%)
Determinants
0 (0.00)
7545 (85.60)
4372 (82.97)
3173 (89.50)
Yes
0 (0.00)
1269 (14.39)
897 (17.02)
372 (10.49)
No
Frequent attender
0 (0.00)
952 (10.80)
584 (11.08)
368 (10.38)
Yes
0 (0.00)
7862 (89.19)
4685 (88.91)
3177 (89.61)
No
Laboratory tests at GP
0 (0.00)
4524 (51.32)
2471 (46.89)
2053 (57.91)
Yes
0 (0.00)
4290 (48.67)
2798 (53.10)
1492 (42.08)
No
Preventive consultation at GP
0 (0.00)
947 (10.74)
516 (9.79)
431 (12.15)
Yes
0 (0.00)
7867 (89.25)
4753 (90.20)
3114 (87.84)
No
aSocial registries. bAnatomical therapeutic chemical codes and International Classification of Diseases 10th edition codes related to diabetes, cardiovascular disease, and
chronic obstructive pulmonary disease. Social registries. bAnatomical therapeutic chemical codes and International Classification of Diseases 10th edition codes related to diabetes, cardiovascular disease, and
chronic obstructive pulmonary disease. bAnatomical therapeutic chemical codes and International Classification of Diseases 10th edition codes related to diabetes, cardiovascular disease, and
chronic obstructive pulmonary disease. cAdministrative codes from the general practitioner. invitation and the frequency of GP appointments, being
registered with a preventive consultation, or having a partner
that also consented to the study. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 8
(page number not for citation purposes) Acceptance of the First Digital Invitation The Poisson regressions showed that a higher rate of acceptance
of
the
first
digital
invitation
was
associated
with
sociodemographic factors, including higher age, income, and
educational attainment (Table 4). A higher rate of acceptance
was also associated with being female, employed, born in
Denmark, and cohabiting. Patients not diagnosed with or in
treatment for T2DM, CVD, or COPD were more likely to accept
the first digital invitation than patients in treatment. Similarly,
the acceptance rate was higher among patients who had seen
their GP or who had registered 1 or more laboratory tests at
their GP within 2 years of giving consent. We found no
association between the likelihood of accepting the first digital The CHAID analysis showed that age was the strongest predictor
of accepting the first digital invitation followed by the
educational attainment in patients below the age of 50 years
and income in patients above the age of 50 years (Figure 2). The CHAID showed large subgroup differences in acceptance
rates. Of patients below the age of 40 years, with secondary
school as the highest educational attainment, 15.92% accepted
the first digital invitation. By contrast, the acceptance rate among
patients above the age of 50 years, with high income and with
at least a bachelor-level education was 68.58 %. http://www.jmir.org/2019/1/e11658/ XSL•FO
RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Table 4. Analysis of associations between acceptance of the first digital invitation and sociodemographic determinants, medical treatment, and heal
care usage. Acceptance of the First Digital Invitation Model 3 (minimally adjusted)
Model 2 (adjusted for age and sex)
Model 1 (crude)
Sample size (N)
Determinants
P value
IRR (95% CI)
P value
IRR (95% CI)
P value
IRRa (95% CI)
Ageb (years)
—
1 (0)
—
1 (0)
—c
1 (0)
2653
29-39
.001
1.08 (1.06-1.10)
.001
1.08 (1.06-1.10)
.001
1.08 (1.06-1.10)
3026
40-49
.001
1.20 (1.18-1.22)
.001
1.20 (1.18-1.22)
.001
1.20 (1.18-1.22)
3135
50-60
Sexb
—
1 (0)
—
1 (0)
—
1 (0)
4435
Female
.001
0.94 (0.93-0.95)
.001
0.94 (0.93-0.95)
.001
0.94 (0.93-0.95)
4379
Male
Country of originb
—
1 (0)
—
1 (0)
—
1 (0)
7831
Denmark
.001
0.81 (0.79-0.84)
.001
0.84 (0.81-0.86)
.001
0.81 (0.79-0.84)
549
Western
.001
0.81 (0.79-0.84)
.001
0.84 (0.81-0.86)
.001
0.81 (0.79-0.84)
416
Non-Western
Cohabitationd
—
1 (0)
—
1 (0)
—
1 (0)
2242
Single
.001
1.05 (1.03-01.07)
.001
1.07 (1.05-01.08)
.001
1.08 (1.06-01.10)
6554
Cohabiting
Partner in projecte
—
1 (0)
—
1 (0)
—
1 (0)
4752
Yes
.70
1.00 (0.99-1.02)
.001
0.96 (0.95-0.98)
.001
0.96 (0.95-0.98)
4044
No
Educational attainmentf
—
1 (0)
—
1 (0)
—
1 (0)
1707
Secondary school
.001
1.09 (1.05-1.14)
.001
1.09 (1.05-1.13)
.001
1.08 (1.04-1.12)
356
High school
.001
1.10 (1.08-1.12)
.001
1.10 (1.08-1.12)
.001
1.09 (1.07-1.12)
3803
Vocational education
.001
1.16 (1.14-1.19)
.001
1.17 (1.14-1.19)
.001
1.16 (1.14-1.19)
2365
Higher education
Employment statusd
—
1 (0)
—
1 (0)
—
1 (0)
6610
Employed
.05
0.97 (0.94-1.00)
.03
0.96 (0.93-1.00)
.089
0.97 (0.94-1.00)
430
Self-employed
.03
0.95 (0.91-1.00)
.001
0.93 (0.89-0.97)
.001
0.92 (0.88-0.96)
272
Benefits
.001
0.94 (0.92-0.96)
.001
0.90 (0.88-0.92)
.001
0.90 (0.88-0.92)
1146
Social welfare
.005
0.92 (0.87-0.97)
.001
0.85 (0.82-0.89)
.001
0.84 (0.80-0.87)
251
Other
Family incomed
—
1 (0)
—
1 (0)
—
1 (0)
2047
Low
.001
1.05 (1.02-1.07)
.001
1.07 (1.05-1.10)
.001
1.08 (1.06-1.10)
2147
Middle-low
.001
1.06 (1.04-1.09)
.001
1.11 (1.09-1.14)
.001
1.13 (1.11-1.16)
2235
Middle-high
.001
1.08 (1.05-1.10)
.001
1.14 (1.12-1.17)
.001
1.20 (1.17-1.22)
2255
High
Prescriptions and diagnosesd
—
1 (0)
—
1 (0)
—
1 (0)
1736
Treatment
.04
1.02 (1.00-1.04)
.02
1.02 (1.00-1.04)
.001
0.97 (0.95-0.99)
7078
No treatment
Attendance at general practitioner (GP)d Table 4. Analysis of associations between acceptance of the first digital invitation and sociodemographic determinants, medical treatment, and health
care usage. care usage. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 9 (page number not for citation purposes) |
|
|
| p
(page number not for citation purposes) Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 9
(page number not for citation purposes) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 10
(page number not for citation purposes) Acceptance of the First Digital Invitation Sample s
Determinants
Ageb (years)
2653
29-39
3026
40-49
3135
50-60
Sexb
4435
Female
4379
Male
Country of originb
7831
Denmark
549
Western
416
Non-Western
Cohabitationd
2242
Single
6554
Cohabiting
Partner in projecte
4752
Yes
4044
No
Educational attainmentf
1707
Secondary school
356
High school
3803
Vocational education
2365
Higher education
Employment statusd
6610
Employed
430
Self-employed
272
Benefits
1146
Social welfare
251
Other
Family incomed
2047
Low
2147
Middle-low
2235
Middle-high
2255
High
Prescriptions and diagnosesd
1736
Treatment
7078
No treatment
Attendance at general practitioner (G
7545
Yes http://www.jmir.org/2019/1/e11658/ XSL•FO
RenderX XSL•FO
RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Model 3 (minimally adjusted)
Model 2 (adjusted for age and sex)
Model 1 (crude)
Sample size (N)
Determinants
P value
IRR (95% CI)
P value
IRR (95% CI)
P value
IRRa (95% CI)
.001
0.95 (0.93-0.98)
.001
0.93 (0.91-0.95)
.001
0.91 (0.89-0.93)
1269
No
Frequent attender to GPd
—
1 (0)
—
1 (0)
—
1 (0)
952
Yes
.07
1.02 (1.00-1.05)
.007
1.03 (1.01-1.06)
.30
1.01 (0.99-1.04)
7862
No
Laboratory tests at GPd
—
1 (0)
—
1 (0)
—
1 (0)
4524
Yes
.001
0.95 (0.94-0.97)
.001
0.95 (0.94-0.97)
.001
0.93 (0.91-0.94)
4290
No
Preventive consultation at GPd
—
1 (0)
—
1 (0)
—
1 (0)
947
Yes
.66
0.99 (0.97-1.02)
.94
1.00 (0.98-1.02)
.001
0.96 (0.94-0.98)
7867
No
aIncidence rate ratio. bModel 3 adjustments: no adjustments. cReference category. dModel 3 adjustments: age, sex, country of origin, and education. eModel 3 adjustments: cohabitation. fModel 3 adjustments: age, sex, and country of origin. fModel 3 adjustments: age, sex, and country of origin. http://www.jmir.org/2019/1/e11658/ XSL•FO
RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Figure 2. Chi-square automatic interaction detection analysis of the acceptance of the first digital invitation. GP: general practitioner. Acceptance of the Second Digital Invitation
Acceptance of the Second Digital Invitation Among Acceptance of the Second Digital Invitation Among
Consenters (N=3545) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 11
(page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ Main Findings In this study, 40.22% of our sample accepted the first digital
invitation and 30.19% accepted both digital invitations. That
is, among those who accepted the first digital invitation, 75.06%
also accepted the second. The rates of acceptance of both digital
invitations were higher among women, elderly patients, patients
of higher SES, and patients not diagnosed with or in treatment
for T2DM, COPD, or CVD. Patients who had seen their GP
within the past 2 years were also more likely to accept the digital
invitations. The frequency of GP appointments, registering for
a preventive consultation, or having a partner who had accepted
the first digital invitation showed no association with the
acceptance of either invitation. In the subpopulation of patients
(N=2661) who accepted the first digital invitation, women,
patients of relatively high age and SES, and patients who had
not seen their GP for a period of 2 years before giving their
consent showed a higher rate of acceptance of the second
invitation. No other health care usage, including having had a
health check within the previous 2 years or being diagnosed
with or in treatment for T2DM, COPD, or CVD showed any
association with the rate of acceptance. Low patient uptake of stepwise models for preventing chronic
diseases seems to be the current norm. This trend may be
attributed to a combination of a recent overall increase in the
use of preventive health checks in primary care and a decrease
in response rates to research studies in general [34,35]. A recent
Dutch study of a stepwise prevention model showed an uptake
rate of 29% in patients aged 45 to 70 years, whereas an
Australian study had an initial uptake rate of 31% in patients
aged 40 to 64 years [11,36]. A feasibility study to this study
showed an uptake rate of 63% using paper-based invitations,
with a link to a Web-based questionnaire and an enclosed hard
copy questionnaire and return envelope [37]. Two other Danish
studies of stepwise models showed uptake rates of 55% in a
general population aged 30 to 49 years and 30% in a population
of social housing residents aged 45 to 70 years [38,39]. Both
Danish studies used a proactive approach by which paper-based
invitations indicated a prebooked time and date—a method
which has been shown to garner increased response rates in a
previous study [40]. Acceptance of the Second Digital Invitation Acceptance of the Second Digital Invitation In the entire study population, the rate of acceptance of the
second digital invitation showed associations similar to the ones
in the analysis of the first digital invitation. The only differences
were higher rates of acceptance among frequent GP attenders
and no association with attendance or nonattendance to the GP. The similarity also applied to the CHAID analysis, in which
age was shown to be the strongest predictor, followed by
educational attainment in the age below 50-years bracket and
income in the age above 50-years bracket. The CHAID analysis
showed a rate of acceptance of 8.37% in patients below the age
of 40, with secondary school as their highest educational
attainment and income below 50% of the median. By contrast,
among patients aged more than 50 years, with an income of
more than 50% above the median and at least a bachelor-level
education, 60.40% accepted the second digital invitation. Among the patients who accepted the first digital invitation, the
Poisson regressions indicated associations between the rate of
acceptance of the second digital invitation and most
sociodemographic variables. The rate of acceptance increased
with age, educational attainment, and income. Being female,
employed, and born in Denmark also correlated positively with
rates of acceptance. We saw higher rates of acceptance among
patients who had not seen their GP within 2 years before
consenting to the study. No other variables describing health
status or health care usage showed an association with rates of
acceptance—that includes having received a health check during
a period of 2 years before consenting to the study. The CHAID analysis showed that age was the strongest predictor
of acceptance of the second digital invitation among those who http://www.jmir.org/2019/1/e11658/ J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 11
(page number not for citation purposes) XSL•FO
RenderX XSL•FO
RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al evidence of the association between uptake of stepwise models
and medical treatment or health care usage is scarce and largely
inconclusive. This is presumably because of a general lack of
health and health care information on nonresponders. Nonetheless, the results of medical treatment and health care
usage in this study differ somewhat from previous studies. We
show slightly higher rates of acceptance of the digital invitations
among patients not diagnosed with or in treatment for T2DM,
COPD, or CVD. Acceptance of the Second Digital Invitation This is in line with a comparable Danish study
[38], whereas other studies report either higher uptake among
patients with chronic diseases [42,43] or no association [44,45]. The inconsistency in results could be explained by different
definitions of medical treatment. Similarly, different definitions
of preventive services could explain why we see no association
between acceptance of the digital invitations and use of
preventive consultations at the GP, whereas other studies suggest
an association [38]. We saw no association between acceptance
of either invitation and having had a health check during a period
of 2 years leading up to consent; however, other studies have
consistently found that prior use of health checks seems to
increase the likelihood of getting another health check [46-48]. Interestingly, we found an association between not having had
a GP appointment during the previous 2 years and a higher rate
of acceptance of the second digital invitation among those who
also accepted the first digital invitation. This may suggest that
true compliers to the study (ie, patients who would not have
taken up the offer had they not been invited) are more likely to
also accept the second digital invitation than always-users (ie,
patients who always respond to invitations to participate in
preventive services) [49]. As we saw no association between
the rates of acceptance of the second digital invitation and other
variables of health care utilization, this result should be
interpreted with great care and examined further in future
studies. accepted the first digital invitation and the only predictor in
patients younger than 50 years. In patients older than 50 years,
income followed age as the second strongest predictor. Specifically, when it came to the patients older than 50 years,
with a middle-low or middle-high income, the CHAID analysis
revealed that nonattendance at the GP and not receiving medical
treatment during a period of 2 years before consenting were
both strong predictors of whether or not participants accepted
the second digital invitation. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 12
(page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ Main Findings Furthermore, the associations between
acceptance of digital invitations and socioeconomic determinants
are in line with the evidence from other Danish and European
studies of health checks [6,38,41]. However, the CHAID
analyses showed that the differences in rates of acceptance
among the SES groups in our study are larger than those
observed in the 2 Danish studies mentioned above [38,39]. The Efficacy of Digital Recruitment To our knowledge, this is the first study to report on the rates
of acceptance of digital invitations to participate in a stepwise
model for prevention of chronic diseases. Moreover, it is most
likely the first to report on digital recruitment to a health
intervention using digital invitations. We cannot establish
whether rates of acceptance would have been different if
recruitment had been paper-based, as we did not include a
random subpopulation, which received paper-based invitations. To our knowledge, comparisons of digital and paper-based
invitations sent by regular mail have only been reported once
in a randomized study by Ebert et al [19]. This study showed
that 50- to 59-year-old responders to the digital invitation were
more likely to be of higher SES than their counterparts who
responded to paper invitations. However, no differences were
seen in those aged 30 to 39 years. The overall rate of acceptance
of digital invitations and paper-based invitations was
comparable. The rate of acceptance of digital invitations in this
study and the results reported by Ebert et al may suggest slightly
lower overall acceptance rates and slightly larger SES
differences when using digital invitations. However, the rate of
acceptance of digital invitations combined with Web-based data
collection may resemble the emergence of the combined
paper-based invitation and Web-based data-collection approach, http://www.jmir.org/2019/1/e11658/ XSL•FO
RenderX XSL•FO
RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Larsen et al data from the primary care sector on specific tasks performed
as part of a consultation, such as taking a blood sample, a
spirometry, or having a preventive consultation, may be
incomplete and more prone to human error and should as such
be interpreted with care. Another strength is the combination
of DAG and CHAID analyses to establish a both theory-driven
and data-driven analytical approach. The DAG established
adjustments to the Poisson regression models based on a
theoretical and evidence-based causal model. The CHAID used
the data from this study and identified the strongest predictors
of attendance. However, residual confounding and collider bias
cannot be eliminated in the regressions because of both a rather
complex causal model, conditional independence between
exposures,
and
the
unavailability
of
a
number
of
exposures—especially health-risk behaviors as well as cognitive
and psychological parameters [6]. Acknowledgments The project received funding from the Region of Southern Denmark and the Danish Ministry of Health. The funders had no role
in the analysis or in the interpretation of the data, writing of the manuscript, or the decision to submit it for publication. Authors' Contributions All authors participated in the design of the study. LBL analyzed and interpreted the patient data and was the main contributor
to the manuscript. All authors contributed to the interpretation of patient data and the manuscript. All authors read and approved
the final manuscript. Conclusions To our knowledge, this is the first study to report on the rates
of acceptance of digital invitations to participate in a stepwise
model for prevention of chronic diseases. We show acceptance
rates of 40% for a first digital invitation and 30% for a second
digital invitation, the rates being higher among women, elderly
patients, and patients of higher SES; patients not diagnosed
with, or in treatment for T2DM, COPD, and/or CVD; and
patients having attended the GP within a period of 2 years before
consent. A total of 75% of those who accepted the first digital
invitation also accepted the second. On one hand, the 2 digital
invitations seem to deepen the sociodemographic differences
in acceptance compared with a single digital invitation; on the
other hand, patients who had not consulted a GP during a period
of 2 years before the study, and who were not receiving medical
treatment, showed a significantly higher rate of acceptance of
the second invitation when it came to those who had accepted
the first digital invitation. This suggests that compliers are more
prone to accepting the second digital invitation than
always-users are. In future studies, multimodal recruitment
approaches, which complement digital invitations, are warranted
to increase the rates of acceptance among harder-to-reach
subpopulations. Nonetheless, more studies of digital invitations are needed to
determine if the acceptance rates seen in this study and in the
study by Ebert et al could be expected from future studies as
well. Similarly, more research is needed to determine whether
a multimodal recruitment approach, including digital invitations
to personal digital mailboxes will reach other subpopulations
more effectively than digital invitations only. Strengths and Limitations The main strengths of this study relate to the high validity of
the registries of Statistics Denmark and the random sampling
of patients from a large number of GP clinics. Especially, the
health and social registries are of high quality, with few missing
cases and up-to-date information that has been registered either
immediately before study commencement or during another
specified period before study commencement [54]. Thus, the
impact is most likely negligent because of the strong association
between acceptance of the digital invitations and SES. All direct
contacts with the GP are most likely both valid and complete
as this type of information is automatically registered onto
patients’ personal health insurance cards. Other administrative Efficacy of Digital Recruitment Finally, the invitations sent
to the digital mailbox and the digital support system were in
Danish language only, which may have had a negative impact
on the rate of acceptance among people originating from outside
of Denmark. where the advent of Web-based data collection methods
precipitated an initial drop in the rate of acceptance [34]. In addition, digital invitations sent to personal digital mailboxes
seem to be an especially suitable and low-cost recruitment
method for patients of high SES. It is well known that risk
factors of chronic lifestyle-related diseases are clustered in low
SES populations [50]. To generate population health effects,
stepwise models for the prevention of chronic diseases may
have to employ other low-tech recruitment approaches, which
complement digital invitations. Results from the health check
program of the British National Health Service indicate that
uptake may increase over time, with a clear focus on the
hardest-to-reach populations [51]. Thus, it would appear that
the lower uptake among patients of lower SES found in this as
well as in many other studies can be eliminated, or even
inverted, by a focused recruitment effort aimed at deprived
communities and outreach services [52,53]. However, at present,
digital recruitment is only applicable in a few countries, among
others the Nordic countries of Denmark, Norway, and Sweden. When digital mail gets more widespread, the results from the
described recruitment procedure and intervention may be well
applicable in other settings as well. Multimedia Appendix 2 Data on the acceptance of the second digital invitation in the entire study population. [PDF File (Adobe PDF File), 290KB-Multimedia Appendix 2] Data on the acceptance of the second digital invitation in the entire study population. [PDF File (Adobe PDF File), 290KB-Multimedia Appendix 2] References Patients' perceptions and experiences of cardiovascular disease and diabetes
prevention programmes: a systematic review and framework synthesis using the Theoretical Domains Framework. Soc Sci
Med 2016 May;156:192-203. [doi: 10.1016/j.socscimed.2016.03.015] [Medline: 27043372] 8. Shaw R, Holland C, Pattison H, Cooke R. Patients' perceptions and experiences of cardiovascular disease and diabetes
prevention programmes: a systematic review and framework synthesis using the Theoretical Domains Framework. Soc Sci
Med 2016 May;156:192-203. [doi: 10.1016/j.socscimed.2016.03.015] [Medline: 27043372] y
j
9. Piepoli M, Hoes A, Agewall S, Albus C, Brotons C, Catapano AM, ESC Scientific Document Group. 2016 European
Guidelines on cardiovascular disease prevention in clinical practice: the Sixth Joint Task Force of the European Society of
Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives
of 10 societies and by invited experts)Developed with the special contribution of the European Association for Cardiovascular
Prevention & Rehabilitation (EACPR). Eur Heart J 2016 Dec 01;37(29):2315-2381 [FREE Full text] [doi:
10.1093/eurheartj/ehw106] [Medline: 27222591] y
j
9. Piepoli M, Hoes A, Agewall S, Albus C, Brotons C, Catapano AM, ESC Scientific Document Group. 2016 European
Guidelines on cardiovascular disease prevention in clinical practice: the Sixth Joint Task Force of the European Society of
Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives
of 10 societies and by invited experts)Developed with the special contribution of the European Association for Cardiovascular
Prevention & Rehabilitation (EACPR). Eur Heart J 2016 Dec 01;37(29):2315-2381 [FREE Full text] [doi:
10.1093/eurheartj/ehw106] [Medline: 27222591] 11. van den Brekel-Dijkstra K, Rengers A, Niessen M, de Wit NJ, Kraaijenhagen R. Personalized prevention approach with
use of a web-based cardiovascular risk assessment with tailored lifestyle follow-up in primary care practice--a pilot study. Eur J Prev Cardiol 2016 Mar;23(5):544-551. [doi: 10.1177/2047487315591441] [Medline: 26080811] 11. van den Brekel-Dijkstra K, Rengers A, Niessen M, de Wit NJ, Kraaijenhagen R. Personalized prevention approach with
use of a web-based cardiovascular risk assessment with tailored lifestyle follow-up in primary care practice--a pilot study. Eur J Prev Cardiol 2016 Mar;23(5):544-551. [doi: 10.1177/2047487315591441] [Medline: 26080811] ( )
12. Marshall T, Westerby P, Chen J, Fairfield M, Harding J, Westerby R, et al. The Sandwell Project: a controlled evaluation
of a programme of targeted screening for prevention of cardiovascular disease in primary care. BMC Public Health 2008
Feb 25;8:73 [FREE Full text] [doi: 10.1186/1471-2458-8-73] [Medline: 18298863] 12. Marshall T, Westerby P, Chen J, Fairfield M, Harding J, Westerby R, et al. Conflicts of Interest None declared. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 13
(page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ XSL•FO
RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al References 1. Krogsbøll LT, Jørgensen KJ, Grønhøj Larsen C, Gøtzsche PC. General health checks in adults for reducing morbidity and
mortality from disease: Cochrane systematic review and meta-analysis. Br Med J 2012 Nov 20;345:e7191 [FREE Full text]
[doi: 10.1136/bmj.e7191] [Medline: 23169868] 2. Si S, Moss J, Sullivan T, Newton S, Stocks N. Effectiveness of general practice-based health checks: a systematic review
and meta-analysis. Br J Gen Pract 2014 Jan;64(618):e47-e53 [FREE Full text] [doi: 10.3399/bjgp14X676456] [Medline:
24567582] 3. Prochazka A, Caverly T. General health checks in adults for reducing morbidity and mortality from disease: summary
review of primary findings and conclusions. JAMA Intern Med 2013 Mar 11;173(5):371-372. [doi:
10.1001/jamainternmed.2013.3187] [Medline: 23318544] j
4. Capewell S, Graham H. Will cardiovascular disease prevention widen health inequalities? PLoS Med 2010 Aug
24;7(8):e1000320 [FREE Full text] [doi: 10.1371/journal.pmed.1000320] [Medline: 20811492] j
p
5. Gøtzsche P, Jørgensen K, Krogsbøll L. General health checks don't work. Br Med J 2014;348:g3680. [doi: 10.1136/bmj.g3680] 5. Gøtzsche P, Jørgensen K, Krogsbøll L. General health checks don't work. Br Med J 2014;348:g3680. [doi: 10.1136/bmj.g3680]
6. Dryden R, Williams B, McCowan C, Themessl-Huber M. What do we know about who does and does not attend general
health checks? Findings from a narrative scoping review. BMC Public Health 2012 Aug 31;12:723 [FREE Full text] [doi:
10 1186/1471 2458 12 723] [M dli
22938046] 6. Dryden R, Williams B, McCowan C, Themessl-Huber M. What do we know about who does and does not attend general
health checks? Findings from a narrative scoping review. BMC Public Health 2012 Aug 31;12:723 [FREE Full text] [doi:
10.1186/1471-2458-12-723] [Medline: 22938046] 7. Bender A, Jørgensen T, Pisinger C. Is self-selection the main driver of positive interpretations of general health checks? The Inter99 randomized trial. Prev Med 2015 Dec;81:42-48. [doi: 10.1016/j.ypmed.2015.07.004] [Medline: 26190370] 7. Bender A, Jørgensen T, Pisinger C. Is self-selection the main driver of positive interpretations of general health checks? The Inter99 randomized trial. Prev Med 2015 Dec;81:42-48. [doi: 10.1016/j.ypmed.2015.07.004] [Medline: 26190370] 7. Bender A, Jørgensen T, Pisinger C. Is self-selection the main driver of positive interpretations of general health checks? The Inter99 randomized trial. Prev Med 2015 Dec;81:42-48. [doi: 10.1016/j.ypmed.2015.07.004] [Medline: 26190370]
8. Shaw R, Holland C, Pattison H, Cooke R. Patients' perceptions and experiences of cardiovascular disease and diabetes
prevention programmes: a systematic review and framework synthesis using the Theoretical Domains Framework. Soc Sci 8. Shaw R, Holland C, Pattison H, Cooke R. Multimedia Appendix 3 Data on the acceptance of the second digital invitation among those who accepted the first digital invitation. [PDF File (Adobe PDF File), 187KB-Multimedia Appendix 3] [PDF File (Adobe PDF File), 187KB-Multimedia Appendix 3] J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 14
(page number not for citation purposes) Multimedia Appendix 1 Invitations (in Danish). [PDF File (Adobe PDF File), 310KB-Multimedia Appendix 1] JOURNAL OF MEDICAL INTERNET RESEARCH Scand J Public Health 2011 Jul;39(7 Suppl):91-94. [d
10 1177/1403494810394715] [Medline: 21775362] 25. Petersson F, Baadsgaard M, Thygesen L. Danish registers on personal labour market affiliation. Scand J Public Health
2011 Jul;39(7 Suppl):95-98. [doi: 10.1177/1403494811408483] [Medline: 21775363] 26. Baadsgaard M, Quitzau J. Danish registers on personal income and transfer
Suppl):103-105. [doi: 10.1177/1403494811405098] [Medline: 21775365] 26. Baadsgaard M, Quitzau J. Danish registers on personal income and transfer payments. Scand J Public Health 2011 Jul;39(7
Suppl):103-105. [doi: 10.1177/1403494811405098] [Medline: 21775365] 27. Pottegård A, Schmidt S, Wallach-Kildemoes H, Sørensen HT, Hallas J, Schmidt M. Data resource profile: the Danish
national prescription registry. Int J Epidemiol 2017 Dec 01;46(3):798-798f [FREE Full text] [doi: 10.1093/ije/dyw213]
[Medline: 27789670] 28. Schmidt M, Schmidt S, Sandegaard J, Ehrenstein V, Pedersen L, Sørensen HT. The Danish National Patient Registry: a
review of content, data quality, and research potential. Clin Epidemiol 2015;7:449-490 [FREE Full text] [doi:
10.2147/CLEP.S91125] [Medline: 26604824] 29. Andersen J, Olivarius N, Krasnik A. The Danish national health service register. Scand J Public Health 2011 Jul;39(7
Suppl):34-37. [doi: 10.1177/1403494810394718] [Medline: 21775348] OECD. What are the equivalence scales Internet URL: http://www.oecd.org/els/soc/OECD-Note-Equivale 30. OECD. What are the equivalence scales Internet URL: http://www.oecd.org/els/soc/OECD-Note-E
[accessed 2018-11-20] [WebCite Cache ID 7450EgjQd] 30. OECD. What are the equivalence scales Internet URL: http://www.oecd.org/els/soc/OECD-Note-EquivalenceScales.pdf
[accessed 2018-11-20] [WebCite Cache ID 7450EgjQd] 31. Vedsted P, Fink P, Sørensen HT, Olesen F. Physical, mental and social factors associated with frequent attendance in Danish
general practice. A population-based cross-sectional study. Soc Sci Med 2004 Aug;59(4):813-823. [doi:
10.1016/j.socscimed.2003.11.027] [Medline: 15177837] j
32. DAGitty. Direct acyclic graph (DAG) on the determinants of attendance at health checks URL: http://www.dagitty.net
[accessed 2018-09-27] [WebCite Cache ID 6zFVbmCxc] 33. Kass G. An Exploratory Technique for Investigating Large Quantities of Categorical Data. Applied Statistics
1980;29(2):119-127. [doi: 10.2307/2986296] 34. van Gelder MM, Bretveld R, Roeleveld N. Web-based questionnaires: the future in epidemiology? Am J Epidemiol 2010
Dec 01;172(11):1292-1298. [doi: 10.1093/aje/kwq291] [Medline: 20880962] 34. van Gelder MM, Bretveld R, Roeleveld N. Web-based questionnaires: the future in epidemiology? Am J Epidemiol 2010
Dec 01;172(11):1292-1298. [doi: 10.1093/aje/kwq291] [Medline: 20880962] 35. Christensen AI, Ekholm O, Davidsen M, Juel K. Viden. Copenhagen: Statens Institut for Folkesundhed; 2012. Health and
morbidity in Denmark 2010 - and developments since 1987 URL: https://viden.sl.dk/artikler/voksne/socialt-udsatte/
sundhed-og-sygelighed-2010/ 35. Christensen AI, Ekholm O, Davidsen M, Juel K. Viden. Copenhagen: Statens Institut for Folkesundhed; 2012. Health and
morbidity in Denmark 2010 - and developments since 1987 URL: https://viden.sl.dk/artikler/voksne/socialt-udsatte/
sundhed-og-sygelighed-2010/ 36. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 15
(page number not for citation purposes) References The Sandwell Project: a controlled evaluation
of a programme of targeted screening for prevention of cardiovascular disease in primary care. BMC Public Health 2008
Feb 25;8:73 [FREE Full text] [doi: 10.1186/1471-2458-8-73] [Medline: 18298863] 13. Marshall T, Caley M, Hemming K, Gill P, Gale N, Jolly K. Mixed methods evaluation of targeted case finding for
cardiovascular disease prevention using a stepped wedged cluster RCT. BMC Public Health 2012;12:908 [FREE Full text]
[doi: 10.1186/1471-2458-12-908] [Medline: 23101763] 13. Marshall T, Caley M, Hemming K, Gill P, Gale N, Jolly K. Mixed methods evaluation of targeted case finding for
cardiovascular disease prevention using a stepped wedged cluster RCT. BMC Public Health 2012;12:908 [FREE Full text]
[doi: 10.1186/1471-2458-12-908] [Medline: 23101763] 14. Badenbroek IF, Stol DM, Nielen MM, Hollander M, Kraaijenhagen RA, de Wit GA, et al. Design of the INTEGRATE
study: effectiveness and cost-effectiveness of a cardiometabolic risk assessment and treatment program integrated in primary
care. BMC Fam Pract 2014 May 09;15:90 [FREE Full text] [doi: 10.1186/1471-2296-15-90] [Medline: 24884779] 14. Badenbroek IF, Stol DM, Nielen MM, Hollander M, Kraaijenhagen RA, de Wit GA, et al. Design of the INTEGRATE
study: effectiveness and cost-effectiveness of a cardiometabolic risk assessment and treatment program integrated in primary
care. BMC Fam Pract 2014 May 09;15:90 [FREE Full text] [doi: 10.1186/1471-2296-15-90] [Medline: 24884779] 15. Maindal H, Støvring H, Sandbaek A. Effectiveness of the population-based Check your health preventive programme
conducted in primary care with 4 years follow-up [the CORE trial]: study protocol for a randomised controlled trial. Trials
2014 Aug 29;15:341 [FREE Full text] [doi: 10.1186/1745-6215-15-341] [Medline: 25169211] 15. Maindal H, Støvring H, Sandbaek A. Effectiveness of the population-based Check your health preventive programme
conducted in primary care with 4 years follow-up [the CORE trial]: study protocol for a randomised controlled trial. Trials
2014 Aug 29;15:341 [FREE Full text] [doi: 10.1186/1745-6215-15-341] [Medline: 25169211] J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 14
(page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ XSL•FO
RenderX XSL•FO
RenderX JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al 16. Bruun Larsen L, Soendergaard J, Halling A, Thilsing T, Thomsen J. A novel approach to population-based risk stratification,
comprising individualized lifestyle intervention in Danish general practice to prevent chronic diseases: Results from a
feasibility study. Health Informatics J 2017 Dec;23(4):249-259. [doi: 10.1177/1460458216645149] [Medline: 27245672] y
y
( )
17. Larsen LB, Sonderlund AL, Sondergaard J, Thomsen JL, Halling A, Hvidt NC, et al. Targeted prevention in primary care
aimed at lifestyle-related diseases: a study protocol for a non-randomised pilot study. BMC Fam Pract 2018;19:124. [doi:
10.1186/s12875-018-0820-8] ]
18. Craig P, Dieppe P, Macintyre S, Michie S, Nazareth I, Petticrew M, Medical Research Council Guidance. Developing and
evaluating complex interventions: the new Medical Research Council guidance. Br Med J 2008 Sep 29;337:a1655 [FREE
Full text] [doi: 10.1136/bmj.a1655] [Medline: 18824488] 19. Ebert JF, Huibers L, Christensen B, Christensen MB. Paper- or web-based questionnaire invitations as a method for data
collection: cross-sectional comparative study of differences in response rate, completeness of data, and financial cost. J 19. Ebert JF, Huibers L, Christensen B, Christensen MB. Paper- or web-based questionnaire invitations as a method for data
collection: cross-sectional comparative study of differences in response rate, completeness of data, and financial cost. J
Med Internet Res 2018 Jan 23;20(1):e24 [FREE Full text] [doi: 10.2196/jmir.8353] [Medline: 29362206] 20. Digitization Agency. 2018. Monthly statistics on access to digital mail in Denmark Internet URL: https
digital-post/om-loesningen/tal-og-statistik/ [accessed 2018-09-27] [WebCite Cache ID 72jZRhJGB g
p
g
g
j
21. Pedersen K, Andersen J, Søndergaard J. General practice and primary health care in Denmark. J Am Board Fam Med 2012
Mar;25(Suppl 1):S34-S38 [FREE Full text] [doi: 10.3122/jabfm.2012.02.110216] [Medline: 22403249] 21. Pedersen K, Andersen J, Søndergaard J. General practice and primary health care in Denmark. J Am Board Fam Med 2012
Mar;25(Suppl 1):S34-S38 [FREE Full text] [doi: 10.3122/jabfm.2012.02.110216] [Medline: 22403249]
22. tof.sundhedsmappe.dk. Odense: Dansk Almen Medicinsk KvalitetsEnhed (DAK-E) URL: https://tof.sundhedsmappe.dk/
[accessed 2018-09-27] Mar;25(Suppl 1):S34-S38 [FREE Full text] [doi: 10.3122/jabfm.2012.02.110216] [Medline: 22403249]
22. tof.sundhedsmappe.dk. Odense: Dansk Almen Medicinsk KvalitetsEnhed (DAK-E) URL: https://tof.sundhedsmappe.dk/
[accessed 2018-09-27] [
]
23. Pedersen C. The Danish civil registration system. Scand J Public Health 2011 Jul;39(7 Suppl):22-25. [doi:
10.1177/1403494810387965] [Medline: 21775345] sen V, Rasmussen A. Danish education registers. Scand J Public Health 2011 Jul;39(7 Suppl):91-94. [doi: 24. Jensen V, Rasmussen A. Danish education registers. Scand J Public Health 2011 Jul;39(7 Suppl):91-94. [doi:
10.1177/1403494810394715] [Medline: 21775362] Jensen V, Rasmussen A. Danish education registers. JOURNAL OF MEDICAL INTERNET RESEARCH Introduction to Danish (nationwide) registers on health and social
issues: structure, access, legislation, and archiving. Scand J Public Health 2011 Jul;39(7 Suppl):12-16. [doi:
10.1177/1403494811399956] [Medline: 21898916] JOURNAL OF MEDICAL INTERNET RESEARCH Utilisation of preventative health check-ups in the UK: findings from individual-level
repeated cross-sectional data from 1992 to 2008. BMJ Open 2013 Dec 23;3(12):e003387 [FREE Full text] [doi:
10.1136/bmjopen-2013-003387] [Medline: 24366576] 47. Labeit A, Peinemann F, Baker R. Utilisation of preventative health check-ups in the UK: findings from individual-level
repeated cross-sectional data from 1992 to 2008. BMJ Open 2013 Dec 23;3(12):e003387 [FREE Full text] [doi:
10.1136/bmjopen-2013-003387] [Medline: 24366576] 48. Koopmans B, Nielen MM, Schellevis FG, Korevaar JC. Non-participation in population-based disease prevention programs
in general practice. BMC Public Health 2012 Oct 09;12:856 [FREE Full text] [doi: 10.1186/1471-2458-12-856] [Medline:
23046688] 49. Gruber J, Arnold B, Reygadas F, Hubbard A, Colford J. Estimation of treatment efficacy with com
effects (CACE) in a randomized stepped wedge trial. Am J Epidemiol 2014;179(9):1134-1142. 50. Meader N, King K, Moe-Byrne T, Wright K, Graham H, Petticrew M, et al. A systematic review on the clustering and
co-occurrence of multiple risk behaviours. BMC Public Health 2016 Dec 29;16:657 [FREE Full text] [doi:
10.1186/s12889-016-3373-6] [Medline: 27473458] 51. Robson J, Dostal I, Madurasinghe V, Sheikh A, Hull S, Boomla K, et al. NHS Health Check comorbidity and management:
an observational matched study in primary care. Br J Gen Pract 2017 Feb;67(655):e86-e93 [FREE Full text] [doi:
10.3399/bjgp16X688837] [Medline: 27993901] jgp
52. Woringer M, Cecil E, Watt H, Chang K, Hamid F, Khunti K, et al. Evaluation of community provision of a preventive
cardiovascular programme - the National Health Service Health Check in reaching the under-served groups by primary
care in England: cross sectional observational study. BMC Health Serv Res 2017 Dec 14;17(1):405 [FREE Full text] [doi:
10.1186/s12913-017-2346-5] [Medline: 28615019] 53. Roberts DJ, de Souza VC. A venue-based analysis of the reach of a targeted outreach service to deliver opportunistic
community NHS Health Checks to 'hard-to-reach' groups. Public Health 2016 Aug;137:176-181. [doi:
10.1016/j.puhe.2016.03.004] [Medline: 27062066] 53. Roberts DJ, de Souza VC. A venue-based analysis of the reach of a targeted outreach service to deliver opportunistic
community NHS Health Checks to 'hard-to-reach' groups. Public Health 2016 Aug;137:176-181. [doi:
10.1016/j.puhe.2016.03.004] [Medline: 27062066] j p
54. Thygesen L, Daasnes C, Thaulow I, Brønnum-Hansen H. Introduction to Danish (nationwide) registers on health and social
issues: structure, access, legislation, and archiving. Scand J Public Health 2011 Jul;39(7 Suppl):12-16. [doi:
10.1177/1403494811399956] [Medline: 21898916] j p
54. Thygesen L, Daasnes C, Thaulow I, Brønnum-Hansen H. JOURNAL OF MEDICAL INTERNET RESEARCH Laws RA, Fanaian M, Jayasinghe UW, McKenzie S, Passey M, Davies GP, et al. Factors influencing participation in a
vascular disease prevention lifestyle program among participants in a cluster randomized trial. BMC Health Serv Res 2013
May 31;13:201 [FREE Full text] [doi: 10.1186/1472-6963-13-201] [Medline: 23725521] 37. Bruun Larsen L, Soendergaard J, Halling A, Thilsing T, Thomsen J. A novel approach to population-based risk stratification,
comprising individualized lifestyle intervention in Danish general practice to prevent chronic diseases: Results from a
feasibility study. Health Informatics J 2017 Dec;23(4):249-259. [doi: 10.1177/1460458216645149] [Medline: 27245672] 38. Bjerregaard A, Maindal H, Bruun N, Sandbæk A. Patterns of attendance to health checks in a municipality setting: the
Danish 'Check Your Health Preventive Program'. Prev Med Rep 2017 Mar;5:175-182 [FREE Full text] [doi:
10.1016/j.pmedr.2016.12.011] [Medline: 28050340] J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 15
(page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al 39. Larsen LB, Sandbaek A, Thomsen JL, Bjerregaard A. Uptake of health checks by residents from the Danish social housing
sector - a register-based cross-sectional study of patient characteristics in the 'Your Life - Your Health' program. BMC
Public Health 2018 May 02;18(1):585 [FREE Full text] [doi: 10.1186/s12889-018-5506-6] [Medline: 29720162] y
40. Norman P. Applying the health belief model to the prediction of attendance at health checks in general practice. Br J Clin
Psychol 1995 Sep;34 ( Pt 3):461-470. [Medline: 8845784] 41. Bender A, Jørgensen T, Helbech B, Linneberg A, Pisinger C. Socioeconomic position and participation in baseline and
follow-up visits: the Inter99 study. Eur J Prev Cardiol 2014 Jul;21(7):899-905. [doi: 10.1177/2047487312472076] [Medline:
23233551] 42. Brunner-Ziegler S, Rieder A, Stein KV, Koppensteiner R, Hoffmann K, Dorner TE. Predictors of participation in preventive
health examinations in Austria. BMC Public Health 2013 Dec 05;13:1138 [FREE Full text] [doi: 10.1186/1471-2458-13-1138]
[Medline: 24308610] 43. Wilhelmsen L, Ljungberg S, Wedel H, Werkö L. A comparison between participants and non-participants in a primary
preventive trial. J Chronic Dis 1976 May;29(5):331-339. [Medline: 939796] 44. Lakerveld J, Ijzelenberg W, van Tulder MW, Hellemans IM, Rauwerda JA, van Rossum AC, et al. Motives for (not)
participating in a lifestyle intervention trial. BMC Med Res Methodol 2008 Apr 10;8:17 [FREE Full text] [doi:
10.1186/1471-2288-8-17] [Medline: 18402683] 45. Jacobsen B, Thelle D. The Tromsø Heart Study: responders and non-responders to a health questionnaire, do they differ? Scand J Soc Med 1988;16(2):101-104. [Medline: 3387934] 46. Thorogood M, Coulter A, Jones L, Yudkin P, Muir J, Mant D. Factors affecting response to an invitation to attend for a
health check. J Epidemiol Community Health 1993 Jun;47(3):224-228 [FREE Full text] [Medline: 8350036]
47. Labeit A, Peinemann F, Baker R. Utilisation of preventative health check-ups in the UK: findings from individual-level
d
i
l d
f
1992
2008 BMJ O
2013 D
23 3(12) 003387 [FREE F ll
] [d i 46. Thorogood M, Coulter A, Jones L, Yudkin P, Muir J, Mant D. Factors affecting response to an invitation to attend for a
health check. J Epidemiol Community Health 1993 Jun;47(3):224-228 [FREE Full text] [Medline: 8350036] g
,
,
,
,
,
g
p
health check. J Epidemiol Community Health 1993 Jun;47(3):224-228 [FREE Full text] [Medline: 8350036]
47. Labeit A, Peinemann F, Baker R. http://www.jmir.org/2019/1/e11658/ J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 16
(page number not for citation purposes) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 17
(page number not for citation purposes) Abbreviations ATC: anatomical therapeutic chemical
CHAID: chi-square automatic interaction detection
COPD: chronic obstructive pulmonary disease
CVD: cardiovascular disease
DAG: direct acyclic graph
EPR: electronic patient record
FEV1: forced expiratory volume in one second
FVC: forced vital capacity
GP: general practitioner
ICD-10: International Classification of Diseases 10th Edition
IRR: incidence rate ratio
SES: socioeconomic status J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 16
(page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al T2DM: type 2 diabetes mellitus T2DM: type 2 diabetes mellitus Edited by G Eysenbach; submitted 22.07.18; peer-reviewed by J Ebert, J Apolinário-Hagen; comments to author 19.09.18; revised
version received 27.09.18; accepted 29.09.18; published 21.01.19 Please cite as: Please cite as:
Larsen LB, Sondergaard J, Thomsen JL, Halling A, Sønderlund AL, Christensen JR, Thilsing T
Digital Recruitment and Acceptance of a Stepwise Model to Prevent Chronic Disease in the Danish Primary Care Sector: Cross-Sectional
Study
J Med Internet Res 2019;21(1):e11658
URL: http://www.jmir.org/2019/1/e11658/
doi: 10.2196/11658
PMID: 30664466 ©Lars Bruun Larsen, Jens Sondergaard, Janus Laust Thomsen, Anders Halling, Anders Larrabee Sønderlund, Jeanette Reffstrup
Christensen, Trine Thilsing. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 21.01.2019. This is an open-access article distributed under the terms of the Creative Commons Attribution License
(https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic
information, a link to the original publication on http://www.jmir.org/, as well as this copyright and license information must be
included. http://www.jmir.org/2019/1/e11658/ http://www.jmir.org/2019/1/e11658/ XSL•FO
RenderX
|
https://openalex.org/W4220863350
|
https://pureadmin.qub.ac.uk/ws/files/312092631/FPsychiatry1.pdf
|
English
| null |
Unraveling the Complexity of Cardiac Distress: A Study of Prevalence and Severity
|
Frontiers in psychiatry
| 2,022
|
cc-by
| 9,642
|
Queen's University Belfast - Research Portal:
Link to publication record in Queen's University Belfast Research Portal Queen s University Belfast - Research Portal:
Link to publication record in Queen's University Belfast Research Portal y
Link to publication record in Queen's University Publisher rights
Copyright 2022 the authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/),
which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. g
Copyright 2022 the authors. py g
This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/
which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited Take down policy
Th
R
h P Take down policy
The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to
ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the
Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Open Access
This research has been made openly available by Queen's academics and its Open Research team. We would love to hear how access to
this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Published in:
Frontiers in psychiatry Published in:
Frontiers in psychiatry Published in:
Frontiers in psychiatry Document Version:
Publisher's PDF, also known as Version of record General rights g
Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other
copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated
with these rights. Take down policy
The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to
ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the
Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Unraveling the Complexity of Cardiac Distress: A Study of Prevalence
and Severity
Jackson, A. C., Rogerson, M. C., Amerena, J., Smith, J., Hoover, V., Alvarenga, M. E., Higgins, R. O., Grande,
M. R. L., Ski, C. F., Thompson, D. R., & Murphy, B. M. (2022). Unraveling the Complexity of Cardiac Distress: A
Study of Prevalence and Severity. Frontiers in psychiatry, 13, Article 808904.
https://doi.org/10.3389/fpsyt.2022.808904 Unraveling the Complexity of Cardiac Distress: A Study of Prevalence
and Severity
Jackson, A. C., Rogerson, M. C., Amerena, J., Smith, J., Hoover, V., Alvarenga, M. E., Higgins, R. O., Grande,
M. R. L., Ski, C. F., Thompson, D. R., & Murphy, B. M. (2022). Unraveling the Complexity of Cardiac Distress: A
Study of Prevalence and Severity. Frontiers in psychiatry, 13, Article 808904. https://doi.org/10.3389/fpsyt.2022.808904 Unraveling the Complexity of Cardiac Distress: A Study of Prevalence
and Severity
Jackson, A. C., Rogerson, M. C., Amerena, J., Smith, J., Hoover, V., Alvarenga, M. E., Higgins, R. O., Grande,
M. R. L., Ski, C. F., Thompson, D. R., & Murphy, B. M. (2022). Unraveling the Complexity of Cardiac Distress: A
Study of Prevalence and Severity. Frontiers in psychiatry, 13, Article 808904.
https://doi.org/10.3389/fpsyt.2022.808904 Jackson, A. C., Rogerson, M. C., Amerena, J., Smith, J., Hoover, V., Alvarenga, M. E., Higgins, R. O., Grande,
M. R. L., Ski, C. F., Thompson, D. R., & Murphy, B. M. (2022). Unraveling the Complexity of Cardiac Distress: A
Study of Prevalence and Severity. Frontiers in psychiatry, 13, Article 808904. https://doi.org/10.3389/fpsyt.2022.808904 Unraveling the Complexity of Cardiac
Distress: A Study of Prevalence and
Severity
Alun C. Jackson 1,2,3*, Michelle C. Rogerson 1, John Amerena 4,5, Julian Smith 6,7,
Valerie Hoover 8, Marlies E. Alvarenga 1,9,10,11, Rosemary O. Higgins 1,12,13,
Michael R. Le Grande 1,14, Chantal F. Ski 1,15,16, David R. Thompson 1,16,17 and
Barbara M. Murphy 1,2,18 Alun C. Jackson 1,2,3*, Michelle C. Rogerson 1, John Amerena 4,5, Julian Smith 6,7,
Valerie Hoover 8, Marlies E. Alvarenga 1,9,10,11, Rosemary O. Higgins 1,12,13,
Michael R. Le Grande 1,14, Chantal F. Ski 1,15,16, David R. Thompson 1,16,17 and
Barbara M. Murphy 1,2,18 1 Australian Centre for Heart Health, Melbourne, VIC, Australia, 2 Center on Behavioral Health, Faculty of Health, Deakin
University, Geelong, VIC, Australia, 3 Behavioral Health, University of Hong Kong, Pokfulam, Hong Kong SAR, China,
4 Barwon Health, Geelong, VIC, Australia, 5 Deakin School of Medicine, University Hospital Geelong, Geelong, VIC, Australia,
6 Department of Cardiothoracic Surgery, Monash Health, Melbourne, VIC, Australia, 7 Department of Surgery, School of
Clinical Sciences, Monash Health, Melbourne, VIC, Australia, 8 Psychiatry and Behavioral Sciences, Stanford Women’s Heart
Health Clinic, Stanford, CA, United States, 9 School of Health and Life Sciences, Federation University Australia, Berwick,
VIC, Australia, 10 Victorian Heart Institute, Melbourne, VIC, Australia, 11 Monash Health & Department of Medicine, Monash
University, Melbourne, VIC, Australia, 12 Department of Psychology, Deakin University, Geelong, VIC, Australia, 13 Department
of Physiotherapy, University of Melbourne, Melbourne, VIC, Australia, 14 Centre for Behaviour Change, Melbourne School of
Psychological Sciences, University of Melbourne, Melbourne, VIC, Australia, 15 Integrated Care Academy, University of
Suffolk, Ipswich, United Kingdom, 16 Department of Psychiatry, University of Melbourne, Melbourne, VIC, Australia, 17 School
of Nursing and Midwifery, Queen’s University Belfast, Belfast, United Kingdom, 18 Department of Psychology, University of
Melbourne, Melbourne, VIC, Australia Edited by:
Melissa Thong,
German Cancer Research Center
(DKFZ), Germany Reviewed by:
Gianluca Serafini,
San Martino Hospital (IRCCS), Italy
Daniela Doege,
German Cancer Research Center
(DKFZ), Germany Introduction: While much research attention has been paid to anxiety and depression
in people who have had a recent cardiac event, relatively little has focused on the
broader concept of cardiac distress. Cardiac distress is a multidimensional construct
that incorporates but extends beyond common mood disorders such as anxiety and
depression. In the present study we assessed the prevalence, severity and predictors of
a broad range of physical, affective, cognitive, behavioral and social symptoms of cardiac
distress. This is the first study to investigate cardiac distress in this comprehensive way. *Correspondence:
Alun C. Jackson
alun.jackson@
australianhearthealth.org.au Specialty section:
This article was submitted to
Psychological Therapy and
Psychosomatics,
a section of the journal
Frontiers in Psychiatry Method: A sample of 194 patients was recruited from two hospitals in Australia. Eligible
participants were those who had recently been hospitalized for an acute cardiac event. Data were collected at patients’ outpatient clinic appointment ∼8 weeks after their
hospital discharge. Using a questionnaire developed through a protocol-driven 3-step
process, participants reported on whether they had experienced each of 74 issues and
concerns in the past 4 weeks, and the associated level of distress. They also provided
sociodemographic and medical information. Regression analyses were used to identify
risk factors for elevated distress. Received: 04 November 2021
Accepted: 23 February 2022
Published: 31 March 2022 Accepted: 23 February 2022
Published: 31 March 2022 Open Access
Thi
h Open Access
This research has been made openly available by Queen's academics and its Open Research team. We would love t
this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Download date:24. Oct. 2024 ORIGINAL RESEARCH
published: 31 March 2022
doi: 10.3389/fpsyt.2022.808904 Unraveling the Complexity of Cardiac
Distress: A Study of Prevalence and
Severity
Alun C. Jackson 1,2,3*, Michelle C. Rogerson 1, John Amerena 4,5, Julian Smith 6,7,
Valerie Hoover 8, Marlies E. Alvarenga 1,9,10,11, Rosemary O. Higgins 1,12,13,
Michael R. Le Grande 1,14, Chantal F. Ski 1,15,16, David R. Thompson 1,16,17 and
Barbara M. Murphy 1,2,18 INTRODUCTION and self-concept have been reported (12), as have worries about
getting back to one’s previous sense of self (13). These are often
tied to changes in roles and role function (8, 12), including
loss of the ability to provide (11) or be the “breadwinner” or
“homemaker” (7). Impacts on intimate relationships are also
a concern for some survivors (13), including concerns about
resuming sexual activity due to fear of causing another heart
attack (7, 14). Challenges navigating the health system are
also evident, including difficulties in obtaining information and
advice (13), and concerns about having to rely on help from
health professionals (9). The psychosocial impacts of an acute cardiac event have gained
increasing recognition in the past two decades. It is now well-
accepted that both anxiety and depression are common after
heart attack and heart surgery (1–3). In a recent Australian
study involving over 900 patients admitted to hospital after
acute myocardial infarction (AMI) or to undergo coronary artery
bypass graft surgery (CABGS), over 40% had elevated anxiety and
over 20% had symptoms of depression in the period shortly after
hospital discharge, while 31% had anxiety or depression at 6–12
months post-event (3). These rates are up to four times higher
than in the general population. The cardiac event also triggers difficulties in coping with
change (9), including difficulties adjusting to limitations in
everyday life (6) and living with pain (7), resistance to being on
medications (9), hypervigilance regarding bodily sensations (7),
concerns about making lifestyle changes (7, 15), and difficulties
associated with resuming work or being unable to work (7). Some
survivors express difficulty in accepting the diagnosis and the
disease itself (6). For some, the trauma associated with the cardiac
event can trigger the resurfacing of past traumas or unresolved
grief, with concomitant intrusive thoughts and nightmares (7). Fear about the future (8, 11, 13) and concerns about having to
reprioritise goals and life plans in a foreshortened future (8, 11)
have also been reported. Concerns about having another heart
attack and fear of dying can also emerge (7, 10), as survivors are
forced to confront their mortality, possibly for the first time in
their life (7, 8). There is also growing evidence that acute cardiac events are
often experienced as traumatic, thereby predisposing survivors to
experiencing post-traumatic stress symptoms (PTSS) or disorder
(PTSD). Citation: Jackson AC, Rogerson MC,
Amerena J, Smith J, Hoover V,
Alvarenga ME, Higgins RO,
Grande MRL, Ski CF, Thompson DR
and Murphy BM (2022) Unraveling the
Complexity of Cardiac Distress: A
Study of Prevalence and Severity. Front. Psychiatry 13:808904. doi: 10.3389/fpsyt.2022.808904 Results: Across the 74 issues and concerns, prevalence ratings ranged from a high of
66% to a low of 6%. The most commonly endorsed items were within the domains of
dealing with symptoms, fear of the future, negative affect, and social isolation. Common
experiences were “being physically restricted” (66%), “lacking energy” (60%), “being
short of breath” (60%), “thinking I will never be the same again” (57%), and “not sleeping
well” (51%). While less prevalent, “not having access to the health care I need,” “being March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org Jackson et al. Unraveling the Complexity of Cardiac Distress concerned about my capacity for sexual activity,” and “being unsupported by family and
friends” were reported as highly distressing (74, 64, and 62%) for those experiencing
these issues. Having a mental health history and current financial strain were key risk
factors for elevated distress. Conclusion and Implications: Specific experiences of distress appear to be highly
prevalent in people who have had a recent cardiac event. Understanding these
specific fears, worries and stressors has important implications for the identification and
management of post-event mental health and, in turn, for supporting patients in their
post-event cardiac recovery. Keywords: psychocardiology, cardiac distress, anxiety, stress, depression, fear of progression, secondary
prevention Frontiers in Psychiatry | www.frontiersin.org INTRODUCTION The traumatic components of a cardiac event include its
abruptness, the risk of death, and a sense of helplessness and loss
of control during and after the event (4). It has been suggested
that around 12% of patients experience acute coronary syndrome
(ACS)-induced PTSD (5). But is there more to a cardiac event than just anxiety,
depression and PTSD? Qualitative studies involving cardiac
event survivors and clinicians who support them provide greater
insight into the breadth of experiences, concerns and worries
expressed during convalescence after an acute cardiac event and
therefore provide a nuanced understanding of cardiac distress. One qualitative study of survivors concluded that they experience
“being forced into a demanding life-shaking journey” (6), while
another study of clinicians described it as “a lonely journey, an
existential crisis” (7). It appears that the acute cardiac event can
trigger the beginning of a completely new life chapter, involving
new and difficult emotions, changes in self-concept and identity,
and other unexpected challenges, fears and concerns. These challenging emotions, changes and experiences that
follow an acute cardiac event can all be conceptualized as “cardiac
distress.” We have defined this multi-dimensional construct in
previous publications: Cardiac distress is a persistent negative emotional state rather
than a transient state; involving multiple psychosocial domains;
that challenges a patient’s capacity to cope with living with their
heart condition, the treatment of the condition, and the resultant
changes to daily living; and challenges the person’s sense of self and
future orientation (16, 17). Reported experiences gleaned from qualitative studies include
a range of emotions, such as feelings of uncertainty (8–
10), vulnerability (6), loneliness and fear of being alone (7),
hopelessness and helplessness (6, 7), anger and resentment (7),
sadness, grief and loss (7, 8). Specific losses include loss of
independence (7, 11), loss of health and physical strength (8, 11),
and loss of control (7). Similarly, various changes in self-identify Importantly cardiac distress spans multiple psychosocial
domains, thereby incorporating patients’ responses to physical,
affective,
cognitive,
behavioral
and
social
symptoms
and
experiences related to their cardiac event and their recovery (16). March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 2 Unraveling the Complexity of Cardiac Distress Jackson et al. factors
(high
blood
pressure,
high
cholesterol,
obesity,
and positive family history), and significant comorbidity
(diabetes,
musculoskeletal
conditions,
chronic
obstructive
pulmonary
disorder,
cancer,
stroke,
and
dementia). Participant Recruitment A sample of 194 patients was recruited from two hospitals
in Australia, one in metropolitan Melbourne (Monash Health)
and one in regional Victoria (Barwon Health, Geelong). The
procedure at each of the two hospitals differed slightly. At Barwon Health, the majority of participants were recruited
while they were inpatients at the University Hospital Geelong
following their hospital admission for either AMI, CABGS or
PCI. At this contact, the Research Nurse provided eligible
patients with a brief explanation of the study and asked if they
would be willing to participate. Interested patients were then
provided with the consent form. Consent was obtained to re-
contact participants via telephone ∼6–8 weeks later to complete
the questionnaire. A smaller number of participants were
recruited during their attendance at cardiac rehabilitation (CR). At Monash Health, participants were recruited at the time
of their appointment at the Cardiothoracic Preadmission Clinic,
prior to their hospital admission for CABGS, or in the Cardiac
Care Unit (CCU) for those with AMI, PCI, and other cardiac
conditions. At this contact, the Research Nurse provided eligible
patients with a brief explanation of the study and asked if
they would be willing to participate. Interested patients were
then provided with the consent form. Consent was obtained
to re-contact participants at their routine 6–8-week follow-
up appointment. INTRODUCTION Psychosocial data included living alone (Y/N), partner status
(partnered/unpartnered), presence of a close confidante (Y/N),
loss of a close relative or friend in the past 12 months (Y/N;
defined as “recent bereavement”), having been diagnosed with
a mental health disorder prior to the cardiac event (Y/N,
defined as “mental health history”), and financial strain reported
on a 5-point Likert scale from 0 “no financial strain” to 4
“extreme financial strain.” The questionnaire was prepared
in both hardcopy form for mailout and return, and via the
REDCap
(Research
Electronic
Data
Capture)
system
for
online completion. The definition also highlights both present and future concerns,
thereby taking into account impacts on current self-perception as
well as fears about the future (16). The definition also highlights both present and future concerns,
thereby taking into account impacts on current self-perception as
well as fears about the future (16). Current understanding of the prevalence and correlates
of cardiac distress has been limited by a lack of quantitative
studies on this topic. While several qualitative studies have
been undertaken to explore survivors’ post-event experiences,
as outlined earlier, no quantitative studies have investigated the
broad range of cardiac distress experiences across multiple
psychosocial
domains,
and
incorporating
both
current
and future-orientations. Demographic, Medical, and Psychosocial
Questions Questions regarding demographic, medical and psychosocial
characteristics were also included. Demographic information
included age, sex, country of birth, education, and employment
status. Medical information included event type, cardiac
rehabilitation
(CR)
attendance
(Y/N),
cardiovascular
risk Inclusion and Exclusion Criteria Eligible participants were those who have had an acute coronary
event, namely acute myocardial infarction (AMI), percutaneous
coronary intervention (PCI), coronary artery bypass graft surgery
(CABGS), valve issues, heart rhythm disturbance, spontaneous
coronary artery dissection (SCAD), or cardiac arrest in the
previous 12 months and who attended an outpatient clinic
at participating hospitals. Patients who did not have adequate
English language proficiency to read and understand the PICF
and questionnaire were excluded. Item Generation and Distress
Questionnaire Preparation Q
p
A broad pool of items was generated following a strict protocol
which has been described in full elsewhere (17). In brief,
the process involved three key steps. First, items included in
instruments to measure cardiac anxiety, depression, quality
of life and other cardiac-related constructs were reviewed, as
were measures of distress used in the oncology and diabetes
settings, with a view to creating a pool of cardiac distress-related
items. Second, qualitative studies from the cardiac literature
were reviewed to identify relevant constructs and generate
further items. Third, the item pool was reviewed by an expert
multidisciplinary committee of cardiac researchers and clinicians
to identify missing constructs and fine-tune item wording. Following these steps, a set of 74 items addressing various issues
and concerns was generated. These assessed issues and concerns
across seven key conceptual domains, determined a priori by the
project team, namely symptoms, self-perception, concerns about
the future, negative affect, self-management, social functioning
and role functioning. Participants reported on whether or not
they had experienced each of 74 issues or concerns in the
past 4 weeks by responding Yes or No for each item. For
endorsed items, participants then reported on the level of distress
associated with the issue, using a response scale where 0 = “no
distress at all,” 1 = “slight distress,” 2 = “moderate distress,” and
3 = “severe distress.” Aims of the Study In the present study we assessed the prevalence and severity of a
broad range of physical, affective, cognitive, behavioral and social
symptoms of cardiac distress. We also investigated the correlates
of distress by identifying the patient characteristics that predict
these distressing experiences. This is the first study to assess
the prevalence, severity and predictors of cardiac distress in this
comprehensive way. Questionnaire Completion Due to the COVID-19 pandemic and imposed lockdowns,
participant recruitment varied between face-to-face and via
telehealth. Questionnaires were either completed in hard copy March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 3 Unraveling the Complexity of Cardiac Distress Jackson et al. TABLE 1 | Characteristics of participants. TABLE 1 | Characteristics of participants. N
n
%
Sociodemographic characteristics
Sex
Male
194
140
72.2
Female
53
27.3
Prefer not to say
1
0.5
Country of birth
Australia
194
138
71.1
Outside Australia
56
28.9
Marital status
Married/living with partner
194
137
70.6
Divorced/separated
26
13.4
Widowed
15
7.7
Never married
16
8.2
Education
Primary
193
3
1.6
Secondary
83
43.0
Trade or certificate
55
28.5
University degree/post-graduate
52
26.9
Employment status
In workforce
194
98
50.5
Not in workforce
96
49.5
Psychosocial characteristics
Lives alone
189
43
22.8
Recent bereavement
179
43
24.0
Financial strain
179
73
40.8
Mental health history
194
64
33.0
Medical characteristics
Event type
Acute myocardial infarction
194
102
52.6
Coronary artery bypass graft surgery
74
38.1
Percutaneous coronary intervention
57
29.4
Other
41
21.1
Attended cardiac rehabilitation
180
80
44.4
Significant co-morbidity
194
113
58.2
N = 179–194 with variations due to incompletion of some questionnaires. Recent bereavement = having lost of relative or friend in the past 12 months. Financial strain = reports of
moderate, considerable, or extreme financial strain. “Other” event type includes valve issues (n = 21), heart rhythm disturbance (n = 15), SCAD (n = 6), and cardiac arrest (n = 6). TABLE 1 | Characteristics of participants. ion of some questionnaires. Recent bereavement = having lost of relative or friend in the past 12 months. Financial strain = reports of
train. “Other” event type includes valve issues (n = 21), heart rhythm disturbance (n = 15), SCAD (n = 6), and cardiac arrest (n = 6). Social functioning (16 items), Role functioning (five items),
and Self-management (13 items). For prevalence, positively
endorsed items within each domain were added together to
provide domain prevalence scores. Within the “Symptoms”
domain, two sub-domains of “physical symptoms” (seven items)
and “cognitive symptoms” (five items) were also created. Higher domain and sub-domain prevalence scores indicated
endorsement of more items (issues and concerns) within that
domain. For distress severity ratings, a distress severity score
for each domain was calculated by taking the mean severity
ratings of endorsed items within that domain. Ethics Approval This study had ethics approval from the Monash Health
Human Research Ethics Committee (approval number: RES-19-
0000631A – 55979, which covered data collection at both the
Monash Health and the Barwon University Hospital sites. Questionnaire Completion For each domain
and sub-domain, for both prevalence scores and distress severity
scores, bivariate analyses (t-tests) were undertaken to identify
variations in terms of demographic (age, sex, and employment
status), medical (event type, CVD risk factors, comorbidities, and
CR attendance), and psychosocial (mental health history, social
isolation, and financial strain) characteristics. These particular
psychosocial characteristics were selected for examination as they
have been previously demonstrated to be strongly predictive
of persistent or worsening post-event anxiety and depression
(18). Using significant variables from the bivariate analyses,
a series of multivariable linear regression analyses were then or online. Thus, instead of completing the questionnaire while
waiting for their clinic appointments as originally intended,
participants were either directed to the website of the Australian
Center for Heart Health (ACHH) to use an online link to
the REDCap questionnaire or were mailed a hard copy of the
questionnaire for completion at home and return in a reply-
paid envelope to the ACHH. The questionnaire took ∼25 min
to complete. No identifying information was collected as no
participant follow-up was involved. Data Analysis Frequencies were calculated for the prevalence and the distress
severity ratings of each of the 74 issues and concerns. Items
were clustered into the seven pre-determined domains of
Symptoms (15 items), Self-perception (nine items), Concerns
about the future (eight items), Negative Affect (eight items), March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 4 Unraveling the Complexity of Cardiac Distress Jackson et al. TABLE 2 | Prevalence of issues and concerns in rank order. Issue/item
n
%
Being physically restricted
126
65.6
Lacking energy
115
60.3
Being short of breath
115
59.6
Thinking I will never be the same again
110
57.0
Not sleeping well
98
50.5
Thinking about having another heart event
93
48.4
Being irritated by little things
93
48.4
Thinking my condition might get worse
91
46.9
Having chest discomfort
87
45.1
Avoiding situations and activities
86
45.0
Being unable to do things that I know will improve my
health
87
44.8
Having difficulty concentrating
86
44.6
Being overly aware of my heart in my chest
85
44.0
Thinking that I am not the person that I used to be
84
43.8
Not knowing how my family will cope if something
should happen to me
82
42.9
Having difficulty remembering things
78
40.4
Not knowing what the future holds for me
77
40.3
Avoiding activities that make my heart beat faster
76
39.4
Being unsure about how much exercise or physical
activity I should be doing
75
39.3
Having changes in my usual roles
72
37.9
Being tearful more easily than before
72
37.1
Forgetting things more than before
70
36.6
Not knowing what will happen to other people if I die
68
35.4
Having to make difficult lifestyle changes because of
my heart condition
65
33.9
Being emotionally exhausted
63
33.3
Being afraid of dying
64
33.0
N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by >33% of participants shown. As shown in Table 1, participants were mostly male (72%) and
married or partnered (71%). Most (71%) were born in Australia
and for those who were not (n = 56), the majority were born in
the United Kingdom (n = 18), and New Zealand (n = 7). Nearly
half of the participants were no longer in the paid workforce,
and 40% were experiencing financial strain. Most Common Issues and Concerns The most commonly endorsed issues and concerns are shown
in Table 2 and Figure 1, with those items endorsed by over a
third of participants depicted. The data indicate the proportion
of participants who had experienced each of these in the previous
4 weeks. Table 2 shows the items in rank order from highest to
lowest, while Figure 1 shows the same items grouped according
to the seven pre-determined domains. N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by >33% of participants shown. N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by >33% of participants shown. undertaken to identify the key predictors of prevalence and
severity for each domain and sub-domain. For each model
the variables entered included: age (years), sex, event type
(CABGS vs. non-CABGS), living alone status, financial stress,
history of mental illness, bereavement, and employment status. For the financial strain variable, responses of 3 “moderate,” 4
“considerable,” and 5 “extreme financial strain” were combined
to indicate presence of financial strain. CVD risk factors (high
blood pressure, high cholesterol, obesity, positive family history),
various event types (AMI, PCI, and “other”), CR attendance, and
comorbidities (Diabetes Mellitus, musculoskeletal, and “other”
comorbid conditions) were not significant at the bivariate level
and were therefore not entered into the multivariate analyses. As shown in Table 2, the most commonly endorsed items were
being physically restricted, lacking energy, being short of breath,
thinking I will never be the same again, and not sleeping well. Each of these five items was endorsed by ≥50% of participants. A further 10 items were endorsed by between 40 and 50%
of participants, including thinking about having another heart
event, being irritated by little things, thinking my condition
might get worse, having chest discomfort, avoiding situations and
activities, being unable to do things that I know will improve my
health, having difficulty concentrating, being overly aware of my
heart in my chest, thinking that I’m not the person I used to be,
and not knowing how my family will cope if something should
happen to me. A further 11 items were endorsed by between
33 and 40% of participants. The remaining 48 items (from the
full pool of 74 items) were each endorsed by fewer than 33% of
participants (not shown in table). Data Analysis Approximately one
quarter lived alone and for those who lived with others (n = 146),
the majority lived with their partner (n = 83), or their partner and
children (n = 40). All participants had experienced their event in the previous
12 months, with the majority (94%) occurring within the past
3 months (mean = 2.2, SD = 1.9 months). Most participants
had an AMI, CABGS, or PCI, while a smaller number (21%)
reported having experienced other heart issues (valve issues, n
= 21; heart rhythm disturbance, n = 15; SCAD, n = 6; cardiac
arrest, n = 6). More than half (n = 100, 56%) of the participants
did not attend CR, with the most common reasons being people
waiting for CR (42%), declining attendance (21%), and concerns
associated with COVID-19 (14%), including barriers associated
with using telehealth. Just over half the participants (58%) had at
least one significant co-morbidity, most often diabetes (n = 58,
30%) or musculoskeletal conditions (n = 40, 32%). In addition,
cardiovascular risk factors such as hypertension (37%), high
cholesterol (31%), obesity (13%), and obstructive sleep apnoea
(12%) were present. One third of participants had a history
of mental health problems (depression or anxiety), with one
in four having experienced depression and one in five having
experienced anxiety. Participants Three of the four next most distressing
items belonged to the “Social” domain. For both the “Social” and
“Symptoms” domains, three items were rated as moderately or
severely distressing by over 50% of those who experienced these
issues, whereas this was the case for only one item within each of
the “Future,” “Affect,” and “Role functioning” domains. Overall
though, the pattern evidenced in Figure 2 shows that a range of
items across all six domains were identified as highly distressing. Most Distressing Issues and Concerns
While some items were highly prevalent, others were highly
distressing. The items which elicited the highest ratings in
terms of the severity of distress they caused are shown in
Table 3. Not having access to needed health care was identified
as the most distressing issue, being rated as either moderately
or severely distressing by 74% of those who experienced this
issue. Notably though, only 14% of participants actually reported
experiencing this issue. Being concerned about capacity for
sexual activity was the next most distressing issue, being rated
as moderately or severely distressing by 64% of those who
experienced it. However, only around one in five participants
reported experiencing this issue. Being unsupported by family
and friends, being isolated from family and friends, and being
unavailable to family and friends were each reported as highly
distressing albeit not commonly experienced issues. Notably,
“not knowing how my family will cope if something happens Participants As shown in Figure 1, four of the five most commonly
endorsed items belonged to the “Symptoms” domain and
more specifically, the “physical symptoms” sub-domain. Indeed,
endorsement of items within the Symptoms domain was Participants ranged in age from 22 to 90 years, with a mean
(SD) age of 63.7 (11.2) years. Participant sociodemographic,
psychosocial and medical characteristics are shown in Table 1. March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 5 Unraveling the Complexity of Cardiac Distress Jackson et al. FIGURE 1 | Prevalence of issues and concerns in domains. N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by
>33% of participants shown. FIGURE 1 | Prevalence of issues and concerns in domains. N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by
>33% of participants shown FIGURE 1 | Prevalence of issues and concerns in domains. N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by
>33% of participants shown. s and concerns in domains. N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by to me” and “not sleeping well” were rated as both distressing
and common. common, with nine of the symptom-related items each being
endorsed by over a third of participants. Items within the
“Future” domain were the next most commonly endorsed,
with three items from this domain being in the top 10, and
six being endorsed by over a third of participants. Items
in the “Affect” domain were the next most prevalent, with
four being endorsed by over a third of participants. In
contrast, items within the domains of “Social functioning,” “Self-
management,” “Self-perception,” and “Role functioning” were the
least commonly endorsed. In terms of the experience of “severe distress,” having bad
dreams and nightmares was identified as the most severely
distressing for those who experienced it; 28% of those who
experienced bad dreams and nightmares reported that this
was “severely distressing.” Being unsupported by family and
friends and being unable to work were also commonly identified
as causing severe distress amongst those who experienced
these issues. As shown in Figure 2, the two most distressing issues
belonged to the “Self-management” and “Self-perception”
domains, although other items within these two domains were
rated as less distressing. Frontiers in Psychiatry | www.frontiersin.org Predictors of Prevalence and Severity
Ratings Results of the multivariate regression analyses are shown in
Table 4. In terms of prevalence ratings, financial strain was a significant
predictor for all domains, and history of mental illness was a
significant predictor for all but two domains. Living alone was March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 6 Unraveling the Complexity of Cardiac Distress Jackson et al. TABLE 3 | Most distressing issues and concerns in rank order. TABLE 3 | Most distressing issues and concerns in rank order. Prevalence
Level of distress caused
Moderate
Severe
Moderate or severe
Not having access to the health care I need
14.0
66.7
7.4
74.1
Being concerned about my capacity for sexual activity
22.0
47.6
16.7
64.3
Being unsupported by my friends or family
6.8
38.5
23.1
61.6
Being isolated from family and friends
15.3
44.8
13.8
58.6
Not knowing how my family will cope if something happens to me
42.9
39.0
18.3
57.3
Being unavailable to my family and friends
17.3
45.5
9.1
54.6
Not sleeping well
50.5
43.3
10.3
53.6
Having more pain than I expected to have
22.8
36.4
15.9
52.3
Having bad dreams and nightmares
24.0
23.9
28.3
52.2
Not being able to cope effectively with my heart condition
27.1
40.4
11.5
51.9
Lacking energy
60.3
36.0
15.8
51.8
Becoming a burden to my family
31.4
38.3
13.3
51.6
Not knowing what the future holds for me
40.3
42.9
7.8
50.7
Being unsure about how much exercise I should be doing
39.3
45.3
5.3
50.6
Being emotionally exhausted
33.3
42.9
6.3
49.2
Being unable to deal with stress
32.8
38.7
9.7
48.4
Being unable to do things that will improve my health
44.8
41.4
6.9
48.3
Being unable to plan for the future
24.9
39.6
8.3
47.9
Not being able to return to work or continue working
23.7
25.6
22.2
47.8
Thinking that I’m not the person I used to be
43.8
40.5
7.1
47.6
Not having anyone to talk to who understands my difficulties
17.9
35.3
11.8
47.1
Being afraid of dying
33.0
35.9
10.9
46.8
Feeling lonely
24.5
38.3
8.5
46.8
Lacking purpose and meaning in life
27.1
28.8
17.3
46.1
Having difficulty making decisions
25.0
4.2
4.2
45.9
N = 189–194 with variations due to incompletions in some questionnaires. Only 25 most distressing items shown. Predictors of Prevalence and Severity
Ratings relating to cardiac symptoms, self-perception, concerns about
the future, negative affect, social functioning, role functioning,
and self-management. The most commonly endorsed items were
within the domains of dealing with symptoms, fear of the future,
negative affect, and social functioning, and included “being
physically restricted,” “lacking energy,” “being short of breath,”
“thinking I will never be the same again,” and “not sleeping
well.” While less prevalent, “not having access to the health
care I need,” “being concerned about my capacity for sexual
activity,” and “being unsupported by family and friends” were
reported as highly distressing for those experiencing these issues. The study has also identified key demographic and psychosocial
predictors of distress. In doing so, the study has expanded our
understanding of the multi-faceted nature of distress caused by a
cardiac event. predictive of prevalence ratings for the “Cognitive symptoms”
sub-domain and the “Social” domain. Females had significantly
higher prevalence for the “Symptoms,” “Future,” “Affect,” and
“Social” domains, and the “Physical symptoms” sub-domain,
while those who had undergone CABGS had higher prevalence
for the “Symptoms” domain and the “Physical symptoms” sub-
domain. Bereavement was associated with higher prevalence for
the Symptoms domain [aOR = 3.82 (95% CI 1.19–12.33), p =
0.025], while employment status was not significantly associated
with prevalence ratings for any of the domains (not shown
in table). In terms of severity ratings, history of mental illness was a
significant predictor across all but one domain, while financial
strain was a significant predictor for the “Affect” and “Self-
management” domains. Younger age was associated with higher
severity ratings for the “Social” domain [aOR = 0.99 (95% CI
0.97–1.00), p = 0.016; not shown in table]. Female sex, event
type, living alone, and employment status were not significantly
associated with severity ratings for any of the domains. Many of the highly prevalent issues and concerns identified by
these cardiac event survivors related to physical symptoms and
the fear that they would not ever be the same again. Concerns
about loss of health have been identified previously in qualitative
studies (6, 8, 11), as have concerns about loss of one’s previous
sense of self (13). Many of these concerns about symptoms
and physical adjustment are addressed and normalized in CR,
thereby reinforcing the need to increase the rate of referral to and
attendance at CR as a crucial step in recovery. DISCUSSION The findings of the present study document the prevalence and
severity of cardiac distress across 74 items within seven domains March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 7 Unraveling the Complexity of Cardiac Distress Jackson et al. FIGURE 2 | Issues and concerns rated as severely or moderately distressing. N = 189–194 with variations due to incompletions in some questionnaires. Dark color
indicates rating of “severe” distress, light color indicates rating of “moderate distress.” Only 25 most distressing items shown. FIGURE 2 | Issues and concerns rated as severely or moderately distressing. N = 189–194 with variations due to incompletions in some questionnaires. Dark color
indicates rating of “severe” distress, light color indicates rating of “moderate distress.” Only 25 most distressing items shown. Concerns about the future were also highly prevalent. These
included fears about worsening symptoms, having a recurrent
event and, ultimately, fears about death. Again, these issues
have been identified in previous qualitative studies (7, 8, 10,
11, 13). Additionally, future-related items were also identified
as highly distressing, particularly those centering on the issue of
an uncertain future. Previous qualitative studies have similarly
highlighted the distress caused by uncertainty and the inherent
inability to plan for the future post-cardiac event (8–11). disturbance, including poor sleep quality and nightmares, and
concerns about how patients’ families will cope, were both highly
endorsed and very distressing. These issues are very important
to address, to enhance patients’ physical and emotional recovery. We have argued elsewhere for the need for screening for sleep
disorders in the recovery phase during CR (19) so that survivors
are given relevant and timely assistance with this issue. Similarly,
health professionals engaging with cardiac patients in recovery
need to be alert to survivors’ concerns about family and, indeed,
to the importance of knowing about the degree of connectedness
survivors have with their family or within their community. Those experiencing difficulties in this area should be considered
for referral to a family-oriented service. y
p
p
While recognizing the highly prevalent nature of physical
symptoms in particular, this study highlights the importance of
assessing not just what people experience, but how distressing
these issues are perceived to be. Not having access to needed
health care was identified as the most distressing issue, extending
previous qualitative findings regarding worries about obtaining
professional help and advice (9, 13). Frontiers in Psychiatry | www.frontiersin.org DISCUSSION Given that the present
study was conducted during the COVID-19 pandemic, with
concomitant restrictions in face-to-face healthcare delivery and
pivoting to telehealth support options, it is perhaps not surprising
that this issue was identified as highly distressing. Also extending
previous qualitative studies (7, 14), ours is the first to highlight
concerns about sexual capacity as amongst the most distressing of
all issues faced by cardiac event survivors. Concern about future
sexual capacity reflects not only a person’s concern with physical
functioning but represents an important dimension of personal
identity that is under threat, and a concern with acceptance of
potential limitations, whether psychological or physical in origin. It is notable that women experienced more of the issues
and concerns listed than did men, with female sex a significant
predictor of high prevalence across several domains. This is
consistent with women’s previously reported higher levels of
anxiety and depression (20) and may also be due to women being
more expressive than men and highly attuned to identifying
and acknowledging their psychosocial concerns. Although female
sex was not predictive of distress severity ratings, this may be
due to the fact that these analyses were undertaken by domain
rather than for individual items. Consistently, it was somewhat
surprising that few age differences were identified and, again,
this may be due to a lack of fine-tuned comparisons, which was
outside the scope of the present study. As may be expected, people with a mental health history
experienced more of the issues and concerns, and also rated them
as more distressing. Those with a mental health history tend to The present findings highlight a number of issues that can
be addressed in cardiac rehabilitation. Difficulties due to sleep March 2022 | Volume 13 | Article 808904 8 Unraveling the Complexity of Cardiac Distress Jackson et al. TABLE 4 | Significant predictors of distress prevalence and severity ratings. DISCUSSION Predictor variables
Female sex
CABGS
Live alone
Financial Strain
History of mental illness
aOR (95%CI)
p
aOR (95%CI)
p
aOR (95%CI)
p
aOR (95%CI)
p
aOR (95%CI)
p
Prevalence ratings
Symptoms
4.29 (1.33–13.87)
0.015
2.97 (1.33–13.87)
0.042
6.95 (2.46–19.67)
<0.001
5.81 (1.95–17.33)
0.002
Sympt-Phys
1.89 (1.02–3.49)
0.042
1.98 (1.14–3.43)
0.015
2.27 (1.31–3.93)
0.004
2.15 (1.21–3.81)
0.009
Sympt-Cog
1.92 (1.08–3.44)
0.027
1.90 (1.17–3.07)
0.009
2.01 (1.21–3.34)
0.007
Future
2.45 (1.01–5.97)
0.049
3.73 (1.69–8.22)
0.001
Affect
2.17 (1.06–4.47)
0.035
3.64 (1.91–6.90)
<0.001
5.12 (2.60–10.08)
<0.001
Social
3.82 (1.21–12.07)
0.023
6.29 (1.86–21.29)
0.003
6.66 (2.42–18.30)
<0.001
5.58 (1.92–16.12)
0.002
Self-perception
3.84 (1.92–7.71)
<0.001
2.47 (1.17–5.22)
0.018
Role function
2.44 (1.57–3.80)
<0.001
Self-manage
8.38 (3.33–21.10)
<0.001
3.11 (1.17–8.30)
0.024
Severity ratings
Symptoms
1.42 (1.18–1.72)
<0.001
Sympt-Phys
1.42 (1.15–1.76)
0.002
Sympt-Cog
1.45 (1.11–1.89)
0.007
Future
1.35 (1.08–1.69)
0.009
Affect
1.25 (1.02–1.54)
0.032
1.40 (1.13–1.74)
0.002
Social
1.53 (1.25–1.96)
<0.001
Self-perception
1.45 (1.12–1.87)
<0.001
Role function
1.59 (1.15–2.19)
0.005
Self-manage
1.29 (1.06–1.57)
0.012
Full multivariable linear regression models for each rating included: age (years), sex, CABGS status, live alone status, financial stress, history of mental illness, and bereavement in
past 12 months. aOR, adjusted odds ratio; CABGS, coronary artery bypass graft surgery; Symptoms, symptoms domain; Sympt-Phys, Physical symptoms subdomain; Sympt-Cog,
Cognitive symptoms subdomain; Future, Concerns about the future domain; Affect, Negative Affect domain; Social, Social functioning domain; Self-Perception, Self-perception domain;
Role Function, Role functioning domain; Self-Manage, Self-management domain. TABLE 4 | Significant predictors of distress prevalence and severity ratings. Full multivariable linear regression models for each rating included: age (years), sex, CABGS status, live alone status, financial stress, history of mental illness, and bereavement in
past 12 months. aOR, adjusted odds ratio; CABGS, coronary artery bypass graft surgery; Symptoms, symptoms domain; Sympt-Phys, Physical symptoms subdomain; Sympt-Cog,
Cognitive symptoms subdomain; Future, Concerns about the future domain; Affect, Negative Affect domain; Social, Social functioning domain; Self-Perception, Self-perception domain;
Role Function, Role functioning domain; Self-Manage, Self-management domain. Full multivariable linear regression models for each rating included: age (years), sex, CABGS status, live alone status, financial stress, history of mental illness, and bereavement in
past 12 months. aOR, adjusted odds ratio; CABGS, coronary artery bypass graft surgery; Symptoms, symptoms domain; Sympt-Phys, Physical symptoms subdomain; Sympt-Cog,
Cognitive symptoms subdomain; Future, Concerns about the future domain; Affect, Negative Affect domain; Social, Social functioning domain; Self-Perception, Self-perception domain;
Role Function, Role functioning domain; Self-Manage, Self-management domain. DISCUSSION symptoms, raising concerns for this group in terms of their
cardiac recovery. Consistently, a recent study found that death
of a spouse or partner during the year prior to a first AMI is
associated with an increased risk of recurrent AMI and cardiac-
related death (24), highlighting bereavement as a red flag for
compromised cardiac recovery. have a lower base of resilience and coping and therefore will
experience more cardiac-related issues and find them inherently
more distressing. Previous anxiety/depression is a known risk
factor for poor post-cardiac event mental health outcomes (3, 18),
underscoring the importance of identifying these patients early
on and targeting them for mental health support. The present study also identified other known correlates of
persistent anxiety and depression such as financial strain (3,
18), and social isolation (3, 18). Indeed, financial strain was
a consistent predictor of prevalence ratings across all domains
and sub-domains, highlighting this patient characteristic as an
important and easily assessed “red flag” for poor post-event
mental health recovery, as we have noted previously (3, 18). Feelings of isolation have been exacerbated by the COVID–
related mandatory lockdowns and restrictions, and have brought
this issue into sharp focus internationally (21). Poignantly, of
those who reporting feeling unsupported by family and friends,
over 60% found this severely or moderately distressing, with
this distress likely heightened by the COVID-19 lockdowns
(21). Social isolation as a barrier to good recovery has been
identified as a contributor to persistent or worsening mental
health post cardiac event (3, 22) as well as conferring a higher
risk of premature death (23). Those who had experienced a
recent bereavement endorsed more items related to cognitive Frontiers in Psychiatry | www.frontiersin.org DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation. Limitations Some study limitations should be acknowledged. First, while
the present results confirm our definition of cardiac distress
as multifactorial and non-transient, a stronger case for the
latter point may have been made if the questionnaire had been
administered further into convalescence. The 8-week assessment
point was chosen to coincide with routine follow-up clinic
appointments, in order to optimize participation. However,
at this relatively early point of recovery it is possible that
symptoms of the cardiac blues, typical during the early post-
event adjustment period (25), may not yet have resolved. Future longitudinal studies to investigate the trajectories of the
components of distress are needed. Second, due to the large
number of distress items assessed, we identified predictors for
domains only, rather than for individual items. More fine-
tuned analysis to investigate item correlates would provide a
more nuanced understanding of sex and age-related differences March 2022 | Volume 13 | Article 808904 9 Unraveling the Complexity of Cardiac Distress Jackson et al. routine screening for cardiac distress in primary care and
cardiac rehabilitation settings. The present findings further our
understanding of the relative importance of various post-event
issues and concerns, thereby providing useful clinical guidance
for health professionals working in cardiac rehabilitation and
psychocardiology settings. in particular, but was outside the scope of the present study. Again, this could be the focus of more targeted hypothesis
testing within specific distress domains and/or for specific
patient groups. Third, being undertaken during the COVID-
19 pandemic, the study findings may have been impacted by
transient and extraneous stressors that were not measured. Despite these limitations, the study was strengthened by the
inclusion of consecutive series of cardiac patients from two large
hospitals, representing both metropolitan and regional areas
of Australia. FUNDING This study was partially funded by the Angela Anita Reid bequest
to the Australian Centre for Heart Health. This study was partially funded by the Angela Anita Reid bequest
to the Australian Centre for Heart Health. ETHICS STATEMENT The present study extends the findings of earlier qualitative
studies by quantifying common issues and concerns experienced
by cardiac event survivors and providing assessment of the
level of distress caused by these issues and concerns. It is the
first study to explore cardiac distress in this way. The findings
highlight the importance of investigating both endorsement
of these issues as well as ratings of distress severity in our
attempts to understand cardiac distress. The fact that issues such
as not being able to access suitable health care and concern
about sexual functioning were highly distressing although not
highly endorsed, shows the necessity for measuring both, and
not simply assuming that high prevalence of an issue corelates
with a high level of distress and vice versa. In terms of
clinical implications, the findings underscore the importance of
providing the opportunity for patients to express the specific
nature of their worries and concerns, and to reveal the impacts
of these on their psychological wellbeing. Patients expressing
high levels of distress should be given the opportunity for
psychocardiology-informed counseling to support them in their
recovery. Indeed, the findings point to the need for implementing This study had ethics approval from the Monash Health
Human Research Ethics Committee (approval number: RES-19-
0000631A – 55979). Written informed consent for participation
was not required for this study in accordance with the national
legislation and the institutional requirements. AUTHOR CONTRIBUTIONS BM, MR, ML, and AJ undertook data analysis and data
interpretation. AJ, MR, and BM drafted the manuscript. JA,
JS, VH, MA, RH, CS, and DT contributed to and approved
the manuscript. All authors contributed to study design,
questionnaire development, and data collection. All authors
contributed to the article and approved the submitted version. BM, MR, ML, and AJ undertook data analysis and data
interpretation. AJ, MR, and BM drafted the manuscript. JA,
JS, VH, MA, RH, CS, and DT contributed to and approved
the manuscript. All authors contributed to study design,
questionnaire development, and data collection. All authors
contributed to the article and approved the submitted version. REFERENCES 6. Simonÿ CP, Dreyer P, Pedersen BD, Birkelund R. It is not just a minor thing - a
phenomenological-hermeneutic study of patients’ experiences when afflicted
by a minor heart attack and participating in cardiac rehabilitation. Scand J
Caring Sci. (2017) 31:232–40. doi: 10.1111/scs.12334 6. Simonÿ CP, Dreyer P, Pedersen BD, Birkelund R. It is not just a minor thing - a
phenomenological-hermeneutic study of patients’ experiences when afflicted
by a minor heart attack and participating in cardiac rehabilitation. Scand J
Caring Sci. (2017) 31:232–40. doi: 10.1111/scs.12334 1. Lichtman
JH,
Froelicher
ES,
Blumenthal
JA,
Carney
RM,
Doering
LV, Frasure-Smith N. Depression as a risk factor for poor prognosis
among patients with acute coronary syndrome: systematic review and
recommendations:
a
scientific
statement
from
the
American
Heart
Association, on behalf of the American Heart Association Statistics
Committee of the Council on Epidemiology and Prevention and the Council
on Cardiovascular and Stroke Nursing. Circulation. (2014) 129:1350–69. doi: 10.1161/CIR.0000000000000019 1. Lichtman
JH,
Froelicher
ES,
Blumenthal
JA,
Carney
RM,
Doering
LV, Frasure-Smith N. Depression as a risk factor for poor prognosis
among patients with acute coronary syndrome: systematic review and
recommendations:
a
scientific
statement
from
the
American
Heart
Association, on behalf of the American Heart Association Statistics
Committee of the Council on Epidemiology and Prevention and the Council
on Cardiovascular and Stroke Nursing. Circulation. (2014) 129:1350–69. doi: 10.1161/CIR.0000000000000019 7. Higgins RO, Murphy BM, Nicholas A, Worcester MU, Lindner H. Emotional
and adjustment issues faced by cardiac patients seen in clinical practice: a
qualitative survey of experienced clinicians. J Cardiopulm Rehabil Prev. (2007)
27:291–7. doi: 10.1097/01.HCR.0000291296.62894.e5 8. Doiron-Maillet N, Meagher-Stewart D. The uncertain journey: women’s
experiences following a myocardial infarction. Can J Cardiovasc Nurs. (2003) 13:14–23. 2. Celano CM, Daunis DJ, Lokko HN, Campbell KA, Huffman JC. Anxiety
disorders and cardiovascular disease. Curr Psychiatry Rep. (2016) 18:101–101. doi: 10.1007/s11920-016-0739-5 2. Celano CM, Daunis DJ, Lokko HN, Campbell KA, Huffman JC. Anxiety
disorders and cardiovascular disease. Curr Psychiatry Rep. (2016) 18:101–101. doi: 10.1007/s11920-016-0739-5 9. Wang W, Thompson DR, Chair SY, Twinn SF. Chinese couples’ experiences
during convalescence from a first heart attack: a focus group study. J Adv Nurs. (2008) 61:307–15. doi: 10.1111/j.1365-2648.2007.04529.x 3. Murphy B, Le Grande M, Alvarenga M, Worcester M, Jackson A. Anxiety
and depression after a cardiac event: prevalence and predictors. Front Psychol. (2020) 10:3010. doi: 10.3389/fpsyg.2019.03010 3. Murphy B, Le Grande M, Alvarenga M, Worcester M, Jackson A. REFERENCES Anxiety
and depression after a cardiac event: prevalence and predictors. Front Psychol. (2020) 10:3010. doi: 10.3389/fpsyg.2019.03010 10. Iles-Smith H, Deaton C, Campbell M, Mercer C, McGowan L. The
experiences of myocardial infarction patients readmitted within six months of
primary percutaneous coronary intervention. J Clin Nurs. (2017) 26:3511–8. doi: 10.1111/jocn.13715 4. Vilchinsky N, Ginzburg K, Fait K, Foa EB. Cardiac-disease-induced PTSD
(CDI-PTSD): a systematic review. Clin Psychol Rev. (2017) 55:92–106. doi: 10.1016/j.cpr.2017.04.009 4. Vilchinsky N, Ginzburg K, Fait K, Foa EB. Cardiac-disease-induced PTSD
(CDI-PTSD): a systematic review. Clin Psychol Rev. (2017) 55:92–106. doi: 10.1016/j.cpr.2017.04.009 11. Merritt CJ, de Zoysa N, Hutton JM. A qualitative study of younger men’s
experience of heart attack (myocardial infarction). Br J Health Psychol. (2017)
22:589–608. doi: 10.1111/bjhp.12249 5. Edmondson D, Richardson S, Falzon L, Davidson KW, Mills MA,
Neria
Y. Posttraumatic
stress
disorder
prevalence
and
risk
of
recurrence
in
acute
coronary
syndrome
patients:
a
meta-analytic
review. PLoS
ONE. (2012)
7:e38915. doi:
10.1371/journal.pone.00
38915 5. Edmondson D, Richardson S, Falzon L, Davidson KW, Mills MA,
Neria
Y. Posttraumatic
stress
disorder
prevalence
and
risk
of
recurrence
in
acute
coronary
syndrome
patients:
a
meta-analytic
review. PLoS
ONE. (2012)
7:e38915. doi:
10.1371/journal.pone.00
38915 12. Fleury J, Sedikides C, Lunsford V. Women’s experience following a cardiac
event: the role of the self in healing. J Cardiovasc Nurs. (2001) 15:71–82. doi: 10.1097/00005082-200104000-00006 March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 10 Unraveling the Complexity of Cardiac Distress Jackson et al. 13. Thompson DR, Ersser SJ, Webster RA. The experiences of patients and
their partners 1 month after a heart attack. J Adv Nurs. (1995) 22:707–14. doi: 10.1046/j.1365-2648.1995.22040707.x 22. Jackson A, Murphy B. Loneliness, social isolation and cardiovascular risk. Br
J Cardiac Nurs. (2019) 14:1–8. doi: 10.12968/bjca.2019.0093 22. Jackson A, Murphy B. Loneliness, social isolation and cardiovascular risk. Br
J Cardiac Nurs. (2019) 14:1–8. doi: 10.12968/bjca.2019.0093 j
23. Holt-Lunstad J, Smith TB, Baker M. Loneliness and social isolation as risk
factors for mortality: a meta-analytic review. Perspect Psychol Sci. (2015)
10:227–37. doi: 10.1177/1745691614568352 23. Holt-Lunstad J, Smith TB, Baker M. Loneliness and social isolation as risk
factors for mortality: a meta-analytic review. Perspect Psychol Sci. (2015)
10:227–37. doi: 10.1177/1745691614568352 14. Abramsohn EM, Decker C, Garavalia B, Garavalia L, Gosch K, Krumholz HM,
et al. “I’m not just a heart, I’m a whole person here”: a qualitative study to
improve sexual outcomes in women with myocardial infarction. REFERENCES J Am Heart
Assoc. (2013) 2:e000199. doi: 10.1161/JAHA.113.000199 24. Wei D, Janszky I, Ljung R, Leander K, Chen H, Fang F, et al. Bereavement in
the year before a first myocardial infarction: Impact on prognosis. Eur J Prev
Cardiol. (2021) 28:1229–34. doi: 10.1177/2047487320916958 15. Gregory S, Bostock Y, Backett-Milburn K. Recovering from a heart attack:
a qualitative study into lay experiences and the struggle to make lifestyle
changes. Fam Pract. (2006) 23:220–5. doi: 10.1093/fampra/cmi089 25. Murphy B, Higgins R, Jackson A. Anxiety, depression and psychological
adjustment after an acute cardiac event. In: D Byrne and M Alvarenga,
editors, Handbook of Psychocardiology. Singapore: Springer (2016). p. 57. doi: 10.1007/978-981-287-206-7_57 16. Jackson AC, Murphy BM, Thompson DR, Ski CF, Alvarenga ME, Le Grande
MR, et al. What is cardiac distress and how should we measure it? Br J Cardiac
Nurs. (2018) 13:286–93. doi: 10.12968/bjca.2018.13.6.286 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. 17. Jackson A, Rogerson M, Le Grande M, Thompson D, Ski C, Alvarenga M,
et al. Protocol for the development and validation of a measure of persistent
psychological and emotional distress in cardiac patients: the Cardiac Distress
Inventory. BMJ Open. (2020) 10:e034946. doi: 10.1136/bmjopen-2019-034946 18. Murphy BM, Elliott PC, Ludeman D, Judd F, Humphreys J, Edington J, et al. ’Red flags’ for anxiety and depression after an acute cardiac event: 6-month
longitudinal study in regional and rural Victoria. Eur J Prev Cardiol. (2014)
21:1079–89. doi: 10.1177/2047487313493058 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. 19. Le Grande M, Neubeck L, Murphy BM, McIvor D, Lynch D, McLean H,
et al. Screening for obstructive sleep apnoea in cardiac rehabilitation: a
position statement from the Australian Centre for Heart Health and the
Australian Cardiovascular Rehabilitation Association. Eur J Prev Cardiol. (2017) 23:1466–75. doi: 10.1177/2047487316652975 Copyright © 2022 Jackson, Rogerson, Amerena, Smith, Hoover, Alvarenga, Higgins,
Grande, Ski, Thompson and Murphy. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). Copyright © 2022 Jackson, Rogerson, Amerena, Smith, Hoover, Alvarenga, Higgins,
Grande, Ski, Thompson and Murphy. 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. Frontiers in Psychiatry | www.frontiersin.org March 2022 | Volume 13 | Article 808904 REFERENCES 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. 20. O’Neil A, Russell JD, Murphy B. How does mental health impact
women’s heart health? Heart, Lung and Circulation. (2020) 30:59–68. doi: 10.1016/j.hlc.2020.05.111 21. Murphy BM, Higgins RO, Jackson AC. The mental health burden of COVID-
19 on cardiac healthcare workers and their patients. Br J Cardiac Nurs. (2020)
15:1–8. doi: 10.12968/bjca.2020.0129 March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 11
|
https://openalex.org/W2640860722
|
https://europepmc.org/articles/pmc5428246?pdf=render
|
English
| null |
The blood labyrinthine barrier in the human normal and Meniere’s disease macula utricle
|
Scientific reports
| 2,017
|
cc-by
| 8,358
|
Gail Ishiyama1, Ivan A. Lopez2, Paul Ishiyama2, Harry V. Vinters3 & Akira Ishiyama2 iyama1, Ivan A. Lopez2, Paul Ishiyama2, Harry V. Vinters3 & Akira Ishiyama2 The ultrastructural organization of the blood labyrinthine barrier (BLB) was investigated in the
human vestibular endorgan, the utricular macula, using postmortem specimens from individuals
with documented normal auditory and vestibular function and surgical specimens from patients with
intractable Meniere’s disease. Transmission electron microscopic analysis of capillaries located in
the normal human utricular stroma showed vascular endothelial cells with few pinocytotic vesicles,
covered by a smooth and uniform basement membrane surrounded by pericyte processes. Meniere’s
disease specimens revealed differential ultrastructural pathological changes in the cellular elements of
the microvasculature. With moderate degeneration of the BLB, there were numerous vesicles within
the vascular endothelial cells (VECs), with increased numbers at the abluminal face, pericyte process
detachment and disruption of the perivascular basement membrane surrounding the VECs. With severe
degeneration of the BLB, there was severe vacuolization or frank apparent necrosis of VECs and loss
of subcellular organelles. A higher severity of BLB degenerative changes was associated with a higher
degree of basement membrane thickening and edematous changes within the vestibular stroma. This
study presents the first ultrastructural analysis of the capillaries constituting the BLB in the human
vestibular macula utricle from normal and Meniere’s disease. In the inner ear, the term blood labyrinthine barrier (BLB) refers to the barrier between the vasculature and the
inner ear fluids, either endolymph or perilymph1, 2. The BLB is critical for the maintenance of the inner ear fluid
ionic homeostasis and for the prevention of the entry of deleterious substances into the inner ear3. By studying the
penetration of dyes and drugs from the systemic circulation into the fluids of the inner ear, it can be shown that
the BLB is selective and that the composition of the inner ear fluids is regulated and markedly different from that
of blood or of other fluids such as cerebrospinal fluid2. l
pl
Understanding the dynamics of the BLB is important to develop therapeutic drug delivery systems to the
inner ear to block or enhance the BLB inflammatory response. The BLB in the cochlea is well studied in animal
models2, 4, 5. Initial transmission electron microscopy (TEM) studies demonstrated that the BLB in the guinea pig
stria vascularis and spiral ligament is composed of vascular endothelial cells (VECs) surrounded by a basement
membrane6. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 20 October 2016
Accepted: 20 February 2017
Published: xx xx xxxx The blood labyrinthine barrier in
the human normal and Meniere’s
disease macula utricle Received: 20 October 2016
Accepted: 20 February 2017
Published: xx xx xxxx Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 Gail Ishiyama1, Ivan A. Lopez2, Paul Ishiyama2, Harry V. Vinters3 & Akira Ishiyama2 More recent studies have noted that, in addition, the intrastrial BLB is composed of pericytes and
perivascular-resident macrophage-like melanocytes7–11. The VECs line the interior surface of the blood vessels,
connected by tight junctions, forming an interface between circulating blood and the rest of the vessel wall. Pericytes are the only cell type to intimately connect with VECs as they lie embedded within the endothelial
basement membrane. The perivascular-resident macrophage-like melanocytes have foot processes on the outer
surface of capillaries and these cells may play a similar role as astrocytes in the brain to maintain the integrity
of the barrier and may play a role in inflammatory responses3, 12. The BLB within the perilymph-filled areas that
are part of the blood-perilymph barrier include the cochlear spiral ligament, spiral limbus, modiolus, osseous
spiral lamina, vestibular stromal vasculature and the sub-epithelial dark cell area13. The vestibular BLB unit in
the rodent is similar to the rodent strial BLB unit at the light microscopic level: composed of vascular endothelial
cells, pericytes and perivascular-resident macrophage-like melanocytes10. 1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, USA. 2Department of Head and Neck Surgery, David Geffen School of Medicine, University of California, Los Angeles, USA. 3Department of Pathology & Laboratory Medicine (Neuropathology), David Geffen School of Medicine, University
of California, Los Angeles, USA. Correspondence and requests for materials should be addressed to G.I. (email:
gishiyama@mednet.ucla.edu) Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. The BLB in normal capillaries. Capillary located in the stroma of the macula utricle from a normal
subject (62-year-old male). (a) Low magnification view. The lumen (lu) of the capillary is smooth and
vascular endothelial cells (vec) exhibit normal morphology. The perivascular basement membrane (pbm)
and extracellular matrix (asterisk) are also normal. (b) high magnification view from (a), vec show normal
cytoplasm. Tight junctions show a normal organization (tj). Bar (a) = 2 μm, (b) = 300 nm. Figure 1. The BLB in normal capillaries. Capillary located in the stroma of the macula utricle from a normal
subject (62-year-old male). (a) Low magnification view. The lumen (lu) of the capillary is smooth and
vascular endothelial cells (vec) exhibit normal morphology. The perivascular basement membrane (pbm)
and extracellular matrix (asterisk) are also normal. (b) high magnification view from (a), vec show normal
cytoplasm. Tight junctions show a normal organization (tj). Gail Ishiyama1, Ivan A. Lopez2, Paul Ishiyama2, Harry V. Vinters3 & Akira Ishiyama2 Bar (a) = 2 μm, (b) = 300 nm. Recent studies have implicated loss of the integrity of the BLB in several inner ear pathologies including acous-
tic trauma, autoimmune inner ear disease, and presbycusis3, 7, 8, 14–16. Meniere’s disease is a disabling syndrome
of fluctuating hearing loss, episodic vertigo, and ear fullness, the etiology and pathophysiology of which remain
poorly understood. Endolymphatic hydrops, a ballooning of the endolymph fluid, was demonstrated in postmor-
tem human temporal bone studies on patients with Meniere’s disease17, 18. Endolymphatic hydrops is character-
ized by an excess of endolymph; however, the mechanism whereby vertigo and hearing loss occur is unclear19–21. MRI studies have demonstrated that within patients with Meniere’s disease, the degree of hydrops correlates with
the degree of hearing loss22. Hypothesized mechanisms for hearing loss in Meniere’s disease include a decrease
in cochlear blood flow to one-third as a direct effect of hydrops23; however, cochlear blood flow measurement in
subjects with Meniere’s disease did not differ from control groups24. While pathophysiology of the BLB has been
proposed in Meniere’s disease, no prior studies have evaluated the BLB ultrastructure in normal humans or in
Meniere’s disease. Of note, Meniere’s disease is a uniquely human disease and there are no animal models which
replicate the disease.h p
The normal vascular anatomy of the cochlea has been well documented using light, TEM and scanning EM of
corrosion cast of the cochlear vasculature25. To our knowledge, there are no previous ultrastructural studies of the
human vestibular stromal capillaries. We have previously documented pathological alterations of the vestibular
crista ampullaris sub-epithelial basement membrane in patients with intractable Meniere’s disease26. The utricular
stroma demonstrated thickening of the basement membrane, fibroblast vacuolization and fibrillary deposition
close to the blood vessels. However, the ultrastructural morphology of the human vestibular BLB has not yet been
investigated. We report the first ultrastructural study of the normal human BLB of capillaries located underneath
the utricular sensory epithelia and compare the results with those from Meniere’s patients. The BLB in subepithelial capillaries of the utricular stroma in Meniere’s disease.
We exam The BLB in subepithelial capillaries of the utricular stroma in Meniere s disease. We examined
the ultrastructural changes of the capillaries located within the stroma of the macula utricle from Meniere’s dis-
ease patients. There were cases with mild or severe (ischemic) morphological alterations in the VECs. The VECs
exhibited abundant vesicular transport and cytoplasmic vacuolization, pseudopodic protrusions into the lumen,
microvilli-like projections, and swelling and degeneration of the organelles. In some cases, there were complete
degenerative changes and necrosis of the VECs. The perivascular basement membrane, similarly, ranged from
normal appearance to severely vacuolated and thickened. VECs with active vesicular transport exhibited hyper-
trophic Golgi complexes and markedly increased vesicle formation. Figure 3a shows a relatively intact and smooth
lumen of a capillary, which demonstrates mild alterations (39-year-old with delayed endolymphatic hydrops, with
history of profound hearing loss as a child with intractable vertigo spells despite medications). The VECs demon-
strate some vacuoles and a prominent increase in vesicular transport facing the abluminal side, with some vesicles
opening onto the abluminal area (Fig. 3b). The perivascular basement membrane is relatively intact, with uniform
thickness. The VEC show mild vacuolar changes. There appears to be detachment of a pericyte process (Fig. 3b),
and there are mild edematous changes within the basement membrane. Figure 4 shows the cross section of a capillary located in the stroma of the macula utricle from an 82-year-old
male Meniere’s patient with Tumarkin falls and progressive severe loss of caloric function. The VECs exhibit
swelling and numerous cytoplasmic vesicles, with narrowed and uneven capillary lumen (Fig. 4a). At higher
magnification, vesicles in the endothelial cell cytoplasm are lined up polarized to the abluminal membrane. There
are chained or coalesced pinocytotic vesicles forming tubular channel-like formations. Tight junctions joining
VECs appear to be intact with no ultrastructural changes apparent. The perivascular basement membrane is
mildly disorganized, and the extracellular matrix demonstrates mild edema. The endothelial cell lumen demon-
strates multiple areas of microvilli- like projections into the lumen, and protrusion of pseudopodic expansion of
the endothelial cell. There is abundant abluminal oriented vesicular transport, facing the basement membrane
with some vesicles opening onto the basement membrane. Other areas of the lumen epithelium demonstrate
deep invaginations that can be seen ending in micropinocytic vesicles. At higher magnification (Fig. 4b) apparent
activation of a Golgi complex within the VEC can be seen. Results
Th The normal human BLB in the capillaries of the utricle stroma. The normal human BLB consists
of vascular endothelial cells (VECs) surrounded by a perivascular basement membrane with pericyte processes
embedded within the basement membrane that surrounds the VECs (Fig. 1a). Figure 1a shows a cross section of
a blood vessel (low magnification) located in the stroma underneath the utricular macula sensory epithelia from
a 62-year-old individual with normal vestibular or auditory function. The capillaries of the vestibular stroma
are continuous, and the endothelial lining is without fenestrations. Higher magnification view of the normal
human BLB (Fig. 1b) shows the VECs connected by tight junctions. The capillary endothelial surface facing the
lumen is smooth and uniform, and without many pseudopodic extensions; the VECs contain organelles, with
numerous mitochondria and a paucity of endocytic vesicles or vacuoles. There are only a few endocytic vesicles
and no vacuoles within the cytoplasm of VECs (Fig. 1b). Another component of the BLB, the perivascular base-
ment membrane, is a uniform structure with a homogeneous matrix (Fig. 1a and b). The perivascular basement
membrane has an even thickness and intermingles uniformly within the abluminal portion of the VECs and per-
icytes (Fig. 1b). It completely surrounds the VECs and pericyte dendritic processes, and there is no rarefaction or
duplication of the basement membrane. Pericytes have dendritic processes that surround VECs (not seen in this
micrograph). There is a paucity of vacuoles or vesicles within the VEC or the pericyte. Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 2 www.nature.com/scientificreports/ Figure 2. The BLB in the macula utricle from a normal subject, older age (86-year-old female). Note the normal
appearance of the VECs (vec), the basement membrane (bm), extracellular matrix (ecm), tight junctions (tj),
and capillary lumen (lu) are also normal. The pericyte (p) cytoplasm appears normal. Bar = 2.5 μm. Figure 2. The BLB in the macula utricle from a normal subject, older age (86-year-old female). Note the normal
appearance of the VECs (vec), the basement membrane (bm), extracellular matrix (ecm), tight junctions (tj),
and capillary lumen (lu) are also normal. The pericyte (p) cytoplasm appears normal. Bar = 2.5 μm. Figure 2. The BLB in the macula utricle from a normal subject, older age (86-year-old female). Note the normal
appearance of the VECs (vec), the basement membrane (bm), extracellular matrix (ecm), tight junctions (tj),
and capillary lumen (lu) are also normal. Results
Th The pericyte (p) cytoplasm appears normal. Bar = 2.5 μm. The capillary ultrastructure in a normal aging utricle. Figure 2 shows a cross section of a blood vessel
(low magnification) located in the stroma underneath the utricular macula sensory epithelia from an 86-year-old
individual with normal vestibular function. The VEC demonstrates few vesicles, and the tight junctions remain
intact. The basement membrane is smooth, but mildly thickened in comparison with the capillary basement
membrane from a younger subject. A pericyte process with normal basement membrane is easily identified. The BLB in subepithelial capillaries of the utricular stroma in Meniere’s disease.
We exam Figure 5 shows the cross section of a capillary located in the stroma of the macula utricle from a 40-year-old
individual with congenital deafness, intractable vertigo spells associated with unilateral aural fullness, and caloric
paresis. The VEC is degenerated, with loss of organelles, thinning and extensive vacuolization. There is vacuoli-
zation of the extracellular space. The basement membrane is thickened, edematous, and irregular. Figure 6 shows
the cross section of a capillary located in the stroma of the macula utricle from a 56-year-old with Meniere’s
disease with a history of aural fullness, tinnitus, deafness and intractable vertigo spells. The putative endothelial
cell demonstrates diffuse vacuolization and loss of organelles. The perivascular basement membrane is thickened
and irregular, and the vacuolizations appear to be increased in the boundary. The extracellular perivascular space
exhibits vacuolizations and edema. Figure 7 shows the cross section of a capillary located in the stroma of the
macula utricle from a 67-year-old individual with Meniere’s disease characterized by intractable vertigo spells, Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. The BLB in Meniere’s disease (39-year-old). Capillary located in the utricle stroma underneath the
vestibular sensory epithelia. (a) Low magnification view. VECs (vec) show an almost normal appearance, lumen
is normal (lu), the extracellular matrix shows edema. (b) High magnification view -insert from (a). Abundant
open caveolar flask-like structures that almost touch the abluminal membrane of the vascular endothelial
cell (vec) were observed. The border of the basal membrane (pbm), is almost normal, a pericyte process
(pp) is detached. Thick arrowhead points to a large vacuole in the vascular endothelial cell cytoplasm. Bar in
(a) = 5 μm, (b) = 2 μm. Figure 3. The BLB in Meniere’s disease (39-year-old). Capillary located in the utricle stroma underneath the
vestibular sensory epithelia. (a) Low magnification view. VECs (vec) show an almost normal appearance, lumen
is normal (lu), the extracellular matrix shows edema. (b) High magnification view -insert from (a). Abundant
open caveolar flask-like structures that almost touch the abluminal membrane of the vascular endothelial
cell (vec) were observed. The border of the basal membrane (pbm), is almost normal, a pericyte process
(pp) is detached. Thick arrowhead points to a large vacuole in the vascular endothelial cell cytoplasm. Bar in
(a) = 5 μm, (b) = 2 μm. Figure 4. A BLB capillary in Meniere’s disease (82-year-old). The BLB in subepithelial capillaries of the utricular stroma in Meniere’s disease.
We exam Capillary located in the utricle stroma underneath
the vestibular sensory epithelia. (a) Low magnification view. The vascular endothelial cell (vec) is swollen
with degeneration of organelles and debris within the cytoplasm, the lumen (lu) is uneven and narrowed
with pseudopodic and microvilli-like projections, the perivascular basement membrane (bm) exhibits mild
thickening. (b) High magnification view (from fig a) of the vascular endothelial cell (vec). The tight junction (tj)
appears normal, bm: basement membrane. Bar (a) = 1 μm, (b) = 0.5 μm. Figure 4. A BLB capillary in Meniere’s disease (82-year-old). Capillary located in the utricle stroma underneath
the vestibular sensory epithelia. (a) Low magnification view. The vascular endothelial cell (vec) is swollen
with degeneration of organelles and debris within the cytoplasm, the lumen (lu) is uneven and narrowed
with pseudopodic and microvilli-like projections, the perivascular basement membrane (bm) exhibits mild
thickening. (b) High magnification view (from fig a) of the vascular endothelial cell (vec). The tight junction (tj)
appears normal, bm: basement membrane. Bar (a) = 1 μm, (b) = 0.5 μm. Tumarkin falls, and unilateral deafness. There is severe perivascular basement membrane disorganization, and
diffuse edema in the extracellular matrix. The VECs are severely swollen, with apparent necrosis and no identifi-
able subcellular organelles. Tumarkin falls, and unilateral deafness. There is severe perivascular basement membrane disorganization, and
diffuse edema in the extracellular matrix. The VECs are severely swollen, with apparent necrosis and no identifi-
able subcellular organelles. Discussionh The stroma in the normal utricle is rich in capillaries, with less abundant capillaries in the deeper portion of the
sensory endorgan areas, and an abundance of capillaries in the subepithelial region. There is a particularly rich
network of fine capillaries beneath the basement membrane of the sensory areas. In the capillaries of the human
vestibular stroma, the VECs are thin walled (2–4 μm) with a paucity of pinocytotic vesicles, and pericytes engulf
the VECs via dendritic processes. In Iurato et al.27 study of the capillaries of the mammalian crista ampullaris, the
flat endothelial cells are noted to be about 0.1 to 1.4 μm, with the outer surface of the VEC in close contact with
the basement membrane, and the inner surface facing the capillary lumen. There are no fenestrations in the VECs Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. A BLB capillary with moderate degenerative changes in Meniere’s disease (40-year-old). Vacuolization is seen in vascular endothelial cells (vec), and there is apparent degeneration with loss of
organelles and thinning. The perivascular basement membrane (bm) shows thickening. Lumen (lu). The stroma
shows signs of edema (asterisk). Bar = 2 μm. Figure 5. A BLB capillary with moderate degenerative changes in Meniere’s disease (40-year-old). Vacuolization is seen in vascular endothelial cells (vec), and there is apparent degeneration with loss of
organelles and thinning. The perivascular basement membrane (bm) shows thickening. Lumen (lu). The stroma
shows signs of edema (asterisk). Bar = 2 μm. Figure 5. A BLB capillary with moderate degenerative changes in Meniere’s disease (40-year-old). Vacuolization is seen in vascular endothelial cells (vec), and there is apparent degeneration with loss of
organelles and thinning. The perivascular basement membrane (bm) shows thickening. Lumen (lu). The stroma
shows signs of edema (asterisk). Bar = 2 μm. Figure 6. A BLB capillary with moderate degenerative changes in Meniere’s disease (56-year-old). A capillary
shows moderate vacuolization of the vascular endothelial cell (vec) and pericytes (pc) and mild edema in the
stroma (arrows). The perivascular basement membrane (pbm) is thickened. The stroma shows signs of edema
(asterisk). Bar = 2 μm. Figure 6. A BLB capillary with moderate degenerative changes in Meniere’s disease (56-year-old). A capillary
shows moderate vacuolization of the vascular endothelial cell (vec) and pericytes (pc) and mild edema in the
stroma (arrows). The perivascular basement membrane (pbm) is thickened. The stroma shows signs of edema
(asterisk). Discussionh Bar = 2 μm. in the normal rodent cristae ampullaris with cell junctions forming zonulae adherents. Similarly, in this study
we found that the capillaries of the human vestibular utricular maculae are of continuous type, and the intercel-
lular junctions between VECs are separated by tight junction-like structures. The morphological organization
resembles that found in the inner ear of several animal models28. The basal lamina of the capillaries in the human
utricular stroma is composed of type IV collagen, laminin and other proteins29, interposed between VECs and
pericytes processes.hi in the normal rodent cristae ampullaris with cell junctions forming zonulae adherents. Similarly, in this study
we found that the capillaries of the human vestibular utricular maculae are of continuous type, and the intercel-
lular junctions between VECs are separated by tight junction-like structures. The morphological organization
resembles that found in the inner ear of several animal models28. The basal lamina of the capillaries in the human
utricular stroma is composed of type IV collagen, laminin and other proteins29, interposed between VECs and
pericytes processes.hi y
The site of perilymph production remains controversial, in general it is believed that it is formed as an ultrafil-
trate of blood1, 2 with some contribution from filtration from cerebrospinal fluid (CSF). The CSF may also filtrate
via the vestibular aqueduct or perivascular channels into the perilymph. The differing protein and amino acid
levels of the perilymph, blood serum, and CSF are indicative of the maintenance of ionic and protein composition
as part of the function of the BLB. Blood vessels, which exhibit cationic polyethyleneimine distribution, which Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. Ultrastructure of a BLB capillary with severe degenerative changes in Meniere’s disease (67-year-
old). A capillary located in the macula utricle stroma. The vascular endothelial cell (vec) exhibits degenerative
changes with no subcellular organelles and edematous change. The perivascular basement membrane (bm) is
disorganized and swollen. There is constriction of the lumen (lu) and edema (asterisk). Bar = 2 μm. Figure 7. Ultrastructure of a BLB capillary with severe degenerative changes in Meniere’s disease (67-year-
old). A capillary located in the macula utricle stroma. The vascular endothelial cell (vec) exhibits degenerative
changes with no subcellular organelles and edematous change. The perivascular basement membrane (bm) is
disorganized and swollen. There is constriction of the lumen (lu) and edema (asterisk). Bar = 2 μm. Discussionh identifies anionic sites, following intravenous administration, are indicative of macromolecular transport systems
of the BLB. In the cochlea, anionic sites are present in the basement membrane of Reissner's membrane. In the
vestibule, they are localized in the basement membrane of the capillary wall, and the sensory epithelium30. The
premature rat does not yet have fully developed anionic sites within the crista ampullaris13. Like the blood brain
barrier (BBB), the BLB develops in the early postnatal period of the immature rat. The timing of the development
of the BLB has important implications for ototoxicity of agents delivered during prenatal or postnatal periods and
to premature infants.i p
Intracerebral cortical vessels contain a mean of five caveolae per μm2 in arteriolar endothelium31, much less
than that in non-neural vessels, which likely corresponds with limited transcellular traffic of solutes in BBB cap-
illaries. Caveolae can undergo budding and fission from the plasma membrane, and translocate to the other side. In the case of Meniere’s disease, there appeared to be increased vesicular formations, with localization of vesicles
to the transluminal side. The increased vesicle formation and caveolae-like formations in the VEC, with polariza-
tion to the abluminal side, which would lead one to conclude that there is increased transcytosis, possibly vesicles
being used to traffic cargo across the VECs. During BBB breakdown, there is also an increase in trans-endothelial
channels in VECs32. In the specimens from Meniere’s disease in the present study, the tight junctions appeared to be normal, even
at high magnification microscopy and also even in severely edematous BLB cytoarchitecture. In contrast, BBB
in vasogenic edema appears to be associated with altered tight junction proteins, and separation noted at tight
junctions; however, it is noted that these changes occur late in the course of brain injury32. It is possible that there
is altered expression of tight junction proteins in the VEC of Meniere’s disease, without structural changes that
can be visualized. Tight junctions are not only important for the regulation of paracellular permeability, but also
may be involved in cytoskeletal dynamics and cellular signaling33.h y
y
y
g
g
The tight junctions in normal human BLB are continuous. No apparent abnormalities of the tight junction
were found in the BLB of Meniere’s disease specimens. Discussionh In some cases, the basement membrane appears to be swollen, and in more severe
cases, exhibits rarefaction, duplication, and extreme thickening. The subepithelial basement membrane, which
lies interposed between the epithelium facing the endolymph and the connective stromal tissue within the peri-
lymph, is markedly thickened and under TEM appears to be disorganized fibrils26. Additionally, the composition
of the subepithelial basement membrane is altered: collagen IV expression is diminished46, and the sub-epithelial
basement membrane thickening correlated with neuroepithelial damage and vestibular hair cell loss26. In
Meniere’s disease, both the sub-epithelial basement membrane and the capillary perivascular basement mem-
brane forming the blood-perilymph barrier, exhibit thickening, edema, and disorganization in comparison with
the normal basement membrane which is homogeneous, thin, with a continuous appearance due to a condensed
filamentous pattern and homogeneous matrix.l i
p
g
It has been suggested that Meniere’s disease may be caused by dysfunctional inner ear blood flow47, 48 exacer-
bated by the pathological increase in vascular permeability of the blood-labyrinthine barrier3, 49. In severe cases,
the capillary lumen is severely compromised due to edematous changes in the endothelial cells, and in some cases,
due to apparent debris from necrotic endothelial and pericyte cells. These capillaries are likely to be dysfunctional
to deliver nutrients to the neuroepithelium. The endothelial cell appears to be the earliest cell to exhibit damage. ph
pp
g
The finding of increased vesicular transport in the endothelial cell of the capillaries, VEC degenerative
changes, and thickening of the basement membrane of capillaries in Meniere’s disease raises the question of a
possible inflammatory pathology similar to that proposed as causative in autoimmune inner ear disease. Trune
and Nguyen-Huynh50 reviewed the cascade of events that can lead to increased permeability of the BLB mediated
by inflammatory cytokines and chemokines that strip off the protective glycocalyx, exposing the endothelial cell,
causing a cascade of events leading to loss of BLB integrity and breakdown of stria vascularis blood vessels. While
there are no ultrastructural studies of the BLB in human autoimmune inner ear disease, a human temporal bone
study of Sjogren's disease, an autoimmune disease associated with hearing loss in 25% of patients, demonstrated
thickening of the stria vascularis basement membrane and an associated immunoglobulin deposition in Sjogren's
patients with hearing loss51. Discussionh Using in vivo perilymph sampling of extravasated fluorescein from the vascular com-
ponent, Hirose et al.43 demonstrated higher fluorescein in the perilymph in LPS treated mice. The region of BLB
increased permeability would correspond to capillary beds, which travel through perilymph, filled spaces: spiral
ligament, spiral limbus, modiolus, and osseous spiral lamina, and the subepithelial space within the vestibular
stroma. In both, the uptake of gadolinium or fluorescein is not significant or nonexistent within the endolymph,
indicative that the BLB lining the endolymphatic space is more restrictive than that lining the perilymphatic
space. The studies together provide strong animal model evidence that increased gadolinium uptake within the
perilymph is indicative of BLB permeability increase.hf p
y
p
p
y
The role of cochlear macrophages is controversial, as elimination of macrophages did not alter the effect of
LPS to induce vascular permeability44. In contrast to the cochlea, the vestibular periphery of the C57BL/6J mouse,
exhibited LPS induced activation of perivascular macrophage-like melanocytes, identified using F4/80 stain-
ing, and increased vascular permeability as reflected by fluorescein isothiocyanate infiltration into the vestibular
periphery11. The role of macrophages or perivascular macrophage-like melanocytes in the vascular permeability
seen in the vestibular stromal capillaries from Meniere’s patients is unknown. However, of note, on light micros-
copy, there was no evidence for an infiltrate of inflammatory cells. yil
y
In nearly all Meniere’s cases evaluated, intact VECs of the vestibular BLB exhibited increased vesicular forma-
tion, polarized to the abluminal surface, with edema and vacuolization of the perivascular basement membrane
and of the extracellular matrix. Similar patterns of endothelial cell activation are noted in cerebral edema, which
is also associated with increased permeability of the BBB45. There are two main permeability barriers: the base-
ment membrane and the endothelial cell. Increased endothelial cell vesiculation is reported in hypoxic-ischemic
stroke, hypertensive encephalopathy, and brain trauma. It is important to note that our histopathological studies
are limited, by necessity, to severe intractable stage IV Meniere’s disease associated with profound non-serviceable
hearing. Within this group, there were varying degrees of severity of BLB damage. In some cases, the endothelial
cell had undergone complete necrosis, and in other cases, active vesicular formation is noted within an endothe-
lial cell with relative preservation of subcellular organelles such as mitochondria and endoplasmic reticulum.h p
g
p
The vestibular stromal BLB perivascular basement membrane exhibits thickening, edema, and duplication in
Meniere’s disease inner ear. Discussionh All of the capillaries constituting the BLB of the normal
human vestibular stroma lacked fenestrations and tight junctions were noted in the intercellular space joining
two-endothelial cells, forming a tight barrier which is in alignment with freeze-fracture studies of the guinea pig
blood-perilymphatic barrier34. Of note, the stria vascularis exhibits vastly differing permeability as well as degree
of pinocytosis. Therefore, the vestibular stromal BLB is similar to that of the cochlear modiolus or spiral limbus,
exhibiting a similar morphology to that of the BBB.h Through imaging studies using intravenous gadolinium, there is gathering evidence for blood labyrinthine
barrier compromise in Meniere’s disease. Intravenous gadolinium enhanced magnetic resonance imaging (MRI)
can be used to visualize and quantify endolymphatic hydrops22, 35. Gadolinium, an agent used in magnetic res-
onance imaging (MRI), is taken up into the perilymph, presumably via perfusion through the BLB, specifically
through the blood-perilymphatic barrier. Increased gadolinium enhancement in the perilymph on the ipsilater-
ally inner ear affected with Meniere’s disease has been noted in multiple studies, indicative of breakdown of the
BLB36–39. Of note, in the study of Tagaya et al.37, a high signal intensity ratio correlated with a higher grade (none,
mild, or significant) of hydrops in both the cochlea and the vestibule37. Most recently, Pakdaman et al.40 demon-
strated increased gadolinium uptake in the perilymph in Meniere’s disease ipsilateral and in the contralateral
unaffected ear compared with sudden hearing loss; however, the ratio of affected to unaffected ear in Meniere’s Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 6 www.nature.com/scientificreports/ disease was significantly greater than that of sudden hearing loss. This may be indicative that permeability altera-
tions of the BLB may be one of the primary causes of endolymphatic hydrops or Meniere’s disease. y
p
y
y
p
y
p
Intratympanic injection of lipopolysaccharide (LPS), a substance that breaks down the BLB and also the BBB,
is associated with increased gadolinium perilymphatic enhancement41. A similar increase in gadolinium uptake
in the perilymph has been noted following high intensity impulse noise acoustic trauma42. The role of infiltration
of inflammatory cells is controversial. LeFloc’h et al.41 showed that the reduction of ultrasmall superparamag-
netic iron oxide particles, corresponded with cochlear inflammation, which was hypothesized to be secondary to
macrophage infiltration. However, conclusive identification of a macrophage infiltrate using CD68 immunohis-
tochemistry was negative. Discussionh Specimen
Type of
Tissue
Age
(years)
Gender
Diagnosis
Figure
1
Autopsy
62
Male
Normal
1a,b
2
Autopsy
86
Female
Normal
2
3
Surgical
39
Male
Meniere’s
3a,b
4
Surgical
82
Male
Meniere’s
4a,b
5
Surgical
40
Female
Meniere’s
5
6
Surgical
56
Male
Meniere’s
6
7
Surgical
67
Female
Meniere’s
7
Table 1. Specimens (utricle) used in the present study. Autopsy: Temporal bones collected between 6 to 8 hrs. postmortem, Normal: indicates normal vestibular and auditory function. Table 1. Specimens (utricle) used in the present study. Autopsy: Temporal bones collected between 6 to 8 hr
postmortem, Normal: indicates normal vestibular and auditory function. loss, indicative that this increased permeability likely plays a role in the pathophysiology of Meniere’s disease55. The relation between inner ear autoimmune disease and Meniere’s disease remains to be identified.hh loss, indicative that this increased permeability likely plays a role in the pathophysiology of Meniere’s disease55. The relation between inner ear autoimmune disease and Meniere’s disease remains to be identified.hh hi
The diagram in Fig. 8a illustrates schematically the normal BLB. The BLB is maintained by tight junctions
between vascular endothelial cells which form a continuous barrier without fenestrations, and are covered by
a basement membrane with pericytes intermingled within the basement membrane. Diagram in Fig. 8b, shows
the pathological changes noted in Meniere’s disease inner ear BLB: vascular endothelial cells exhibit edema, with
an increase in vesicles, with apparent transcytosis of macromolecules. There is vacuolization of endothelial cells
and pericytes, and the perivascular basement membrane exhibits thickening and duplication. In some cases, the
pericyte processes are detached, and the capillary lumen is compromised by edematous changes of the endothelial
cells. In conclusion, the BLB dysfunction in the microvasculature of vestibular endorgans obtained from Meniere’s
disease patients likely contributes to edematous changes in the underlying stroma and vacuolization. We hypoth-
esize that alterations in BLB could lead to secondary changes in the vestibular sensory epithelia. Discussionh There is only one report on the ultrastructure of the strial cells in the MRL-Fas/lpr
mouse model of autoimmune disease52, capillary ultrastructure was found to be normal i.e. revealed no alterations
in tight junctions of VECs or alterations in the basement membrane, however, there were signs of hydropic gener-
ation in cells surrounding the strial capillaries. In contrast, in the C3H/lpr autoimmune mouse, extensive leakage
of ferritin into the perivascular tissues was noted, and a corresponding thickening of the basement membrane of
the stria capillaries53. Other studies, however, are indicative that thickening of the basement membrane of the cap-
illaries is non-specific, and has been noted in the aging mouse stria vascularis capillary basement membrane as
well54. In no previous study in human or mouse, however, there are adequate evaluation of the VECs in the BLB of
the peripheral vestibular system to clearly document whether or not there is increase in vesicles or breakdown of
tight junctions or other changes. In a recent MRI study, it was noted that the permeability of the capillaries in the
inner ear of Meniere’s disease patients was much significantly greater than that in sudden sensorineural hearing Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 7 www.nature.com/scientificreports/ Figure 8. Diagram to represent the BLB in (a) normal capillary and (b) Meniere’s disease capillary. In Meniere’s
disease, there are excessive vesicular formation (ves), abluminally concentrated, with degenerative changes
noted early on in the endothelial cell (vec). The perivascular basement membrane (pbm) is thickened and
edematous. Tight junctions (tj) are relatively preserved. Pericytes (p) exhibit vacuolization, and pericyte process
detachment. Figure 8. Diagram to represent the BLB in (a) normal capillary and (b) Meniere’s disease capillary. In Meniere’s
disease, there are excessive vesicular formation (ves), abluminally concentrated, with degenerative changes
noted early on in the endothelial cell (vec). The perivascular basement membrane (pbm) is thickened and
edematous. Tight junctions (tj) are relatively preserved. Pericytes (p) exhibit vacuolization, and pericyte process
detachment. Specimen
Type of
Tissue
Age
(years)
Gender
Diagnosis
Figure
1
Autopsy
62
Male
Normal
1a,b
2
Autopsy
86
Female
Normal
2
3
Surgical
39
Male
Meniere’s
3a,b
4
Surgical
82
Male
Meniere’s
4a,b
5
Surgical
40
Female
Meniere’s
5
6
Surgical
56
Male
Meniere’s
6
7
Surgical
67
Female
Meniere’s
7
Table 1. Specimens (utricle) used in the present study. Autopsy: Temporal bones collected between 6 to 8 hrs. postmortem, Normal: indicates normal vestibular and auditory function. Materials and Methodsh Specimens. The Institutional Review Board (IRB) of UCLA approved this study (IRB protocol # 10-001449). All methods used in this study were in accordance with NIH and IRB guidelines and regulations. Appropriate
informed consent was obtained from each patient before inclusion in the study. Archival temporal bones were
used in the present study. The temporal bone donors were part of a National Institute of Health (NIH) funded
Human Temporal Bone Consortium for Research Resource Enhancement through the National Institute on
Deafness and Other Communication Disorders (NIDCD). The medical history for each of the patients who
had donated their temporal bones was maintained and preserved in a secured electronic database. Two maculae
utricle from 2 individuals with no vestibular or auditory disease were used (62-year-old male, and 82-year-old
female). Five maculae utricle from 5 patients with intractable vertigo spells or Tumarkin falls (vestibular induced
involuntary drop attacks) in Meniere’s disease were studied (Table 1). References Resident macrophages in the cochlear blood-labyrinth barrier and their renewal via migration of bone-marrow-derived cells. Cell Tissue Res 342, 21–30 (2010). 3. Suzuki, M. & Kaga, K. Development of blood-labyrinth barrier in the semicircular canal ampulla of the rat. Hear Res. 129, 27–34
(1999).hl (
)
4. Dengerink, H. A., Wright, J. W. Circulation of the inner ear: III. The physiology of cochlear blood flow: implications for treatment
In Physiology of the Ear. Jahn AF, and Santos-Sacchi J, editors, Raven Press, New York, pp 327–340 (1988). 15. Fetoni, A. R., Picciotti, P. M., Paludetti, G. & Troiani, D. Pathogenesis of presbycusis in animal models: a review. Exp Gerontol. 46,
413–425 (2011).i 16. Yang, Y. et al. Na+/K+-ATPase a1 identified as an abundant protein in the blood-labyrinth barrier that plays an essential role
barrier integrity. PloS One. 6, e16547 (2011). g
y
(
)
17. Yamakawa, K. Hearing organ of a patient who showed Meniere’s symptoms. J Otolaryngol Soc Jpn. 44, 2310–2312 (1938). ll k
b
h
h l
f
’ d
(
f
l
)
d 18. Hallpike, C. S. & Cairns, H. Observations on the pathology of Meniere’s disease: (section of otology). Proc R Soc Med 31, 131
(1938). 19. Merchant, S. N., Adams, J. C. & Nadol, J. B. Jr. Pathophysiology of Meniere’s syndrome: are symptoms caused by endolymphatic
hydrops? Otol Neurotol. 26, 74–81 (2005).h y
p
0. Semaan, M. T., Alagramam, K. N. & Megerian, C. A. The basic science of Meniere’s disease and endolymphatic hydrops. Curr Opin
Otolaryngol Head and Neck Surgery. 13, 301–7 (2005). y g
g y
1. Semaan, M. T. & Megerian, C. A. Contemporary perspectives on the pathophysiology of Meniere’s disease: implications fo
treatment. Curr Opin Otolaryngol Head and Neck Surgery. 18, 393–398 (2010). p
y g
g y
(
)
2. Sepahdari, A. R. et al. Delayed intravenous contrast-enhanced 3D FLAIR MRI in Meniere’s disease: correlation of quantitative
measures of endolymphatic hydrops with hearing. Clinical Imaging 39, 26–31 (2015).l y
p
y
p
g
g g
3. Miller, J. M., Ren, T. Y., Laurikainen, E., Golding-Wood, D. & Nuttal, A. L. Hydrops-induced changes in cochlear blood flow. Ann
Otol Rhinol Laryngol. 104, 476–83 (1995).l y g
4. Selmani, Z., Pyykko, I., Ishaki, H. & Marttila, T. I. Cochlear blood flow measurement in patients with Meniere’s disease and other
inner ear disorders. Acta Otolaryngol Supp. 545, 10–30 (2001).h y g
pp
5. Staging of Meniere’s disease.
d fi
d b
h A
A
d Staging of Meniere’s disease. All subjects with Meniere’s disease had stage IV definite Meniere’s disease as
defined by the American Academy of Otolaryngology- Head and Neck Surgery criteria56 with profound hearing
loss and intractable recurrent vertigo spells despite maximum medical treatment. Patients who had previously
undergone intratympanic gentamicin or endolymphatic shunt surgery were excluded. 8 Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 www.nature.com/scientificreports/ Transmission electron Microscopy. For ultrastructural studies, the specimens are immersed in the fol-
lowing solutions: 2% OsO4 and 2% potassium ferricyanide (EMS, Fort Washington, PA), 0.1% thiocarbohy-
drazide for 1 hr, 2% OsO4 for 30 minutes, uranyl acetate 1% overnight, and 0.1% lead aspartate for 30 minutes
(5 × 3 min washes with double distilled water are made between steps). Tissue was dehydrated in ascending
ethyl alcohols and embedded in resin (Epon®, EMS). Half-micrometer-thin serial sections are made with a dia-
mond knife (Diatome) on an AO/Reichter Ultracut-E ultramicrotome. When the area of interest was visible,
ultrathin 100 nm serial sections were made, and mounted on 200 mesh formvar and carbon coated copper grids
(Polysciences). TEM image acquisition. TEM observations and digital image capture were made using a FEI Tecnai trans-
mission electron microscope T20 TEM −200 KV (Hillsboro, Oregon USA). Morphological analysis was made in
ultrathin sections containing blood vessels through the stroma of the maculae utricle. All sections are systemati-
cally analyzed at low (x 3,500-5000) and higher magnification (x 19,000-25,000). All sections were studied for the
presence of vesicles and tight junctions in the endothelial cells, pericyte cytoplasmic organization, and perivascu-
lar basement membrane alterations (thickening and disruption). References 1. Juhn, S. K., Ryback, L. P. & Prado, S. Nature of blood-labyrinth barrier in experimental conditions. Ann Otol Rhinol Laryngo
135–52 (1981). 2. Juhn, S. K., Rybak, L. P. & Fowlks, W. L. Transport characteristics of blood – perilymph barrier. Am J Otolaryn
3 Shi X Ph
i
h l
f h
hl
i
i
l i
H
i
R
282 10 24 (2011) 2. Juhn, S. K., Rybak, L. P. & Fowlks, W. L. Transport characteristics of blood – perilymph barrier. Am J Otolaryngol. 3, 392–339 (1
3. Shi, X. Physiopathology of the cochlear microcirculation. Hearing Res. 282, 10–24 (2011).l 2. Juhn, S. K., Rybak, L. P. & Fowlks, W. L. Transport characteristics of blood perilymph barrier. Am J Otolaryngol. 3, 392 339 (1982). 3. Shi, X. Physiopathology of the cochlear microcirculation. Hearing Res. 282, 10–24 (2011).l 3. Shi, X. Physiopathology of the cochlear microcirculation. Hearing Res. 282, 10–24 (2011).l y
p
gy
g
4. Juhn, S. K., Hunter, B. A. & Odland, R. M. Blood-labyrinth barrier and fluid dynamics of the inner ear. Int Tinnitus Journal. 7, 72–83
(2001).h 5. Salt, A. N., Melichar, I. & Thalmann, R. Mechanisms of endocochlear potential generation by stria vascularis. Laryngoscope. 97,
984–991 (1987). 6. Sakagami, M., Matunaga, T. & Hashimoto, P. H. Fine structure and permeability of capillaries in the stria vascularis and spiral
ligament of the inner ear of the guinea pig. Cell Tissue Research. 226, 511–522 (1982). g
g
p g
(
)
7. Neng, L., Zhang, F., Kachelmeier, A. & Shi, X. Endothelial cell, pericytes, and perivascular macrophage-type melanocyte interactions
regulate cochlear instrastrial fluid-blood barrier permeability. JARO. 14, 175–85 (2013). gl
y
8. Neng, L. et al. Isolation and culture of endothelial cells, pericytes and perivascular resident macrophage-like melanocytes from the
young mouse ear. Nat Protocols. 8, 709–20 (2013). 9. Shi, X. Cochlear pericytes responses to acoustic trauma and the involvement of hypoxia-inducible factor-1alpha and vascular
endothelial growth factor. Am J Pathol. 174, 1692–1704 (2009). g
0. Zhang, W. et al. Perivascular-resident macrophage-like melanocytes in the inner ear are essential for the integrity of the instrastria
fluid-blood barrier. PNAS USA 109, 10388–93 (2012).l l
11. Zhang, F., Zhang, J., Neng, L. & Shi, X. Characterization and inflammatory response of perivascular resident macrophage-like
melanocytes in the vestibular system. JARO. 14, 635–43 (2013). y
y
12. Shi, X. Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 33. Tsukita, S., Furuse, M. & Itoh, M. Molecular dissection of tight junctions. Cell Struct Function 21, 381–5 (1996).h g
j
34. Jahnke, K. The blood-perilymph barrier. Arch Otorhinolaryngol. 228, 29–34 (1980). h
p
y
p
y g
5. Sepahdari, A. R., Vorasubin, N., Ishiyama, G. & Ishiyama, A. Endolymphatic hydrops reversal following acetazolamide therapy
demonstrated with delayed intravenous contrast-enhanced 3D-FLAIR MRI. AJNR Am J Neuroradiol. 37, 151–154 (2016). y
36. Yamazaki, M., Naganawa, M., Tagaya, M., Kawai, H., Ikeda, M., Sone, M., Teranishi, M., Suzuki, H. & Nakashima, T. Comp
contrast effect on the cochlear perilymph after intratympanic and intravenous gadolinium injection. AJNR 33, 773–778 (2 y
6. Yamazaki, M., Naganawa, M., Tagaya, M., Kawai, H., Ikeda, M., Sone, M., Teranishi, M., Suzuki, H. & Nakashima, T. Comparison o 36. Yamazaki, M., Naganawa, M., Tagaya, M., Kawai, H., Ikeda, M., Sone, M., Teranishi, M., Suzuki, H. & Nakashima, T. Comparison of
contrast effect on the cochlear perilymph after intratympanic and intravenous gadolinium injection. AJNR 33, 773–778 (2012). T
k
T
h
d l
h
h d
d bl
d l b
h
b
’ d ft
7. Tagaya, M., Yamazaki, M. & Teranishi, M. Endolymphatic hydrops and blood-labyrinthine barrier in Meniere’s disease. Acta
Otolaryngol. 131, 474–479 (2011). y g
38. Barath, K., Schuknecht, B., Naldi, A. M., Schrepter, T., Bockish, C. J. & Hegemann, S. C. Detection and grading of endolymphatic
hydrops in Meniere’s disease using MR imaging. AJNR Am J Neuroradiol. 35, 1387–92 (2014). 9. Ishiyama, G., Lopez, I. A., Sepahdari, A. R. & Ishiyama, A. Meniere’s disease: histopathology, cytochemistry, and imaging. Ann NY
Acad Sci. 1343, 49–57 (2015). 0. Pakdaman, M. N. et al. Blood-labyrinth barrier permeability in Meniere’s disease and idiopathic sudden sensorineural hearing loss
findings on delayed post-contrast 3D-FLAIR MRI. AJNR AM J Neuroradiol. doi:10.3174/ajnr.A4822 (2016).l i
g
y
j
41. LeFloch’h, J. et al. Markers of cochlear inflammation using MRI. J Mag Reson Imaging. 39, 150–161 (2014). l
2. Counter, S. A. et al. Magnetic resonance imaging of membranous labyrinth during in vivo gadolinium uptake in the normal and
lesioned cochlea. Neuroreport. 11, 3979–3983 (2000). p
3. Hirose, K., Hartsock, J. J., Johnson, S., Santi, P. & Salt, A. N. Systemic lipopolysaccharide compromises the blood-labyrinth barrier
and increases entry of serum fluorescein into the perilymph. JARO. 15, 707–719 (2014).hl l
4. Hirose, K., Li, S. Z., Harsock, J., Salt, A. www.nature.com/scientificreports/ The blood perilymph barrier is vulnerable to systemic inflammation but is minimally
affected by cochlear macrophages. 7th International Symposium on Meniere’s disease and Inner Ear Disorders. Abst p13. (2015).f f
y
p
g
y
p
p
45. Nag, S., Manias, J. L., Kapadia, A., Stewart, D. J. Molecular changes associated with the protective effects of angiopoietin-1 du
blood-brain barrier breakdown post-injury. Mol Neurobiol., doi:10.1007/s12035-016-9973-4 (2016). j
y
46. Calzada, A. P., Lopez, I. A., Beltran Parrazal, L., Ishiyama, A. & Ishiyama, G. Cochlin expression in vestibular endorgans obtained
from patients with Meniere’s disease. Cell Tissue Res. 350, 373–84 (2012).h 47. Foster, C. A. & Breeze, R. E. The Meniere’s attack: an ischemia/reperfusion disorder of inner ear sensory tissues. Med Hypotheses. 81
1108–1115 (2013). (
)
48. Nuttall, A. L. Sound-induced cochlear ischemia/hypoxia as a mechanism of hearing loss. Noise Health. 2, 17–32 (1999). yp
g
49. Calabrese, V. et al. Oxidative stress, redox homeostasis and cellular stress response in Meniere’s disease: role of vitagenes. Neurochemical Res. 35, 2208–2217 (2010). 50. Trune, D. R. & Nguyen-Huynh, A. Vascular pathophysiology in hearing disorders. Seminars in Hearing 33, 242–250 (2012). 51. Calzada, A. P., Balaker, A. E., Ishiyama, G., Lopez, I. A. & Ishiyama, A. Temporal bone histopathology and immunoglo 50. Trune, D. R. & Nguyen-Huynh, A. Vascular pathophysiology in hearing disorders. Seminars in Hearing 33, 242–250 (2012). 51. Calzada, A. P., Balaker, A. E., Ishiyama, G., Lopez, I. A. & Ishiyama, A. Temporal bone histopathology and immunoglobulin
deposition in Sjogren’s syndrome. Otol Neurotol. 33, 258–266 (2012). p
j g
y
52. Ruckenstein, M. J., Keithley, E. M., Bennett, T., Powell, H. C., Baird, S. & Harris, J. P. Ultrastructural pathology in the stria vascu
of the MRL-Faslpr mouse. Hearing Res. 131, 22–28 (1999). g
3. Lin, D. W. & Trune, D. R. Breakdown of stria vascularis blood – labyrinthine barrier in C3H/lpr autoimmune disease mice
Otolaryngol Head Neck Surg. 117, 530–534 (1997). y g
g
4. Neng, L., Zhang, L., Yang, F., Lopez, I. A., Dong, M. & Shi, X. Structural changes in the strial blood-labyrinth barrier of aged
C57BL/6 mice. Cell Tissue Res. 361, 685–96 (2015). 5. Pakdaman, M. N., Ishiyama, G., Ishiyama, A., Peng, K. A., Kim, H. J., Pope, W. B., Sepahadari, A. R. Blood-Labyrinth barrie
permeability in Meniere’s disease and idiopathic sudden sensorineural hearing loss: findings on delayed postcontrast 2DFLAIR
MRI., doi:3174/ajnr.A48822.i j
56. Goebel, J. A. Author Contributions G.I., I.A.L., and A.I. conceived the project. G.I., I.A.L., and A.I. wrote the manuscript. I.A.L. and P.I. performed
transmission electron microscopy tissue processing and image collection. I.A.L., G.I., H.V.V. performed image
interpretation and analysis. www.nature.com/scientificreports/ 2015 Equilibrium committee amendment to the 1995 AAO-HNS guidelines for the definition of Meniere’s dis
Otolaryngol H&N Surg. 154, 403–404 (2016). Acknowledgements g
Hearing Health Foundation, grant # 20164266 to Gail Ishiyama. g
Hearing Health Foundation, grant # 20164266 to Gail Ishiyama. References Tange, R. & Hodde, K. C. The microvasculature of the cochlea and the vestibular system as seen in scanning electron microscopy
Clin Otolaryngol. 9, 306 (1984). y g
,
(
)
6. McCall, A., Ishiyama, G., Lopez, I. A., Sunita, B. & Ishiyama, A. Histopathological and ultrastructural analysis of vestibula
endorgans obtained from patients with Meniere’s disease. BMC Ear Nose Throat Disorders. 9, 4 (2009). h
27. Iurato, S. Blood supply, light microscopic feature. CH III, In Iurato, S. Ed. Submicroscopic structure of the inner ear. Pergamon Press,
London, First Ed, pp 18–28 (1976).i pp
8. Wersall, J., Densert, O., Per-Gotthard, L. Studies on fine structure of inner ear vessels. In Darin de Lorenzo, A. J., Editor. University
Park Press, Baltimore, London, Tokyo. pp 43–73 (1973). y
pp
9. Ishiyama, A., Mowry, S. E., Lopez, I. A. & Ishiyama, G. Immunohistochemical distribution of basement membrane proteins in the
human inner ear from older subjects. Hearing Res. 254, 1–14 (2009). j
g
30. Suzuki, M., Kitamura, K. & Nomura, Y. Anionic sites of the basement membrane of the labyrinth. Acta Otolaryngol Suppl. 481,
112–5 (1991). 31. Nag, S., Robertson, D. M. & Dinsdale, H. B. Quantitative estimate of pinocytosis in experimental acute hypertension. Acta
Neuropathol. 46, 107–16 (1979). p
,
(
)
2. Stokum, J. A., Gerzanich, V. & Simard, J. M. Molecular pathophysiology of cerebral edema. J Cerebral Blood Flow & Metabolism. 36
513–538 (2016). Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 9 Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 Additional Informationh Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an
institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 10
|
https://openalex.org/W2056413661
|
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0000655&type=printable
|
English
| null |
Validation of the SCID-hu Thy/Liv Mouse Model with Four Classes of Licensed Antiretrovirals
|
PloS one
| 2,007
|
cc-by
| 9,818
|
Validation of the SCID-hu Thy/Liv Mouse Model with
Four Classes of Licensed Antiretrovirals doi:10.1371/journal.pone.0000655 itation: Stoddart CA, Bales CA, Bare JC, Chkhenkeli G, Galkina SA, et al (2007) Validation of the SCID-hu Thy/Liv Mouse
icensed Antiretrovirals. PLoS ONE 2(8): e655. doi:10.1371/journal.pone.0000655 CA, Bare JC, Chkhenkeli G, Galkina SA, et al (2007) Validation of the SCID-hu Thy/Liv Mouse Model with Four Classes o
S ONE 2(8): e655. doi:10.1371/journal.pone.0000655 PLoS ONE | www.plosone.org INTRODUCTION Academic Editor: Linqi Zhang, AIDS Research Center, Chinese Academy of
Medical Sciences and Peking Union Medical College, China Academic Editor: Linqi Zhang, AIDS Research Center, Chinese Academy of
Medical Sciences and Peking Union Medical College, China Received April 18, 2007; Accepted June 20, 2007; Published August 1, 2007 Copyright: 2007 Stoddart 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 project has been funded in whole or in part with Federal funds
from NIAID, NIH, under contract no. N01-AI-05418. Competing Interests: The authors have declared that no competing interests
exist. Academic Editor: Linqi Zhang, AIDS Research Center, Chinese Academy of
Medical Sciences and Peking Union Medical College, China Validation of the SCID-hu Thy/Liv Mouse Model with
Four Classes of Licensed Antiretrovirals Cheryl A. Stoddart1¤*, Cheryl A. Bales1¤, Jennifer C. Bare1, George Chkhenkeli1¤, Sofiya A. Galkina1¤, April N. Kinkade1, Mary E. Moreno1¤, Jose´ M
Rivera1¤, Rollie E. Ronquillo1¤, Barbara Sloan1¤, Paul L. Black2 1 Gladstone Institute of Virology and Immunology, University of California at San Francisco, San Francisco, California, United States of America,
2 Targeted Interventions Branch, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
Maryland, United States of America Background. The SCID-hu Thy/Liv mouse model of HIV-1 infection is a useful platform for the preclinical evaluation of antiviral
efficacy in vivo. We performed this study to validate the model with representatives of all four classes of licensed
antiretrovirals. Methodology/Principal Findings. Endpoint analyses for quantification of Thy/Liv implant viral load included
ELISA for cell-associated p24, branched DNA assay for HIV-1 RNA, and detection of infected thymocytes by intracellular
staining for Gag-p24. Antiviral protection from HIV-1-mediated thymocyte depletion was assessed by multicolor flow
cytometric analysis of thymocyte subpopulations based on surface expression of CD3, CD4, and CD8. These mice can be
productively infected with molecular clones of HIV-1 (e.g., the X4 clone NL4-3) as well as with primary R5 and R5X4 isolates. To
determine whether results in this model are concordant with those found in humans, we performed direct comparisons of two
drugs in the same class, each of which has known potency and dosing levels in humans. Here we show that second-generation
antiretrovirals were, as expected, more potent than their first-generation predecessors: emtricitabine was more potent than
lamivudine, efavirenz was more potent than nevirapine, and atazanavir was more potent than indinavir. After interspecies
pharmacodynamic scaling, the dose ranges found to inhibit viral replication in the SCID-hu Thy/Liv mouse were similar to those
used in humans. Moreover, HIV-1 replication in these mice was genetically stable; treatment of the mice with lamivudine did
not result in the M184V substitution in reverse transcriptase, and the multidrug-resistant NY index case HIV-1 retained its drug-
resistance substitutions. Conclusion. Given the fidelity of such comparisons, we conclude that this highly reproducible mouse
model is likely to predict clinical antiviral efficacy in humans. Citation: Stoddart CA, Bales CA, Bare JC, Chkhenkeli G, Galkina SA, et al (2007) Validation of the SCID-hu Thy/Liv Mouse Model with Four Classes of
Licensed Antiretrovirals. PLoS ONE 2(8): e655. INTRODUCTION occurs with both molecular clones and clinical isolates of HIV-1 in
3–5 weeks [12–16]. This depletion includes loss of CD4+CD8+
(double-positive, DP) immature cortical thymocytes and a decrease
in the CD4/CD8 ratio in the thymic medulla. There is evidence
for both indirect, apoptotic destruction of uninfected thymocytes
and
direct
infection
and
destruction
of
CD32CD4+CD82
intrathymic T-progenitor cells, which severely disrupts thymocyte
maturation [17,18]. Infection of human thymus with HIV-1
induces IFN-a secretion from plasmacytoid dendritic cells, which
upregulates MHC class I (MHC-I) expression on DP thymocytes,
where expression is normally low [19]. The SCID-hu mouse model, in which human lymphoid organs are
implanted into severe-combined immunodeficient (SCID) mice, was
designed by McCune et al. [1] to provide a small animal model for
the study of human hematopoiesis. It has also facilitated study of the
pathogenesis of HIV-1 in human hematolymphoid organs [2–7] and
evaluation of anti-HIV-1 compounds in vivo [2,8,9]. In this model,
SCID mice are implanted with a variety of human fetal organs,
including bone, liver, thymus, lymph node, and spleen. The fetal
implants become tolerant of the mouse environment, and re-
ciprocally, growth of the human tissue is permitted by the
immunocompromised status of the recipient SCID mouse [6]. The administration of nucleoside and nonnucleoside reverse
transcriptase (RT) inhibitors to these mice results in dose-dependent The SCID-hu Thy/Liv mouse, first reported by Namikawa et
al. in 1990 [10], is generated by coimplanting human fetal thymus
and liver beneath the mouse kidney capsule. In a highly
reproducible manner, these organs fuse, become vascularized,
and grow into a stable organ termed ‘‘Thy/Liv,’’ reaching a total
mass of 100–3006106 human cells in 18 weeks. The Thy/Liv
implant reproduces the differentiation, proliferation, and function
of human hematopoietic progenitor cells derived from the fetal
liver within the human thymus. The implants possess histologically
normal cortical and medullary compartments that sustain multi-
lineage human hematopoiesis for 6–12 months [10,11], generating
a continuous source of CD4-expressing thymocytes that can serve
as target cells for HIV-1 infection and replication. Importantly for
a model of antiviral chemotherapy, 50–60 SCID-hu Thy/Liv mice
can be made with tissues from a single fetal donor, and the Thy/
Liv implant is amenable to experimental manipulation and
infection with HIV-1. Viruses The following reagents were obtained through the AIDS Research
and Reference Reagent Program, Division of AIDS, NIAID, NIH:
pNL4-3 [38] from Malcolm Martin and HIV-1 Ba-L [39] from
Suzanne Gartner, Mikulas Popovic, and Robert Gallo. A working
stock of NL4-3 was prepared in 293T cells by calcium phosphate
transfection. A T-20-sensitive NL4-3 (NL4-3 D36G) was altered
by site-directed mutagenesis to match the consensus sequence at
amino acid position 36 (aspartic acid replaced by glycine) [40,41]. The R5X4 AZT-resistant clinical HIV-1 isolate JD [42] was
kindly provided by Mike McCune, and the multidrug-resistant
(MDR) R5X4 NY index case isolate [43] by Hiroshi Morhi and
Martin Markowitz. The clinical isolates were prepared and titrated
by limiting dilution in phytohemagglutin-activated peripheral
blood mononuclear cells. The SCID-hu Thy/Liv model has also been employed by us and
by others for in vivo studies of HIV-1 pathogenesis. The roles of HIV-
1 nef [24–26], other accessory genes [24], and the Rev–Rev
responsive element [27] have been explored as well as the expression
of HIV-1 coreceptors (CXCR4 and CCR5) on thymocytes [28,29]
and the effects of coreceptor usage on viral tropism and pathogenesis
[16,28,30–32]. The CXCR4-utilizing (X4) strain NL4-3 replicates
quickly and extensively in thymocytes in the cortex and medulla,
causing significant thymocyte depletion [16]. In contrast, the CCR5-
utilizing (R5) strain Ba-L initially infects stromal cells, including
macrophages, in the thymic medulla without any obvious pathologic
consequences for 3–4 weeks, after which the infection slowly spreads
through the thymocyte populations and results in slowly progressing
thymocyte depletion after 6 weeks. In addition, three R5 viruses
isolated from rapid AIDS progressors lacking X4 strains were much
less pathogenic than NL4-3 in SCID-hu Thy/Liv mice, suggesting
that R5 virus-mediated rapid disease progression is associated with
host, not viral, factors [30]. The lab-adapted LAI/IIIB isolate and its
associated infectious molecular clones (e.g., HXB2) fail to replicate in
Thy/Liv implants, and this impairment has been mapped to
a specific amino acid change in the crown region of V3 in Env
[33,34]. The model has also been used to study immune
reconstitution and recovery of hematopoietic colony-forming activity
in HIV-1-infected implants after treatment of mice with potent
combination antiretroviral therapy [35–37]. Antiretroviral drugs and mouse dosing The antiretroviral drugs lamivudine (3TC), emtricitabine [(–)-
FTC], nevirapine, efavirenz, indinavir, and atazanavir were kindly
supplied by Opendra Sharma and the AIDS Reagent Program. Dosing solutions were prepared in sterile water for injection or
Dulbecco’s phosphate-buffered saline, and groups of 5–8 mice
were treated by oral gavage (200 ml per dose) with an 18-gauge62-
inch feeding needle twice daily at 7:00 AM and 6:00 PM for the
duration of the infection period (2–6 weeks depending on the
HIV-1 isolate). Enfuvirtide (T-20) was purchased from a pharmacy,
prepared in sterile water, and administered by twice-daily
subcutaneous injection (200 ml per dose). * To whom correspondence should be addressed. E-mail: cheryl.stoddart@ucsf.
edu * To whom correspondence should be addressed. E-mail: cheryl.stoddart@ucsf. edu ¤ Current address: Division of Experimental Medicine, Department of Medicine,
University of California at San Francisco, San Francisco, California, United States of
America The Thy/Liv implants support viral replication after inocula-
tion of HIV-1 by direct injection [3], and thymocyte depletion PLoS ONE | www.plosone.org August 2007 | Issue 8 | e655 1 Validation of SCID-hu Model performed in SCID-hu Thy/Liv mouse cohorts each made with
tissues from a single donor. We observed that the more recent
drugs in each class were significantly more potent than their
predecessors. We conclude from such comparisons with licensed
antiretrovirals that this well-established model is likely to predict
clinical antiviral efficacy in humans for other members of these
classes and possibly other drug classes as well. inhibition of HIV-1 replication, prevention of IFN-a-mediated
MHC-I upregulation, and protection of CD4+ cells within the
implanted human tissue [8,9,20]. The model has been also used to
evaluate new classes of HIV-1 inhibitors, such as bicyclam [21] and
oligonucleotide [22] inhibitors of HIV-1 entry, the nucleoside analog
29-deoxy-39-oxa-49-thiocytidine (dOTC) [20], and an oxime-piper-
idine CCR5 antagonist [23]. This is the first animal model in which
the action of candidate anti-HIV-1 compounds can be tested within
the setting of an intact HIV-1-infected human target organ. MATERIALS AND METHODS We use a standardized protocol described by Rabin et al. [8] for
evaluation of antiviral agents against HIV-1 in the SCID-hu Thy/
Liv mouse model. Drugs can be administered by various routes
(oral, subcutaneous, intraperitoneal, or by mini-osmotic pump)
before or after virus inoculation and continued for 2–6 weeks. Mice are challenged with titrated inocula of various HIV-1
isolates, and endpoint analyses include quantification of viral
protein by p24 ELISA and viral RNA by branched DNA assay,
quantification and isolation of replication-competent virus by
cocultivation after limiting dilution, and assessment of the effects
on human thymocyte subpopulations by flow cytometry [8]. Implantation and inoculation of SCID-hu Thy/Liv
mice Human fetal thymus and liver were obtained through services
provided by a nonprofit organization (Advanced Bioscience
Resources, Alameda, CA) in accordance with federal, state, and
local regulations. Coimplantation of thymus and liver fragments
under the kidney capsule to create SCID-hu Thy/Liv mice and
inoculation of the Thy/Liv implants with HIV-1 was carried out
as described [8,10]. Male C.B-17 SCID (model #CB17SC-M,
homozygous, C.B-Igh-1b/IcrTac-Prkdcscid) mice were obtained at
6–8 weeks of age from Taconic (Germantown, NY), and cohorts
of 50–60 SCID-hu Thy/Liv mice were implanted with tissues
from a single donor. Implants were inoculated 18 weeks after
implantation with 50 ml of stock virus (500–2,000 TCID50) or
RPMI 1640 medium (mock infection) by direct injection after
surgical exposure of the implanted kidney of anesthetized mice. Animal protocols were approved by the UCSF Institutional
Animal Care and Use Committee. Given the potential correlations between virus-host interactions
in the Thy/Liv implant and in humans, we wanted to determine
whether the SCID-hu Thy/Liv mouse model predicts antiretro-
viral efficacy in humans for representatives of all four classes of
currently licensed drugs. Does it, for instance, yield data that
reflect the greater potency of more recently developed drugs than
their first-generation predecessors in each class? In vivo models are
far more stringent than cell culture-based assays for demonstrating
drug activity, particularly in the case of orally delivered drugs as
they must be absorbed by the gastrointestinal tract without
degradation, enter the blood and circulate with sufficient half-lives,
and penetrate solid target organs at concentrations high enough to
be potently efficacious. The greater potency of the second-
generation drugs is likely the result of a combination of greater in
vitro potency and superior pharmacokinetics. In vitro virus-cell
culture systems would likely predict greater potency, but only in
vivo studies can demonstrate sustained plasma half-life or superior
oral bioavailability. To determine such relative potency in vivo,
head-to-head comparisons of two drugs in the same class were PLoS ONE | www.plosone.org Viral load and prophylactic antiviral efficacy are
highly reproducible among mouse cohorts each
made with tissues from a single donor g
The levels of HIV-1 RNA and p24 in the Thy/Liv implants of
untreated- and 3TC-treated SCID-hu Thy/Liv mice were highly
reproducible among seven different mouse cohorts infected with
the same stock of HIV-1 NL4-3 (Figure 1). In seven consecutive
experiments, with mice in a given cohort constructed from human
fetal tissue from a single donor, mean cell-associated HIV-1 RNA
ranged from 5.2–6.3 log10 copies per 106 cells (Figure 1A), and
mean p24 ranged from 640–1,700 pg per 106 cells (Figure 1B). There was also good correspondence between viral RNA and p24 p
g
p
g
p
p
Figure 1. Implant viral loads in untreated- and 3TC-treated SCID-hu Thy/Liv mice are highly reproducible, and 3TC treatment after HIV-1
inoculation is nearly as effective as prophylactic treatment. A, HIV-1 RNA in Thy/Liv implants of untreated mice and mice treated by twice-daily oral
gavage with 3TC at 30 mg/kg per day beginning 1 day before inoculation with NL4-3 (means6SEM) Each experiment was performed in a mouse
cohort made with tissues from a single donor. B, Implant p24 for the same groups as in panel A. C, Implant HIV-1 RNA in mice treated with 3TC
beginning on day 21, day +1, day +3, and day +7 with respect to inoculation with the indicated HIV-1 strains. D, Implant p24 for the same groups as
in panel C. Implants were collected 21 days after inoculation for NL4-3, 14 days for JD, and 42 days for Ba-L. *p#0.05, compared with untreated mice
for 5–7 mice per group. doi:10.1371/journal.pone.0000655.g001 Figure 1. Implant viral loads in untreated- and 3TC-treated SCID-hu Thy/Liv mice are highly reproducible, and 3TC treatment after HIV-1
inoculation is nearly as effective as prophylactic treatment. A, HIV-1 RNA in Thy/Liv implants of untreated mice and mice treated by twice-daily oral
gavage with 3TC at 30 mg/kg per day beginning 1 day before inoculation with NL4-3 (means6SEM) Each experiment was performed in a mouse
cohort made with tissues from a single donor. B, Implant p24 for the same groups as in panel A. C, Implant HIV-1 RNA in mice treated with 3TC
beginning on day 21, day +1, day +3, and day +7 with respect to inoculation with the indicated HIV-1 strains. D, Implant p24 for the same groups as
in panel C. Implants were collected 21 days after inoculation for NL4-3, 14 days for JD, and 42 days for Ba-L. Implant collection and viral load quantification Implant collection and viral load quantification
The Thy/Liv implants were collected from euthanized mice, and
single-cell suspensions were prepared by dispersing the implant
through nylon mesh and processed for p24 ELISA, bDNA assay,
and FACS analysis as described [8,20]. Unless specified otherwise, PLoS ONE | www.ploso August 2007 | Issue 8 | e655 PLoS ONE | www.plosone.org 2 Validation of SCID-hu Model implants were collected 21 days after inoculation with NL4-3,
14 days after JD inoculation, and 42 days after Ba-L inoculation. Statistical analysis Results are expressed as the mean6SEM for each mouse group. Nonparametric statistical analyses were performed by use of the
Mann-Whitney U test. Data for mice in each group were
compared to those for untreated infected mice, and p values
#0.05 were considered statistically significant. Flow cytometry Implant cells were stained with phycoerythrin cyanine dye CY7-
conjugated anti-CD4 (BD Biosciences, San Jose, CA), phycoerythrin
cyanine CY5.5-conjugated anti-CD8 (Caltag Laboratories, Burlin-
game, CA), allophycocyanin cyanine CY7-conjugated anti-CD3
(eBiosciences, San Diego, CA), and phycoerythrin-conjugated anti-
W6/32 (DakoCytomation, Glostrup, Denmark. Cells were fixed and
permeabilized with 1.2% paraformaldehyde and 0.5% Tween 20,
stained with fluorescein isothiocyanate-conjugated anti-p24 (Beck-
man Coulter, Fullerton, CA), and analyzed on an LSR II (BD
Biosciences). After collecting 100,000 total cell events, percentages of
marker-positive (CD4+, CD8+, and DP) thymocytes in the implant
samples were determined by first gating on a live lymphoid cell
population identified by forward- and side-scatter characteristics and
then by CD3 expression. W6/32-positive mean fluorescence
intensity (MFI) of DP thymocytes was determined, and CD4/CD8
ratios were calculated by dividing the percentage of CD4+ cells by
the percentage of CD8+ cells for each individual implant. Head-to-head comparisons of antiretroviral drugs at
multiple dosage levels show relative potency p
g
p
y
In the course of preclinical antiviral drug development, it is often
important to determine whether a newly discovered agent is more
potent than an existing, perhaps licensed, drug in the same class. To
that end, we produced cohorts of SCID-hu Thy/Liv mice that were
large enough for head-to-head comparisons of two drugs at multiple
(typically three) dosage levels against one virus isolate. When the
nucleoside analogs (–)-FTC and 3TC were compared, (–)-FTC was
more potent at the same dosage levels, with greater reductions in
viral RNA at 30 mg/kg per day (p= 0.035) (Figure 4A) and greater
reductions in p24 at 10 mg/kg per day (p= 0.025) and 30 mg/kg
per day (p = 0.009) (Figure 4B). The p24 levels in mice treated with (–
)-FTC at 30 mg/kg per day were 1% that of mice treated with 3TC
at the same dose (1,300 pg per 106 cells in untreated mice versus
1.2 pg per 106 cells for (–)-FTC and 110 pg per 106 cells for 3TC)
(Figure 4B). Good protection from thymocyte depletion was
observed at all three dosage levels for both drugs (Figure 4C). Viral load and prophylactic antiviral efficacy are
highly reproducible among mouse cohorts each
made with tissues from a single donor In the seven experiments, 3TC treatment beginning 1 day
before virus inoculation reduced HIV-1 RNA in the implants by
1.2–2.1 log10 (94%–99%) (Figure 1A) and p24 by 77%–92%
(Figure 1B) compared to untreated mice in the same experiment. Importantly, HIV-1 NL4-3 replication in these mice was
genetically stable over the 21-day infection period; there was no
evidence of the RT M184V substitution in viral RNA amplified by
RT-PCR and sequenced from 32 of 32 Thy/Liv implants
collected from mice treated with 3TC in these seven experiments
(data not shown). The lack of resistance development is most likely
the result of the limited time before virus-mediated thymocyte
depletion becomes severe enough to limit the number of target
cells available for infection. We view the genetic stability of the
viruses over several weeks as an asset because our results are not
confounded by the rapid development of antiviral resistance,
particularly against drugs for which only one amino acid
substitution is sufficient for high-level resistance. Viral load and prophylactic antiviral efficacy are
highly reproducible among mouse cohorts each
made with tissues from a single donor *p#0.05, compared with untreated mice
for 5–7 mice per group. doi:10.1371/journal.pone.0000655.g001 August 2007 | Issue 8 | e655 PLoS ONE | www.plosone.org 3 Figure 2. 3TC treatment of HIV-1 JD-infected SCID-hu Thy/Liv mice inhibits viral replication and protects implants from virus-mediated
thymocyte depletion. Mice were treated by twice-daily oral gavage with 3TC at 300 mg/kg per day beginning on day +1 after inoculation, and Thy/
Liv implants were collected 14, 21, and 28 days after inoculation. Antiviral efficacy was assessed by determining HIV-1 RNA (A), p24 (B), Gag-p24+
thymocytes (C), and MHC-I expression on DP thymocytes (D). Thymocyte protection was assessed by total implant cellularity (E), thymocyte viability
Validation of SCID-hu Model Validation of SCID-hu Model Figure 2. 3TC treatment of HIV-1 JD-infected SCID-hu Thy/Liv mice inhibits viral replication and protects implants from virus-mediated
thymocyte depletion. Mice were treated by twice-daily oral gavage with 3TC at 300 mg/kg per day beginning on day +1 after inoculation, and Thy/
Liv implants were collected 14, 21, and 28 days after inoculation. Antiviral efficacy was assessed by determining HIV-1 RNA (A), p24 (B), Gag-p24+
thymocytes (C), and MHC-I expression on DP thymocytes (D). Thymocyte protection was assessed by total implant cellularity (E), thymocyte viability
(F), percentage of DP thymocytes (G), and CD4/CD8 ratio (H) for 3TC-treated mice versus untreated mice (means6SEM). *p#0.05, compared with
untreated mice for 5–7 mice per group. doi:10 1371/journal pone 0000655 g002 August 2007 | Issue 8 | e655 4 Validation of SCID-hu Model thymocytes (Figure 2C), and IFN-a-induced MHC-I expression on
DP thymocytes (Figure 2D), compared to untreated mice at all
three times. Reductions in viral load by 3TC were accompanied
by virtually complete protection of the Thy/Liv implants from
thymocyte depletion, in terms of total cellularity (Figure 2E),
thymocyte viability (Figure 2F), percentage of DP thymocytes
(Figure 2G), and CD4/CD8 ratio (Figure 2H). Flow cytometric
analysis of representative implants obtained 28 days after in-
oculation with HIV-1 JD showed severe depletion of DP and
CD4+ thymocytes in an untreated mouse and nearly complete
protection from thymocyte depletion in one mouse treated with
3TC (Figure 3). means of implants in the same groups (i.e., lowest for untreated
mice in experiment 6 and highest for those in experiments 3 and
5). Postexposure dosing also reduces viral load and
prevents virus-mediated thymocyte depletion Initiation of 3TC treatment 1 or 3 days after NL4-3 inoculation
was nearly as effective at reducing implant viral RNA (Figure 1C)
and p24 (Figure 1D) as was prophylactic treatment with treatment
beginning 1 day before inoculation. This was also true for mice
inoculated with the clinical R5X4 isolate JD and treated with 3TC
beginning 3 days after inoculation. Treatment of mice inoculated
with the R5 isolate Ba-L, which replicates in Thy/Liv implants
with slower kinetics than does X4 HIV-1 NL4-3 [16], was highly
effective at reducing implant viral RNA and p24 even when
initiation was delayed until 7 days after inoculation. In
another
experiment,
the
nonnucleoside
RT
inhibitor
efavirenz was directly compared with nevirapine. Efavirenz was
approximately
10
times
more
potent
than
nevirapine,
as
demonstrated by similar reductions in viral RNA (Figure 5A)
and p24 (Figure 5B) and by thymocyte protection (Figure 5C) in
groups treated with 300 mg/kg per day of nevirapine compared to
30 mg/kg per day of efavirenz. To determine whether postexposure dosing also prevents
thymocyte depletion, SCID-hu mice were inoculated with HIV-
1 JD and treated twice daily with 3TC at 300 mg/kg per day in
a time-course study in which implants were collected at weekly
intervals up to 28 days after inoculation. By that point, the
implants from untreated mice had become severely depleted of
thymocytes. As expected, 3TC-treated mice had significant
reductions in viral RNA (Figure 2A), p24 (Figure 2B), Gag-p24+ Finally, the second-generation protease inhibitor atazanavir was
3–10 times more potent than indinavir in NL4-3-infected SCID- Figure 3. 3TC treatment protects HIV-1 JD-infected Thy/Liv implants from thymocyte depletion. Flow cytometric analysis of representative
implants stained for CD4 and CD8 obtained 28 days after inoculation with HIV-1 JD shows severe depletion of DP and CD4+ thymocytes in an
untreated mouse and nearly complete protection from thymocyte depletion by 3TC treatment (300 mg/kg per day beginning on day +1 after
inoculation). doi:10.1371/journal.pone.0000655.g003 Figure 3. 3TC treatment protects HIV-1 JD-infected Thy/Liv implants from thymocyte depletion. Flow cytometric analysis of representative
implants stained for CD4 and CD8 obtained 28 days after inoculation with HIV-1 JD shows severe depletion of DP and CD4+ thymocytes in an
untreated mouse and nearly complete protection from thymocyte depletion by 3TC treatment (300 mg/kg per day beginning on day +1 after
inoculation). Figure 3. 3TC treatment protects HIV-1 JD-infected Thy/Liv implants from thymocyte depletion. Postexposure dosing also reduces viral load and
prevents virus-mediated thymocyte depletion Flow cytometric analysis of representative
implants stained for CD4 and CD8 obtained 28 days after inoculation with HIV-1 JD shows severe depletion of DP and CD4+ thymocytes in an
untreated mouse and nearly complete protection from thymocyte depletion by 3TC treatment (300 mg/kg per day beginning on day +1 after
inoculation). doi:10.1371/journal.pone.0000655.g003 doi:10.1371/journal.pone.0000655.g003 PLoS ONE | www.plosone.org August 2007 | Issue 8 | e655 PLoS ONE | www.plosone.org 5 Validation of SCID-hu Model Figure 4. (–)-FTC is more potent than 3TC against HIV-1 NL4-3 in
SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral gavage
with 3TC or (–)-FTC at 10, 30, and 100 mg/kg per day beginning on day
Figure 5. Efavirenz is more potent than nevirapine against HIV-1
NL4-3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral
gavage with nevirapine or efavirenz at the indicated dosage levels
Validation of SCID hu Model Figure 4. (–)-FTC is more potent than 3TC against HIV-1 NL4-3 in
SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral gavage
with 3TC or (–)-FTC at 10, 30, and 100 mg/kg per day beginning on day
–1. Antiviral efficacy was assessed by determining HIV-1 RNA (A) and
p24 (B), and thymocyte protection was assessed by percentage of DP
thymocytes (C) (means6SEM). *p#0.05, compared with untreated mice
for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g004
Figure 5. Efavirenz is more potent than nevirapine against HIV-1
NL4-3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral
gavage with nevirapine or efavirenz at the indicated dosage levels
beginning on day 21. Antiviral efficacy was assessed by determining
HIV-1 RNA (A) and p24 (B), and thymocyte protection was assessed by
percentage of DP thymocytes (C) (means6SEM). *p#0.05, compared
with untreated mice for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g005 Figure 4. (–)-FTC is more potent than 3TC against HIV-1 NL4-3 in
SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral gavage
with 3TC or (–)-FTC at 10, 30, and 100 mg/kg per day beginning on day
–1. Antiviral efficacy was assessed by determining HIV-1 RNA (A) and
p24 (B), and thymocyte protection was assessed by percentage of DP
thymocytes (C) (means6SEM). *p#0.05, compared with untreated mice
for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g004 Figure 5. Efavirenz is more potent than nevirapine against HIV-1
NL4-3 in SCID-hu Thy/Liv mice. Postexposure dosing also reduces viral load and
prevents virus-mediated thymocyte depletion Inhibitory drug dosage levels are comparable
between SCID-hu Thy/Liv mice and humans
The SCID-hu Thy/Liv mouse has the advantage of having
human, not mouse, cells as the targets of antiretroviral uptake and
action, but the pharmacokinetics of antiretrovirals in the mouse
have the potential to be significantly different from those in
humans. Small mammals usually eliminate drugs faster than large
Figure 6. Atazanavir is more potent than indinavir against HIV-1 NL4-
3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral
i h i di
i
i
d
/k
Figure 7. T-20 causes dose-dependent reductions in viral load in HIV-
1 NL4-3 D36G-infected SCID-hu Thy/Liv mice. Mice were treated by
twice-daily subcutaneous injection with T-20 at 10, 30, and 100 mg/kg
per day beginning on day 21. Antiviral efficacy was assessed by
determining cell-associated HIV-1 RNA and p24. Data are expressed as
means6SEM; *p#0.05 for treated mice versus untreated mice by the
Mann-Whitney U test for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g007
Validation of SCID-hu Model Figure 6. Atazanavir is more potent than indinavir against HIV-1 NL4-
3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral
gavage with indinavir or atazanavir at 100, 300, and 1000 mg/kg per
day beginning on day 21. Antiviral efficacy was assessed by
determining HIV-1 RNA (A) and p24 (B), and thymocyte protection
was assessed by percentage of DP thymocytes (C) (means6SEM). *p#0.05, compared with untreated mice for the number of mice
indicated under each bar. doi:10.1371/journal.pone.0000655.g006 Figure 7. T-20 causes dose-dependent reductions in viral load in HIV- Figure 7. T-20 causes dose-dependent reductions in viral load in HIV-
1 NL4-3 D36G-infected SCID-hu Thy/Liv mice. Mice were treated by
twice-daily subcutaneous injection with T-20 at 10, 30, and 100 mg/kg
per day beginning on day 21. Antiviral efficacy was assessed by
determining cell-associated HIV-1 RNA and p24. Data are expressed as
means6SEM; *p#0.05 for treated mice versus untreated mice by the
Mann-Whitney U test for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g007 hu mice, with a 3.1 log10 greater reduction in viral RNA
(p = 0.006) with atazanavir than with indinavir at 300 mg/kg per
day (5.3 log10 copies per 106 cells in untreated mice versus ,1.5
log10 copies per 106 cells for atazanavir and 4.6 log10 copies per
106 cells for indinavir) (Figure 6A). Postexposure dosing also reduces viral load and
prevents virus-mediated thymocyte depletion The p24 levels in mice treated
with atazanavir at 100 mg/kg per day were 5% (p = 0.029) that of
mice treated with indinavir at the same dose (400 pg p24 per 106
cells in untreated mice versus 19 pg p24 per 106 cells for
atazanavir and 380 pg p24 per 106 cells for indinavir) (Figure 6B). There was comparable protection from thymocyte depletion for
the drugs at 100 and 300 mg/kg per day (Figure 6C). Figure 6. Atazanavir is more potent than indinavir against HIV-1 NL4-
3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral
gavage with indinavir or atazanavir at 100, 300, and 1000 mg/kg per
day beginning on day 21. Antiviral efficacy was assessed by
determining HIV-1 RNA (A) and p24 (B), and thymocyte protection
was assessed by percentage of DP thymocytes (C) (means6SEM). *p#0.05, compared with untreated mice for the number of mice T-20 causes dose-dependent reductions in implant
viral load Although we did not perform this experiment as a direct
comparison with another HIV-1 fusion inhibitor, we are including
these data to complete the interspecies scaling analysis described in
the next section with a fourth class of antiretroviral (T-20). Treatment of NL4-3 D36G-infected SCID-hu Thy/Liv mice with
T-20 by twice-daily subcutaneous injection resulted in dose-
dependent reductions of both HIV-1 RNA and p24, reducing viral
RNA by 3.3 log10 copies per 106 cells and p24 to undetectable
levels at 100 and 30 mg/kg per day (Figure 7). Postexposure dosing also reduces viral load and
prevents virus-mediated thymocyte depletion Mice were treated by twice-daily oral
gavage with nevirapine or efavirenz at the indicated dosage levels
beginning on day 21. Antiviral efficacy was assessed by determining
HIV-1 RNA (A) and p24 (B), and thymocyte protection was assessed by
percentage of DP thymocytes (C) (means6SEM). *p#0.05, compared
with untreated mice for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g005 August 2007 | Issue 8 | e655 PLoS ONE | www.plosone.org 6 Figure 7. T-20 causes dose-dependent reductions in viral load in HIV-
1 NL4-3 D36G-infected SCID-hu Thy/Liv mice. Mice were treated by
twice-daily subcutaneous injection with T-20 at 10, 30, and 100 mg/kg
per day beginning on day 21. Antiviral efficacy was assessed by
determining cell-associated HIV-1 RNA and p24. Data are expressed as
means6SEM; *p#0.05 for treated mice versus untreated mice by the
Mann-Whitney U test for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g007
Validation of SCID-hu Model Validation of SCID-hu Model hu mice, with a 3.1 log10 greater reduction in viral RNA
(p = 0.006) with atazanavir than with indinavir at 300 mg/kg per
day (5.3 log10 copies per 106 cells in untreated mice versus ,1.5
log10 copies per 106 cells for atazanavir and 4.6 log10 copies per
106 cells for indinavir) (Figure 6A). The p24 levels in mice treated
with atazanavir at 100 mg/kg per day were 5% (p = 0.029) that of
mice treated with indinavir at the same dose (400 pg p24 per 106
cells in untreated mice versus 19 pg p24 per 106 cells for
atazanavir and 380 pg p24 per 106 cells for indinavir) (Figure 6B). There was comparable protection from thymocyte depletion for
the drugs at 100 and 300 mg/kg per day (Figure 6C). T-20 causes dose-dependent reductions in implant
viral load
Although we did not perform this experiment as a direct
comparison with another HIV-1 fusion inhibitor, we are including
these data to complete the interspecies scaling analysis described in
the next section with a fourth class of antiretroviral (T-20). Treatment of NL4-3 D36G-infected SCID-hu Thy/Liv mice with
T-20 by twice-daily subcutaneous injection resulted in dose-
dependent reductions of both HIV-1 RNA and p24, reducing viral
RNA by 3.3 log10 copies per 106 cells and p24 to undetectable
levels at 100 and 30 mg/kg per day (Figure 7). DISCUSSION The SCID-hu Thy/Liv mouse is an efficient model for the study of
HIV-1 infection and for the preclinical evaluation of potential
antiretrovirals. Here we show that this model provides a validated
platform for reproducible viral infectivity and drug-mediated
reductions in viral load in vivo. We also show that the model is Table 1. Antiretroviral drug dosage levels adjusted for difference in surface area-to-body weight ratio between mice and humans
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Drug
Human dosage regimena
Human dosageb
(mg/kg per day)
Mouse equivalent dosagec
(mg/kg per day)
Calculated reduction in implant
HIV-1 RNAd (log10 copies)
3TC
150 mg twice daily
5.0
62
1.9
(–)-FTC
200 mg once daily
3.3
41
2.3
nevirapine
400 mg once daily
6.7
82
0.9
efavirenz
600 mg once daily
10
123
3.7
indinavir
800 mg three times daily
40
490
1.2
atazanavir
400 mg once daily
6.7
82
1.4
T-20
90 mg twice daily
3.0
37
3.2
aFrom ref. [56]. bBased on 60 kg adult human weight and 20 g mouse weight. cHuman dosage612.3 (fold difference in surface area to body weight between mice and humans; from ref. [45,46]. dCalculated by linear regression of the viral RNA data shown in Figures 3–6 at the equivalent mouse dosage level. doi:10.1371/journal.pone.0000655.t001
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multidrug-resistant NY index case HIV-1 replicates
and depletes thymocytes with kinetics comparable
to HIV-1 NL4-3 A key test of any new class of antiretroviral drug is a demonstration
of potent activity against HIV-1 with known drug-resistance
mutations. We reported the activity of the 3TC analog dOTC
against 3TC-resistant HIV-1 NL4-3/M184V in the SCID-hu
Thy/Liv model [20]. Here we report that the MDR NY index
case HIV-1, which was isolated from a patient with highly rapid
progression to AIDS [43], replicates with kinetics similar to HIV-1
NL4-3 in SCID-hu Thy/Liv mice (Figure 8A–D) resulting in
severe
thymocyte
depletion
by
28 days
after
inoculation
(Figure 8E–H). The lower viral loads for the NY index case
isolate compared to NL4-3 at day 28 are most likely the result of
somewhat faster thymocyte depletion and thus greater loss of
target cells for viral replication. The goal of this study was to validate the SCID-hu Thy/Liv
model for use in drug discovery efforts by determining the efficacy
of different classes of FDA-approved antiretroviral drugs. Antiviral
efficacy in this model is
demonstrated by dose-dependent
reductions in viral load as well as dose-dependent protection of
CD4+ T cells from virus-mediated depletion. Assessment of
thymocyte percentage and viability is particularly important for
proving that the observed antiviral activity is not merely the result
of cytotoxicity to the Thy/Liv implant just as the selectivity index
(ratio of cytotoxic to inhibitory concentration) is important for
assessing antiviral activity in vitro. Additional evidence that HIV-1 replication is genetically stable
within the 28-day infection period was provided by sequence
analysis of implant viral RNA showing that the MDR NY index
case isolate [43] retained the full complement of drug-resistance
amino acid substitutions of the original inoculum (data not shown). These data show that this MDR clinical isolate can readily be used
as a challenge virus in the SCID-hu Thy/Liv model for in vivo
studies of new classes of antiretroviral drugs with activity against
HIV-1 that is resistant to currently licensed antiretrovirals. As new antiretroviral drugs in existing drug classes are
developed to improve upon antiviral potency or to limit in vivo
toxicity, direct comparisons of the existing drug and the new drug
in single SCID-hu mouse cohorts represent a powerful preclinical
tool for the prediction of improved efficacy in human patients. To
accomplish this, we produced cohorts of SCID-hu Thy/Liv mice
that were large enough for comparison of two drugs at three
dosage levels against one virus isolate. Inhibitory drug dosage levels are comparable
between SCID-hu Thy/Liv mice and humans This factor reflects the 12.3-fold difference in surface area-to-body
weight ratio between mice (0.0066 m2/0.02 kg) and humans
(1.6 m2/60 kg); consequently, 12.3 times more drug is required in
the mouse to be comparable to the dose in humans. When
currently approved dosage levels for humans of each antiretroviral
drug are adjusted for this difference, the adjusted dosage levels
were similar to those that cause 0.9–3.7 log10 reductions in viral
RNA in the SCID-hu Thy/Liv mouse (Table 1). Thus, not only is
the model predictive of relative potency within these drug classes,
it might also be useful in determining approximate dosing ranges
for effective use in humans. This factor reflects the 12.3-fold difference in surface area-to-body
weight ratio between mice (0.0066 m2/0.02 kg) and humans
(1.6 m2/60 kg); consequently, 12.3 times more drug is required in
the mouse to be comparable to the dose in humans. When
currently approved dosage levels for humans of each antiretroviral
drug are adjusted for this difference, the adjusted dosage levels
were similar to those that cause 0.9–3.7 log10 reductions in viral
RNA in the SCID-hu Thy/Liv mouse (Table 1). Thus, not only is
the model predictive of relative potency within these drug classes,
it might also be useful in determining approximate dosing ranges
for effective use in humans. Multidrug-resistant NY index case HIV-1 replicates
and depletes thymocytes with kinetics comparable
to HIV-1 NL4-3 For studies of viral
pathogenesis and hematopoietic reconstitution, a relatively small
number of animals can generate sufficient replicate points for
analysis. In contrast, preclinical antiviral evaluations require
groups of animals sufficiently large to demonstrate statistical proof
of efficacy in vivo. Two-drug, three-dose comparisons are made
possible by our unique ability to construct large (50–60-mouse)
SCID-hu cohorts with tissues from a single donor, which permits Inhibitory drug dosage levels are comparable
between SCID-hu Thy/Liv mice and humans The SCID-hu Thy/Liv mouse has the advantage of having
human, not mouse, cells as the targets of antiretroviral uptake and
action, but the pharmacokinetics of antiretrovirals in the mouse
have the potential to be significantly different from those in
humans. Small mammals usually eliminate drugs faster than large
mammals [44], and toxic endpoints for therapeutics administered
systemically to animals scale well between species when doses are
normalized to body surface area [45]. To determine whether the
doses found inhibitory in the SCID-hu Thy/Liv mouse bear any
relation to those used clinically in humans, an interspecies scaling
factor of 12.3 [46] was applied to generate mouse equivalent doses. Figure 6. Atazanavir is more potent than indinavir against HIV-1 NL4-
3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral
gavage with indinavir or atazanavir at 100, 300, and 1000 mg/kg per
day beginning on day 21. Antiviral efficacy was assessed by
determining HIV-1 RNA (A) and p24 (B), and thymocyte protection
was assessed by percentage of DP thymocytes (C) (means6SEM). *p#0.05, compared with untreated mice for the number of mice
indicated under each bar. doi:10 1371/journal pone 0000655 g006 August 2007 | Issue 8 | e655 7 PLoS ONE | www.plosone.org Validation of SCID-hu Model especially valuable for direct, head-to-head comparisons of the
potency and effective dose ranges of existing antiretroviral drugs. In consecutive experiments performed in different SCID-hu
mouse cohorts infected with the same stock of HIV-1 NL4-3, we
observed a range in mean HIV-1 RNA of only 1.1 log10 copies per
106 cells. Reproducible reductions in viral load were also observed
after oral administration of 3TC. Although our standardized
protocol calls for prophylactic treatment 1 day before virus
inoculation to maximize the potential for discerning antiviral
efficacy of new drugs in initial in vivo tests, we show here that
postexposure drug initiation (beginning 1, 3, or 7 days after HIV-1
inoculation) also renders potent antiviral efficacy. Importantly,
potent therapeutic efficacy, in terms of both inhibition of viral
replication and virtually complete protection from virus-mediated
cytopathicity and thymocyte depletion, was also demonstrated in
3TC-treated mice infected with the highly cytopathic R5X4
isolate, JD. In addition, we have minimized one potential
experimental variable by inoculating the mice at a uniform time
point 18 weeks after implantation. DISCUSSION . . dosage levels adjusted for difference in surface area-to-body weight ratio between mice and humans
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 1. Antiretroviral drug dosage levels adjusted for difference in surface area-to-body weight ratio be
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . August 2007 | Issue 8 | e655 PLoS ONE | www.plosone.org 8 Validation of SCID-hu Model Figure 8. Multidrug-resistant (MDR) NY index case HIV-1 replicates and depletes thymocytes with kinetics comparable to HIV-1 NL4-3 in SCID-
hu Thy/Liv mice. Viral replication assessed by determining HIV-1 RNA (A), p24 (B), Gag-p24+ thymocytes (C), and MHC-I expression on DP thymocytes
(D). Thymocyte protection was assessed by total implant cellularity (E), thymocyte viability (F), percentage of DP thymocytes (G), and CD4/CD8 ratio
(H) for NL4-3 versus MDR NY index case HIV-1-infected mice (means6SEM). *p#0.05, compared with untreated mice for 7 mice per group. doi:10.1371/journal.pone.0000655.g008
Validation of SCID-hu Model Figure 8. Multidrug-resistant (MDR) NY index case HIV-1 replicates and depletes thymocytes with kinetics comparable to HIV-1 NL4-3 in SCID-
hu Thy/Liv mice. DISCUSSION Viral replication assessed by determining HIV-1 RNA (A), p24 (B), Gag-p24+ thymocytes (C), and MHC-I expression on DP thymocytes
(D). Thymocyte protection was assessed by total implant cellularity (E), thymocyte viability (F), percentage of DP thymocytes (G), and CD4/CD8 ratio
(H) for NL4-3 versus MDR NY index case HIV-1-infected mice (means6SEM). *p#0.05, compared with untreated mice for 7 mice per group. doi:10.1371/journal.pone.0000655.g008 August 2007 | Issue 8 | e655 PLoS ONE | www.plosone.org 9 Validation of SCID-hu Model six or seven dosing groups of 5–8 mice for optimal statistical
power. Given such group sizes and appropriate controls, it is
possible to assign effective antiviral dose ranges with statistical
precision [8]. Among animal models for HIV-1, this ability is
unique: nonhuman primate models (i.e., SIV- or SHIV-infected
rhesus macaques) cannot provide dosing groups of such size and
cannot achieve the same statistical power and relatively lower cost
per animal. There are several recent reports on the infection of
humanized Rag22/2cc2/2 (RAG-hu) [47–50] and NOD/SCID
BLT mice [51] with HIV-1, but there are no reports yet published
on the evaluation of antiretroviral efficacy in these models, and it
has not yet been demonstrated that 50–60 mice can be generated
with the CD34+ hematopoietic stem cells from a single donor. In
a recent review [52], Manz states that one of the challenges these
CD34+-reconstituted mouse models face is that usually less than 10
mice can be transplanted from one human graft. once-daily dosing [55]. Taken together, these comparisons of
licensed antiretrovirals in each major class suggest that the SCID-
hu Thy/Liv model may also predict clinical antiviral efficacy in
humans. These results extend an earlier validation of the model
with the nucleoside analogs zidovudine and dideoxyinosine [8]. The predictive nature of SCID-hu mouse efficacy is further
supported by the dosage levels required to achieve log order
reductions in implant viral load when adjusted for the difference in
surface area-to-body weight ratio between mice and humans. Upon initial examination, the effective dosage level of 1,000 mg/
kg per day for indinavir in the mice seems overly high until such
an adjustment is made and the very high human daily dosage of
2.4 g (800 mg three times a day) is taken into consideration. Predictiveness of the SCID-hu Thy/Liv model for the behavior
of HIV-1 in humans can be extended beyond antiretroviral
efficacy to simple kinetics of viral replication and virus-mediated
cytopathicity. REFERENCES 1. McCune JM, Namikawa R, Kaneshima H, Shultz LD, Lieberman M, et al. (1988) The SCID-hu mouse: murine model for the analysis of human
hematolymphoid differentiation and function. Science 241: 1632–1639. 14. Stanley SK, McCune JM, Kaneshima H, Justement JS, Sullivan M, et al. (1993)
Human immunodeficiency virus infection of the human thymus and disruption
of the thymic microenvironment in the SCID-hu mouse. J Exp Med 178:
1151–1163. 2. Kaneshima H, Shih CC, Namikawa R, Rabin L, Outzen H, et al. (1991)
Human immunodeficiency virus infection of human lymph nodes in the SCID-
hu mouse. Proc Natl Acad Sci U S A 88: 4523–4527. 15. Aldrovandi GM, Feuer G, Gao L, Jamieson B, Kristeva M, et al. (1993) The
SCID-hu mouse as a model for HIV-1 infection. Nature 363: 732–736. 3. Namikawa R, Kaneshima H, Lieberman M, Weissman IL, McCune JM (1988)
Infection of the SCID-hu mouse by HIV-1. Science 242: 1684–1686. 16. Berkowitz RD, Alexander S, Bare C, Linquist-Stepps V, Bogan M, et al. (1998)
CCR5- and CXCR4-utilizing strains of human immunodeficiency virus type 1
exhibit differential tropism and pathogenesis in vivo. J Virol 72: 10108–10117. 4. Kitchen SG, Zack JA (1998) HIV type 1 infection in lymphoid tissue: natural
history and model systems. AIDS Res Hum Retroviruses 14 Suppl 3: S235–239. 17. Su L, Kaneshima H, Bonyhadi M, Salimi S, Kraft D, et al. (1995) HIV-1-
induced thymocyte depletion is associated with indirect cytopathogenicity and
infection of progenitor cells in vivo. Immunity 2: 25–36. 5. McCune JM (1996) Development and applications of the SCID-hu mouse
model. Semin Immunol 8: 187–196. 6. McCune JM (1997) Animal models of HIV-1 disease. Science 278: 2141–214 6. McCune JM (1997) Animal models of HIV-1 disease. Science 278: 2141–2142. 7. McCune J, Kaneshima H, Krowka J, Namikawa R, Outzen H, et al. (1991) The 18. Jamieson BD, Uittenbogaart CH, Schmid I, Zack JA (1997) High viral burden
and rapid CD4+ cell depletion in human immunodeficiency virus type 1-infected
SCID-hu mice suggest direct viral killing of thymocytes in vivo. J Virol 71:
8245–8253. 7. McCune J, Kaneshima H, Krowka J, Namikawa R, Outzen H, et al. (1991) The
SCID-hu mouse: a small animal model for HIV infection and pathogenesis. Annu Rev Immunol 9: 399–429. 19. Keir ME, Stoddart CA, Linquist-Stepps V, Moreno ME, McCune JM (2002)
IFN-alpha secretion by type 2 predendritic cells up-regulates MHC class I in the
HIV-1-infected thymus. ACKNOWLEDGMENTS We thank Sandra Bridges for longstanding support of the SCID-hu Thy/
Liv model, Opendra Sharma for supplying multiple-gram quantities of
antiretroviral drugs, Tim Schmidt for performing p24 ELISA, and Mike
McCune for helpful discussions. Author Contributions Conceived and designed the experiments: CS PB. Performed the
experiments: CB JB GC SG AK MM JR RR BS. Analyzed the data:
CS MM RR. Wrote the paper: CS. Conceived and designed the experiments: CS PB. Performed the
experiments: CB JB GC SG AK MM JR RR BS. Analyzed the data:
CS MM RR. Wrote the paper: CS. DISCUSSION AZT-resistant (JD) and MDR NY index case R5X4
clinical isolates replicate and cause thymocyte depletion with
kinetics comparable to HIV-1 NL4-3. The data presented here
show that drug-resistant clinical isolates can readily be used as
challenge viruses in the SCID-hu Thy/Liv model for in vivo studies
of new classes of antiretroviral drugs with activity against HIV-1
that is resistant to currently licensed antiretrovirals. In the experiments presented here, the second-generation
antiretrovirals in each drug class were more potent than their
first-generation predecessors. Thus, (–)-FTC was more potent than
3TC, the nonnucleoside RT inhibitor efavirenz was more potent
than nevirapine, and the protease inhibitor atazanavir was more
potent than indinavir. The greater potency of the newer drugs in
SCID-hu Thy/Liv mice is consistent with their observed greater
potency in HIV-1-infected individuals. The nonnucleoside RT
inhibitor (–)-FTC has greater potency than 3TC because the (–)-
FTC-triphosphate is incorporated 10 times more efficiently than
3TC-triphosphate during HIV-1 RT-catalyzed RNA-dependent
DNA synthesis [53]. The 10-fold greater potency of efavirenz
compared to nevirapine in SCID-hu mice is entirely predictive of
the superior oral bioavailability and plasma half-life characteristics
of efavirenz [54], which allow for once-daily dosing in humans as
the current standard of care compared with twice-daily dosing for
nevirapine. Finally, the greater potency of atazanavir over
indinavir in SCID-hu mice can be explained by a combination
of its greater in vitro potency and oral bioavailability, allowing Validation of SCID-hu Model receptor CCR5, is a potent inhibitor of HIV-1 infection in vitro and in vivo. Proc Natl Acad Sci U S A 98: 12718–12723. receptor CCR5, is a potent inhibitor of HIV-1 infection in vitro and in vivo. Proc Natl Acad Sci U S A 98: 12718–12723. 39. Gartner S, Markovits P, Markovitz DM, Kaplan MH, Gallo RC, et al. (1986)
The role of mononuclear phagocytes in HTLV-III/LAV infection. Science 233:
215–219. 24. Aldrovandi GM, Zack JA (1996) Replication and pathogenicity of human
immunodeficiency virus type 1 accessory gene mutants in SCID-hu mice. J Virol
70: 1505–1511. 40. Greenberg ML, Cammack N (2004) Resistance to enfuvirtide, the first HIV
fusion inhibitor. J Antimicrob Chemother 54: 333–340. 25. Aldrovandi GM, Gao L, Bristol G, Zack JA (1998) Regions of human
immunodeficiency virus type 1 nef required for function in vivo. J Virol 72:
7032–7039. J
41. Rimsky LT, Shugars DC, Matthews TJ (1998) Determinants of human
immunodeficiency virus type 1 resistance to gp41-derived inhibitory peptides. J Virol 72: 986–993. 42. Kovalev G, Duus K, Wang L, Lee R, Bonyhadi M, et al. (1999) Induction of
MHC class I expression on immature thymocytes in HIV-1-infected SCID-hu
Thy/Liv mice: evidence of indirect mechanisms. J Immunol 162: 7555–7562. 26. Stoddart CA, Geleziunas R, Ferrell S, Linquist-Stepps V, Moreno ME, et al. (2003) Human immunodeficiency virus type 1 Nef-mediated downregulation of
CD4 correlates with Nef enhancement of viral pathogenesis. J Virol 77:
2124–2133. Thy/Liv mice: evidence of indirect mechanisms. J Immunol 16 43. Markowitz M, Mohri H, Mehandru S, Shet A, Berry L, et al. (2005) Infection
with multidrug resistant, dual-tropic HIV-1 and rapid progression to AIDS:
a case report. Lancet 365: 1031–1038. 27. Valentin A, Aldrovandi G, Zolotukhin AS, Cole SW, Zack JA, et al. (1997)
Reduced viral load and lack of CD4 depletion in SCID-hu mice infected with
Rev-independent clones of human immunodeficiency virus type 1. J Virol 71:
9817–9822. p
44. Mordenti J (1986) Man versus beast: pharmacokinetic scaling in mammals. J Pharm Sci 75: 1028–1040. 28. Berkowitz RD, Beckerman KP, Schall TJ, McCune JM (1998) CXCR4 and
CCR5 expression delineates targets for HIV-1 disruption of T cell differenti-
ation. J Immunol 161: 3702–3710. 45. Freireich EJ, Gehan EA, Rall DP, Schmidt LH, Skipper HE (1966) Quantitative
comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey,
and man. Cancer Chemother Rep 50: 219–244. J
29. Validation of SCID-hu Model Kitchen SG, Zack JA (1997) CXCR4 expression during lymphopoiesis:
implications for human immunodeficiency virus type 1 infection of the thymus. J Virol 71: 6928–6934. p
46. Bast RC, Holland JF, Frei E, American Cancer Society (2000) Cancer medicine
e.5 online. 5th ed. Hamilton, Ont. AtlantaGA: B.C. Decker; American Cancer
Society. y
47. Baenziger S, Tussiwand R, Schlaepfer E, Mazzucchelli L, Heikenwalder M, et
al. (2006) Disseminated and sustained HIV infection in CD34+ cord blood cell-
transplanted Rag2-/-gamma c-/- mice. Proc Natl Acad Sci U S A 103:
15951–15956. J
30. Berkowitz RD, van’t Wout AB, Kootstra NA, Moreno ME, Linquist-Stepps VD,
et al. (1999) R5 strains of human immunodeficiency virus type 1 from rapid
progressors lacking X4 strains do not possess X4-type pathogenicity in human
thymus. J Virol 73: 7817–7822. 48. Berges BK, Wheat WH, Palmer BE, Connick E, Akkina R (2006) HIV-1
infection and CD4 T cell depletion in the humanized Rag2-/-gamma c-/-
(RAG-hu) mouse model. Retrovirology 3: 76. 31. Berkowitz RD, Alexander S, McCune JM (2000) Causal relationships between
HIV-1 coreceptor utilization, tropism, and pathogenesis in human thymus. AIDS Res Hum Retroviruses 16: 1039–1045. 32. Jamieson BD, Pang S, Aldrovandi GM, Zha J, Zack JA (1995) In vivo
pathogenic properties of two clonal human immunodeficiency virus type 1
isolates. J Virol 69: 6259–6264. 49. Gorantla S, Sneller H, Walters L, Sharp JG, Pirruccello SJ, et al. (2007) Human
immunodeficiency virus type 1 pathobiology studied in humanized BALB/c-
Rag2-/-gammac-/- mice. J Virol 81: 2700–2712. 33. Miller ED, Duus KM, Townsend M, Yi Y, Collman R, et al. (2001) Human
immunodeficiency virus type 1 IIIB selected for replication in vivo exhibits
increased envelope glycoproteins in virions without alteration in coreceptor
usage: separation of in vivo replication from macrophage tropism. J Virol 75:
8498–8506. 50. Zhang L, Kovalev GI, Su L (2007) HIV-1 infection and pathogenesis in a novel
humanized mouse model. Blood 109: 2978–2981. 51. Sun Z, Denton PW, Estes JD, Othieno FA, Wei BL, et al. (2007) Intrarectal
transmission, systemic infection, and CD4+ T cell depletion in humanized mice
infected with HIV-1. J Exp Med 204: 705–714. 34. Su L, Kaneshima H, Bonyhadi ML, Lee R, Auten J, et al. (1997) Identification
of HIV-1 determinants for replication in vivo. Virology 227: 45–52. p
52. Manz MG (2007) Human-hemato-lymphoid-system mice: opportunities and
challenges. Immunity 26: 537–541. 53. REFERENCES J Immunol 168: 325–331. 8. Rabin L, Hincenbergs M, Moreno MB, Warren S, Linquist V, et al. (1996) Use
of standardized SCID-hu Thy/Liv mouse model for preclinical efficacy testing of
anti-human immunodeficiency virus type 1 compounds. Antimicrob Agents
Chemother 40: 755–762. 20. Stoddart CA, Moreno ME, Linquist-Stepps VD, Bare C, Bogan MR, et al. (2000) Antiviral activity of 29-deoxy-39-oxa-49-thiocytidine (BCH-10652) against
lamivudine-resistant human immunodeficiency virus type 1 in SCID-hu Thy/
Liv mice. Antimicrob Agents Chemother 44: 783–786. 9. McCune JM, Namikawa R, Shih CC, Rabin L, Kaneshima H (1990) Suppression
of HIV infection in AZT-treated SCID-hu mice. Science 247: 564–566. 10. Namikawa R, Weilbaecher KN, Kaneshima H, Yee EJ, McCune JM (1990)
Long-term human hematopoiesis in the SCID-hu mouse. J Exp Med 172:
1055–1063. 21. Datema R, Rabin L, Hincenbergs M, Moreno MB, Warren S, et al. (1996)
Antiviral efficacy in vivo of the anti-human immunodeficiency virus bicyclam
SDZ SID 791 (JM 3100), an inhibitor of infectious cell entry. Antimicrob Agents
Chemother 40: 750–754. 11. Krowka JF, Sarin S, Namikawa R, McCune JM, Kaneshima H (1991) Human
T cells in the SCID-hu mouse are phenotypically normal and functionally
competent. J Immunol 146: 3751–3756. 22. Stoddart CA, Rabin L, Hincenbergs M, Moreno M, Linquist-Stepps V, et al. (1998) Inhibition of human immunodeficiency virus type 1 infection in SCID-hu
Thy/Liv mice by the G-quartet-forming oligonucleotide, ISIS 5320. Antimicrob
Agents Chemother 42: 2113–2115. p
12. Bonyhadi ML, Rabin L, Salimi S, Brown DA, Kosek J, et al. (1993) HIV induces
thymus depletion in vivo. Nature 363: 728–732. 13. Kaneshima H, Su L, Bonyhadi ML, Connor RI, Ho DD, et al. (1994) Rapid-high,
syncytium-inducing isolates of human immunodeficiency virus type 1 induce
cytopathicity in the human thymus of the SCID-hu mouse. J Virol 68: 8188–8192. 23. Strizki JM, Xu S, Wagner NE, Wojcik L, Liu J, et al. (2001) SCH-C (SCH
351125), an orally bioavailable, small molecule antagonist of the chemokine PLoS ONE | www.plosone.org August 2007 | Issue 8 | e655 10 Validation of SCID-hu Model Validation of SCID-hu Model Feng JY, Shi J, Schinazi RF, Anderson KS (1999) Mechanistic studies show that
(-)-FTC-TP is a better inhibitor of HIV-1 reverse transcriptase than 3TC-TP. FASEB J 13: 1511–1517. 35. Amado RG, Jamieson BD, Cortado R, Cole SW, Zack JA (1999) Reconstitution
of human thymic implants is limited by human immunodeficiency virus
breakthrough during antiretroviral therapy. J Virol 73: 6361–6369. g
g
py
36. Koka PS, Fraser JK, Bryson Y, Bristol GC, Aldrovandi GM, et al. (1998)
Human immunodeficiency virus inhibits multilineage hematopoiesis in vivo. J Virol 72: 5121–5127. 54. Young SD, Britcher SF, Tran LO, Payne LS, Lumma WC, et al. (1995) L-743,
726 (DMP-266): a novel, highly potent nonnucleoside inhibitor of the human
immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents
Chemother 39: 2602–2605. 37. Withers-Ward ES, Amado RG, Koka PS, Jamieson BD, Kaplan AH, et al. (1997) Transient renewal of thymopoiesis in HIV-infected human thymic
implants following antiviral therapy. Nat Med 3: 1102–1109. 55. Robinson BS, Riccardi KA, Gong YF, Guo Q, Stock DA, et al. (2000) BMS-
232632, a highly potent human immunodeficiency virus protease inhibitor that
can be used in combination with other available antiretroviral agents. Antimicrob Agents Chemother 44: 2093–2099. 38. Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, et al. (1986) Production
of acquired immunodeficiency syndrome-associated retrovirus in human and
nonhuman cells transfected with an infectious molecular clone. J Virol 59:
284–291. g
56. De Clercq E (2004) Antiviral drugs in current clinical use. J Clin Virol 30:
115–133. PLoS ONE | www.plosone.org August 2007 | Issue 8 | e655 11
|
https://openalex.org/W2767999634
|
https://europepmc.org/articles/pmc5721167?pdf=render
|
English
| null |
Computational tools for clinical support: a multi-scale compliant model for haemodynamic simulations in an aortic dissection based on multi-modal imaging data
|
Journal of the Royal Society interface
| 2,017
|
cc-by
| 12,879
|
Keywords: y
patient-specific simulation, aortic dissection,
computational fluid dynamics, fluid–structure
interaction, Windkessel model,
moving boundary Computational tools for clinical support:
a multi-scale compliant model for
haemodynamic simulations in an
aortic dissection based on multi-modal
imaging data MB, 0000-0001-5548-3298; SB, 0000-0002-6287-1106; JPG, 0000-0002-2861-0914 MB, 0000-0001-5548-3298; SB, 0000-0002-6287-1106; JPG, 0000-0002-2861-0914 Aortic dissection (AD) is a vascular condition with high morbidity and
mortality rates. Computational fluid dynamics (CFD) can provide insight
into the progression of AD and aid clinical decisions; however, oversimplified
modelling assumptions and high computational cost compromise the accuracy
of the information and impede clinical translation. To overcome these limit-
ations, a patient-specific CFD multi-scale approach coupled to Windkessel
boundary conditions and accounting for wall compliance was developed and
used to study a patient with AD. A new moving boundary algorithm was
implemented to capture wall displacement and a rich in vivo clinical dataset
was used to tune model parameters and for validation. Comparisons between
in silico and in vivo data showed that this approach successfully captures flow
and pressure waves for the patient-specific AD and is able to predict the
pressure in the false lumen (FL), a critical variable for the clinical management
of the condition. Results showed regions of low and oscillatory wall shear stress
which, together with higher diastolic pressures predicted in the FL, may indi-
cate risk of expansion. This study, at the interface of engineering and
medicine, demonstrates a relatively simple and computationally efficient
approach to account for arterial deformation and wave propagation phenom-
ena in a three-dimensional model of AD, representing a step forward in the
use of CFD as a potential tool for AD management and clinical support. http://dx.doi.org/10.1098/rsif.2017.0632 Received: 30 August 2017
Accepted: 13 October 2017 Received: 30 August 2017
Accepted: 13 October 2017 Subject Category:
Life Sciences–Engineering interface
Subject Areas:
biomedical engineering, biomechanics
Keywords:
patient-specific simulation, aortic dissection,
computational fluid dynamics, fluid–structure
interaction, Windkessel model,
moving boundary
Authors for correspondence:
Mirko Bonfanti
e-mail: mirko.bonfanti.15@ucl.ac.uk
Vanessa Dı´az-Zuccarini
e-mail: v.diaz@ucl.ac.uk
Electronic supplementary material is available
online at https://dx.doi.org/10.6084/m9. figshare.c.3917929. Mirko Bonfanti1, Stavroula Balabani1, John P. Greenwood2,3, Sapna Puppala3,
Shervanthi Homer-Vanniasinkam1,3,4 and Vanessa Dı´az-Zuccarini1 Cite this article: Bonfanti M, Balabani S,
Greenwood JP, Puppala S, Homer-
Vanniasinkam S, Dı´az-Zuccarini V. 2017
Computational tools for clinical support:
a multi-scale compliant model for
haemodynamic simulations in an
aortic dissection based on multi-modal
imaging data. J. R. Soc. Interface 14:
20170632. 1Department of Mechanical Engineering, University College London, Torrington Place, London WC1E 7JE, UK
2Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds LS2 9JT, UK
3Leeds Teaching Hospitals NHS Trust, Leeds LS1 3EX, UK g
p
4University of Warwick Medical School & University Hospitals Coventry and Warwickshire NHS Trust,
Coventry CV2 2DX, UK Authors for correspondence:
Mirko Bonfanti
e-mail: mirko.bonfanti.15@ucl.ac.uk
Vanessa Dı´az-Zuccarini
e-mail: v.diaz@ucl.ac.uk Authors for correspondence:
Mirko Bonfanti
e-mail: mirko.bonfanti.15@ucl.ac.uk
Vanessa Dı´az-Zuccarini
e-mail: v.diaz@ucl.ac.uk Electronic supplementary material is available
online at https://dx.doi.org/10.6084/m9. figshare.c.3917929. & 2017 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution
License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original
author and source are credited. 1. Introduction Computational fluid dynamics (CFD) has provided significant insight into the
haemodynamics of many cardiovascular pathologies, being particularly amen-
able to study aortic dissection (AD) due to its complexity and patient-specific
nature [1]. AD is characterized by the separation of the layers of the aortic wall. A tear in the intima layer allows blood to flow within the aortic wall inducing
the formation of two flow channels, the true (TL) and false lumen (FL), separated
by an intimal flap (IF) [2]. Diagnosis, management and treatment of AD are
patient specific and difficult; experts claim that ‘difficulty in diagnosis, delayed
diagnosis or failure to diagnose are so common as to approach the norm for
this disease, even in the best hands. . .’ [3]. Initial management of acute AD focuses
on pain control, heart rate and blood pressure management, followed by surgical
intervention, typically involving stenting of the entry tear. The supra-aortic branches (left and right subclavian arteries (LSA, RSA), left and right common carotid arteries
(LCC, RCC)) and the main visceral branches (coeliac trunk (CT), superior mesenteric artery (SMA), left and right renal arteries (LRA, RRA)) are included. (c) Flow-rate curves
at different locations extracted from 2D PC-MRI data: (A) ascending aorta; (B) RCC and LCC; (C) aortic arch, distal to the LSA; (D,E) flow rate in the TL of the dissected aorta
at two sections along the descending aorta, above and below the diaphragm, respectively; (F) AbAo, proximal to the iliac bifurcation. Although type B dissections (i.e. AD involving only the des-
cending aorta) have lower initial mortality than type A (i.e. AD
of the ascending aorta), they carry a poor long-term prognosis,
with late-term complications reported in 20–50% of cases
within 5 years [4]. the fluid flow by the vessel wall motion and vice versa. In FSI,
high uncertainties regarding the wall thickness and mechanical
properties of the dissected vessel remain [13], complicating
their application to AD studies. Nonetheless, recent FSI work
by our group [14] showed that wall motion has an impact on
clinically relevant haemodynamic parameters for AD, and
thus should be accounted for. Although a reasonable match
between computational and experimental data has been found
in recent attempts to validate simulation results against in vivo
phase-contrast magnetic resonance imaging (PC-MRI) [5,11],
discrepancies attributed to a rigid-wall assumption remained. Hence, the need for reliable compliant models of AD is evident. Detailed characterization of complex intra-aortic haemo-
dynamic parameters via CFD, which currently cannot be
determined in vivo, has the potential to aid clinical decision-
making around AD; for instance, by identifying those prone
to adverse outcomes and supporting clinicians by simulating
different interventional strategies [5–7]. A number of computational studies on AD have been
published in the last decade [8]. CFD modelling approaches
differ significantly across studies, especially regarding the
boundary conditions (BCs) and the treatment of the wall. As
the simulation of the whole vascular system is unpractical
and patient specific, time-varying pressure and flow wave-
forms at all termination branches are usually not available,
different strategies have been developed, e.g. via the coupling
of zero-dimensional (0D) Windkessel models to the outlets of
a three-dimensional (3D) domain, forming together a multi-
scale model (referring to the combination of models of different
dimensions) representing the vascular system [9]. CT data
geometry
RCC
LCC
B
LSA
RSA
C
entry tear
(18.5 mm2)
true
lumen
true
lumen
false
lumen
false
lumen
intimal
flap
inlet
D
B
A
A
C
D, E
F
F
E
600
flow (ml s–1)
inlet
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
0
0.2
0.4
t (s)
0.6
0.8
RCC
LCC
aortic arch
plane D-TL
plane E-TL
AbAo
AbAo
LRA
CT
RRA
SMA
400
200
0
40
20
0
400
200
–200
0
400
200
200
100
–100
0
–200
0
2D PC-MRI
(a)
(b)
(c)
Figure 1. (a) Rendering of the CT data showing the dissected aorta. (b) Three-dimensional model extracted from CT data representing the dissected aorta from the
ascending aorta (inlet) to the abdominal aorta (AbAo). The supra-aortic branches (left and right subclavian arteries (LSA, RSA), left and right common carotid arteries
(LCC, RCC)) and the main visceral branches (coeliac trunk (CT), superior mesenteric artery (SMA), left and right renal arteries (LRA, RRA)) are included. (c) Flow-rate curves
at different locations extracted from 2D PC-MRI data: (A) ascending aorta; (B) RCC and LCC; (C) aortic arch, distal to the LSA; (D,E) flow rate in the TL of the dissected aorta
at two sections along the descending aorta, above and below the diaphragm, respectively; (F) AbAo, proximal to the iliac bifurcation. B
A
C
D, E
F
600
flow (ml s–1)
inlet
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
0
0.2
0.4
t (s)
0.6
0.8
RCC
LCC
aortic arch
plane D-TL
plane E-TL
AbAo
400
200
0
40
20
0
400
200
–200
0
400
200
200
100
–100
0
–200
0
2D PC-MRI
(c) geometry
RCC
LCC
B
LSA
RSA
C
entry tear
(18.5 mm2)
true
lumen
false
lumen
intimal
flap
inlet
D
A
F
E
AbAo
LRA
CT
RRA
SMA
(b) CT data
true
lumen
false
lumen
(a) (a) A AbAo Figure 1. (a) Rendering of the CT data showing the dissected aorta. (b) Three-dimensional model extracted from CT data representing the dissected aorta from the
ascending aorta (inlet) to the abdominal aorta (AbAo). This study aims to tackle some of these challenges
through the development of a novel framework for patient-
specific simulations, combining multi-scale and compliant
AD models as well as dynamic BCs. A flexible and robust
method for tuning Windkessel parameters using non-inva-
sive patient-specific clinical data is presented. Moreover, the
rigid-wall assumption is addressed through the development
of a novel and computationally efficient approach to account
for the vessel wall motion in CFD simulations. Simulation
results are validated against a rich and unique patient-
specific clinical dataset from multiple imaging modalities
such as two-dimensional (2D) PC-MRI, 2D cine-MRI and
computed tomography (CT) scans. However, this coupling is relatively challenging, needing
to be handled appropriately and Windkessel parameters
must be accurately tuned. With the exception of a few studies
[2,5], most often simpler BCs are adopted in AD studies, such
as flow-split [10], constant zero-pressure [11] or pressure
waveforms taken from the literature [12]. rsif.royalsocietypublishing.org The Navier–Stokes (NS) and continuity equations for 3D time-
dependent flows were solved with finite-volume-based CFD
solver ANSYS-CFX 17.0 (ANSYS Inc., PA, USA). The Navier–Stokes (NS) and continuity equations for 3D time-
dependent flows were solved with finite-volume-based CFD
solver ANSYS-CFX 17.0 (ANSYS Inc., PA, USA). Blood
was
modelled
as
incompressible
with
density ¼
1056 kg m23 and non-Newtonian viscosity described by the
Carreau–Yasuda model with parameters taken from Gijsen et al. [16]. Blood flow was considered as laminar, a common assumption
in large arteries [2,12]. BCs applied to the fluid boundaries are
shown schematically in figure 2a. In particular, uniform velocity pro-
files were prescribed at the inlet and LCC and RCC boundaries for
which patient-specific flow-rate waveforms were available from
PC-MRIdata(i.e.QIN(t),QLCC(t),QRCC(t),figure1c).Thecorrespond-
ing mean Reynolds and Womersley numbers, based on the inlet
diameter of the reconstructed aorta, were equal to 1408 and 23,
respectively. The peak Reynolds number was 5275, which is below
the critical Reynolds number for transition to turbulence, based on
a dynamic viscosityof 4 1023 Pa s and following Peacocket al. [17]. 2.2.2. MRI data q
g
p
The NS equations were spatiallyand temporally discretized with
a high-resolution advection scheme [20] and a second-order implicit
backward Euler scheme, respectively, using a uniform time step of
1 ms, good enough for time-step size-independent results. At each anatomical location, a single slice was acquired, for
each of the two pulse sequences. The total acquisition time for
the whole MRI protocol was approximately 80 min. The lumen cross-sectional area (A) was quantified from 2D
cine-MRI sequences using thresholding algorithms available in
IMAGEJ (NIH, MD, USA). The normalized cross-sectional area
variation (DA*) was calculated as 2.2. Clinical dataset
2.2.1. CT scans org
J. R. Soc. Interface 14: 20170632 The entire aorta was imaged with a 16-slice CT scanner (Sensation
16; Siemens AG, Munich, Germany; 120 kV, 380 mA s, rotation
time: 0.5 s, field of view (FOV): 284 mm, slice thickness: 1 mm,
reconstruction kernel: B20F, contrast agent: Ultravist 300; Bayer
AG, Leverkusen, Germany) obtaining 946 slices with in-plane
resolution ¼ 0.55 mm and inter-slice distance ¼ 0.7 mm (figure 1a). g
The geometry of the dissected aorta was extracted using
semi-automated segmentation tools based on thresholding,
implemented in ScanIP (Synopsys Inc., CA, USA). Smoothing
operations were used on the resulting mask to reduce pixellation
artefacts. The IF separating the FL from the TL was identified
based on greyscale-value differences in the two lumina; however,
because it was difficult to clearly resolve its thickness from CT
images, it was approximated as a zero-thickness membrane for
modelling purposes. The aortic branches were cropped perpen-
dicularly to the vessel’s longitudinal axis to provide a flat
surface at the boundaries. The resulting geometry is shown in
figure 1b, representing the dissected aorta: from the ascend-
ing aorta, just distal to the sinotubular junction of the aortic
root, to the abdominal aorta (AbAo) just proximal to the iliac
bifurcation [15]. y
y
g
The remaining outlets of the 3D model were coupled to
three-element Windkessel models (WK3s), shown as electrical
analogues in figure 2a, inset. The flow curve obtained from
PC-MRI at the AbAo was not applied as the outflow BC to
avoid constraining the wave propagation. The flow (Q) and the
mean pressure (P) over these boundaries are related by P ¼ (R1 þ R2)Q R2C dP
dt þ R1R2C dQ
dt ,
ð2:2Þ ð2:2Þ where R1 and R2 represent the proximal and distal resistances, andC
is the compliance of the distal vasculature. R1 is used to absorb the
incoming waves and reduce artificial wave reflections, as shown by
Alastruey et al. [18]. WK3s seem to be the best compromise among
other physiologically relevant 0D outflow models to simulate
the peripheral vasculature [19], and should be used—instead of
purely resistance models—when a significant compliance is located
in the modelled distal vasculature [18]. WK3 parameters must be
tuned to obtain physiological flow/pressure waveforms. The
tuning procedure is described in §2.3.3. 2.2.2. MRI data High spatial and temporal resolution electrocardiogram (ECG)-
gated cine-imaging sequences (2D cine-MRI; slice thickness:
10 mm, repetition time: 3.1 ms, echo time: 1.5 ms, FOV: 375 mm,
in-plane resolution: 1.1 mm, temporal resolution: 40 phases/cardiac
cycle, steady-state free precession) were acquired with an Ingenia
1.5 T MRI scanner (Philips Healthcare, Amsterdam, The Nether-
lands) at several sections perpendicular to the aorta (indicated in
figure 1b). Additional slices (coronal plane) were acquired to
detect IF motion in the distal part of the dissection. Pressure at the model outlet is updated at every solver loop
according to equation (2.2), using as input the current flow rate
calculated at the outlet of the 3D domain. Derivative terms
were discretized with a first-order backward Euler approach
using flow and mean pressure values calculated over the
boundary at the previous time step. A no-slip condition was applied; the IF was assumed to
be rigid. The external aortic wall was allowed to expand and con-
tract with pressure fluctuations using a moving boundary (MB)
technique accounting for aortic compliance, as described in §2.3.2. A no-slip condition was applied; the IF was assumed to
be rigid. The external aortic wall was allowed to expand and con-
tract with pressure fluctuations using a moving boundary (MB)
technique accounting for aortic compliance, as described in §2.3.2. The NS equations were spatiallyand temporally discretized with
a high-resolution advection scheme [20] and a second-order implicit
backward Euler scheme, respectively, using a uniform time step of
1 ms, good enough for time-step size-independent results. Additionally, through-plane phase-contrast velocity mapping
images (2D PC-MRI) were acquired at the same locations for flow
quantification (slice thickness: 6 mm, repetition time: 17 ms, echo
time: 1.9 ms, FOV: 400 mm, in-plane resolution: 1.56 mm, temporal
resolution: 40 phases/cardiac cycle, velocity encoding: 200 cm s21). At each anatomical location, a single slice was acquired, for
each of the two pulse sequences. The total acquisition time for
th
h l
MRI
t
l
i
t l
80
i Additionally, through-plane phase-contrast velocity mapping
images (2D PC-MRI) were acquired at the same locations for flow
quantification (slice thickness: 6 mm, repetition time: 17 ms, echo
time: 1.9 ms, FOV: 400 mm, in-plane resolution: 1.56 mm, temporal
resolution: 40 phases/cardiac cycle, velocity encoding: 200 cm s21). 2.3.1. Flow model and boundary conditions rsif.royalsocietypublishing.org 2.1. Patient information High complexity and the computational demands of fluid–
structure interaction (FSI) simulations means a rigid-wall
approximation is widely used, neglecting the effects exerted on Data from a 77-year-old man with a chronic Stanford type B AD
were studied. The dissection originated approximately 40 mm 2.3. Computational simulation set-up 3 distal
to
the
left
subclavian
artery,
extended
throughout
the length of the descending aorta and terminated about
10 mm distal to the coeliac trunk. From CT scans (figure 1a),
one entry tear (area approx. 18.5 mm2) was located approxi-
mately 10 mm distal to the proximal end of the dissection; no
other communication between the TL and FL was evident from
CT data, confirmed by a reduced flow in the FL observed in
PC-MRI data. 2.3. Computational simulation set-up 2.3.1. Flow model and boundary conditions 2.3.2. Moving boundary method An MB algorithm was employed to account for the compliance of
the aorta, capturing wave transmission effects. Details are pre-
sented in Bonfanti et al. [21] and summarized here. The motion of
the ascending aorta due to the heart motion has been disregarded. DA ¼ A A0
A0
,
ð2:1Þ ð2:1Þ Assuming that the displacement of the aortic wall follows the
local surface-normal direction and is linearly related to the fluid
pressure (figure 2a, inset), the displacement dn (m) of each mesh
node n on the external vessel wall is calculated as where A0 is the minimal cross-sectional area measured during a
cardiac cycle. Velocity
information
was
extracted
from
the
PC-MRI
sequences using the GTFlow software (GyroTools LCC, Zu¨rich,
Switzerland); flow-rate curves measured at different sections are
reported in figure 1c. Flow curves A and B are used as BCs,
whereas C–F are used for validation. A heart rate of 75 bpm and
a stroke volume of 107.6 ml were reported, corresponding to a
cardiac output of 8.1 l min21. dn ¼ pn pext
Kn
nn,
ð2:3Þ dn ¼ pn pext
Kn
nn,
ð2:3Þ ð2:3Þ where pn (Pa) is the pressure at node n, pext (Pa) is the external
pressure set as equal to the mean diastolic pressure over the aortic
wall (in this study 76 mmHg), nn is the unit normal vector in the multi-scale model
0D model
tuning procedure
0D model:
vascular system
pressure (x, t)
0D model:
rigid aorta +
peripheral circulation
target:
Q–
outlet
target:
Psys, Pdia
Csys
Ci, R1
i, R2
i " WK3
Ci, R1
i, R2
i " WK3
multi-scale model:
3D rigid aorta +
0D peripheral circulation
aortic
compliance
estimate
2D-MRI data
vessel-area
variation
multi-scale model:
3D compliant aorta +
0D peripheral circulation
input:
QIN(t)
QRCC(t)
QLCC(t)
1
2
3
4
WK3
RSA
WK3
RRA
WK3
CT
WK3
CT
WK3
SMA
WK3
RRA
WK3
AbAo
WK3
LRA
LR8
LR9
LR10
R2
R1
C
LR12
LR11
LR
L
R
WK3
WK3-AbAo
0
0.3
0.7
1.0
1.4
CA
(mm2 mmHg–1)
WK3
LRA
WK3
SMA
WK3
LSA
WK3
LSA
LR3
LR4
LR2
LR1
LR7
LR5
LR6
WK3
RSA
QRCC
QRCC
QIN
QLCC
QLCC
QIN
A
A
section A-A
dn
dn =
pn
pn – pext
Kn
pext
n
(a)
(b)
(c)
nn
nn
Figure 2. (a) Schematic of the multi-scale model domain and its BCs. 2.3.2. Moving boundary method WK3 indicates the Windkessel models coupled at the outlets (see inset). The colour map
shows the area compliance of the aorta (CA ¼ DA/DP), estimated with patient-specific 2D cine-MRI data. The inset shows a cross-section of the ascending aorta
and a schematic depicting how the displacement of a node n on the vessel wall is related to the pressure pn calculated in the fluid domain according to the MB
method. (b) Schematic of the 0D model (lumped-parameters model) used during the tuning procedure. LR indicates the elementary building block, composed by an
inertance (L) and a resistance (R) (see inset), representing a vessel segment: LR1: ascending aorta and arch, LR2: brachiocephalic trunk, LR3: right subclavian artery,
LR4: right common carotid, LR5: left common carotid, LR6: left subclavian artery, LR7: descending aorta, LR8: coeliac trunk, LR9: superior mesenteric artery,
LR10: right renal artery, LR11: left renal artery, LR12: abdominal aorta. (c) Flowchart of the procedure adopted to tune the parameters of the multi-scale
model. Steps 1–4 are described in more detail in the text. tuning procedure
0D model:
vascular system
pressure (x, t)
0D model:
rigid aorta +
peripheral circulation
target:
Q–
outlet
target:
Psys, Pdia
Csys
Ci, R1
i, R2
i " WK3
Ci, R1
i, R2
i " WK3
multi-scale model:
3D rigid aorta +
0D peripheral circulation
aortic
compliance
estimate
2D-MRI data
vessel-area
variation
multi-scale model:
3D compliant aorta +
0D peripheral circulation
input:
QIN(t)
QRCC(t)
QLCC(t)
1
2
3
4
WK3
LRA
11
(c) (c) tuning procedure 0D model multi-scale model (a) 0D model:
vascular system .org
J. R. Soc. Interface 14: 20170632 2D-MRI data
vessel-area
variation Figure 2. (a) Schematic of the multi-scale model domain and its BCs. WK3 indicates the Windkessel models coupled at the outlets (see inset). The colour map
shows the area compliance of the aorta (CA ¼ DA/DP), estimated with patient-specific 2D cine-MRI data. The inset shows a cross-section of the ascending aorta
and a schematic depicting how the displacement of a node n on the vessel wall is related to the pressure pn calculated in the fluid domain according to the MB
method. (b) Schematic of the 0D model (lumped-parameters model) used during the tuning procedure. 2.3.2. Moving boundary method The ratio R1-to-
Rtot (with Rtot¼ R1 þ R2) was set following Les et al. [23],
and Ri
tot was calculated as Pi= Qi, where Pi is the mean
pressure at WK3 i obtained with the 0D model. Ri
tot was
adjusted to obtain the target flow distribution. Th
0D
d l
i
l
d
i h
b
k
d Mesh displacement equations were solved so as to obtain
an implicit two-way coupling between mesh motion and
fluid dynamics. The compliance Ci was calculated by distributing Csys
among each WK3 i proportionally to Qi. The ratio R1-to-
Rtot (with Rtot¼ R1 þ R2) was set following Les et al. [23],
and Ri
tot was calculated as Pi= Qi, where Pi is the mean
pressure at WK3 i obtained with the 0D model. Ri
tot was
adjusted to obtain the target flow distribution. 2.3.2. Moving boundary method LR indicates the elementary building block, composed by an
inertance (L) and a resistance (R) (see inset), representing a vessel segment: LR1: ascending aorta and arch, LR2: brachiocephalic trunk, LR3: right subclavian artery,
LR4: right common carotid, LR5: left common carotid, LR6: left subclavian artery, LR7: descending aorta, LR8: coeliac trunk, LR9: superior mesenteric artery,
LR10: right renal artery, LR11: left renal artery, LR12: abdominal aorta. (c) Flowchart of the procedure adopted to tune the parameters of the multi-scale
model. Steps 1–4 are described in more detail in the text. outwarddirectionandKn (N m23) is a measure of thewall stiffness at
the location of node n. Under the hypothesis of a circular cross-
section, Kn can be related to the vessel areacompliance CA as follows: (1) A WK3 analogue of the entire vascular system was used to
determine the total system Csys, following the method used
by Les et al. [23]. WK3 parameters Csys, Rsys
1
and Rsys
2
(Rsys
1 =ðRsys
1
þ Rsys
2 Þ ¼ 5:6% [23]) were iteratively varied to
obtain the target Psys and Pdia values, using as input QIN(t). The obtained Csys was equal to 0.99 ml mmHg21. outwarddirectionandKn (N m23) is a measure of thewall stiffness at
the location of node n. Under the hypothesis of a circular cross-
section, Kn can be related to the vessel areacompliance CA as follows: Kn ¼ 2
CA
ffiffiffiffiffiffiffiffiffi
pA0
p
,
ð2:4Þ ð2:4Þ where A0 is the vessel cross-sectional area at the location of node n. CA can be estimated using patient-specific 2D cine-MRI sequences,
as described in §2.3.3. (2) A 0D model representing the vascular system (figure 2b) was
employed to tune R1, R2 and C of each WK3 coupled to the
3D aorta, considered rigid at this stage. The aorta was
divided into segments modelled as 0D building blocks
made by an inertance and a resistance, as shown in figure 2b. (2) A 0D model representing the vascular system (figure 2b) was
employed to tune R1, R2 and C of each WK3 coupled to the
3D aorta, considered rigid at this stage. The aorta was
divided into segments modelled as 0D building blocks
made by an inertance and a resistance, as shown in figure 2b. The compliance Ci was calculated by distributing Csys
among each WK3 i proportionally to Qi. 2.3.5. Mesh
Th
d The extracted AD geometry (figure 2) was discretized with ICEM-
CFD (ANSYS Inc.), adopting a tetrahedral mesh in the core
region and seven prism layers at the walls (external aortic wall and
IF sides). The resulting mesh consisted of about 506 000 elements,
comparable to the grids used in other CFD studies of the aorta [2,14]. In order to select the computational grid, a preliminary mesh
independence study was carried out using the rigid model on
two additional grids, with 267 000 and 1 045 000 elements. Com-
parison between pressure and flow waves obtained at the outlets
with the three meshes (coarse, medium and fine) showed a maxi-
mum difference of 4.8% and 2.5% for flow and pressure,
respectively, between the coarse and medium mesh, and only
1.1% and 0.7% when comparing the medium and fine mesh. Thus, the medium mesh was deemed good enough for simulation
purposes. The effect of the mesh on the results was further ana-
lysed for the compliant model on the coarse and medium grids. The results showed a maximum difference of 1.9% and 1.3% for
the outlets’ flow and pressure waves, respectively. The obtained
TAWSS and OSI distributions over the aortic wall compared well
qualitatively (electronic supplementary material). The difference
between the mean and peak TAWSS and OSI was quantified
over selected regions of interest (i.e. areas around the entry tear
and FL wall, as shown in the electronic supplementary material),
resulting in maximum differences of 7.6% and 5.8% for the peak
and mean TAWSS, respectively, and 6.0% and 10% for the peak
and mean OSI, respectively. These differences give an upper
bound to the errors introduced by using a medium, rather than a
fine, mesh in the model. TL and FL cross-sectional areas were considered in the
calculation of the distensibility, as in [24]. TL and FL cross-sectional areas were considered in the
calculation of the distensibility, as in [24]. y
The
distensibility
of
the
main
aortic
branches
was
calculated from the pressure wave velocity (PWV) as
D ¼ r1 PWV2 using the empirical relationship between
PWV (m s21) and the vessel diameter (d (mm)) from
Reymond et al. [25], PWV ¼ a
db ,
ð2:5Þ ð2:5Þ where a and b are 13.3 and 0.3. Finally, CA (figure 2a) was calculated as CA ¼ D A0, where
A0 is the local vessel cross-sectional area. 2.3.5. Mesh
Th
d By integrating CA
along the vessel length, the compliance associated with the
3D aortic model was obtained (Caorta ¼ 0.41 ml mmHg21). g
(4) The parameters of the WK3 models coupled to the compliant
aorta needed to be re-tuned to obtain physiological pressure
and flow waveforms. Only the compliance attributed to the
peripheral circulation (Cper¼ Csys2 Caorta) was distributed
among the WK3 models. Although Rtot was kept equal to
the one estimated at step 2 for each WK3, R1 was set equal
to r PWV/A0 (i.e. the characteristic impedance of the
coupled 3D vessel) in order to reduce the impedance mis-
match between the 3D compliant model and the 0D WK3,
and thus to minimize the intensity of the non-physiological
waves reflected by the Windkessel models [18,25]. WK3 par-
ameters used for the multi-scale model (compliant 3D aorta
plus 0D WK3 models) are reported in table 2. rsif.royalsocietypublishing.org location
mean flow, Q (ml s21)
source/expression
reference
inlet
134.5
2D PC-MRI
n.a. RCC
12.7
2D PC-MRI
n.a. LCC
8.9
2D PC-MRI
n.a. RSA/LSA
9.8
0:5 ðQIN QRCC QLCC QDescÞa
n.a. CT
20.7
0:330 ðQDesc QAbAoÞa
[22]
SMA
14.0
0:223 ðQDesc QAbAoÞa
[22]
RRA/LRA
14.0
0:223 ðQDesc QAbAoÞa
[22]
AbAo
30.5
2D PC-MRI
n.a. aQDesc ¼ 93:3 ml s1, mean flow at the descending aorta obtained from 2D PC-MRI data (plane E in figure 1). rsif.royalsocietypublishing.org org
J. R. Soc. Interface 14: 20170632 CFD-Post (ANSYS Inc.) and Matlab (Mathworks, MA, USA). Time-averaged haemodynamic indices, such as time-averaged
wall shear stress (TAWSS) and oscillatory shear index (OSI),
were calculated according to Gallo et al. [26]. Table 2. RCR parameters used for the Windkessel models coupled to the
3D compliant model as a result of the tuning procedure. outlet
R1 (mmHg
s ml21)
R2 (mmHg
s ml21)
C (ml
mmHg21)
RSA
1.660
9.977
0.051
LSA
1.518
10.110
0.051
CT
1.588
3.858
0.107
SMA
1.488
6.604
0.072
RRA
2.947
5.102
0.072
LRA
2.162
5.919
0.072
AbAo
0.115
3.608
0.157 3.1. Flow rate and pressure waveforms Comparisons between target pressure and mean flow values
and simulation results are shown via histograms in figure 3
with good agreement between the two datasets (maximum
difference of 1.8%). Flow rates and pressure waveforms obtained with the
multi-scale compliant model at the inlet and at selected outlets
are shown in figure 3. Pressure waveforms from the aortic root
(inlet) to the periphery (AbAo) exhibit typical physiological
features, such as peripheral amplification of systolic and
pulse pressures, and increase in the wave-foot time delay 2.3.3. Model tuning based on patient-specific data The parameters of the combined 3D aortic model and 0D WK3
models were specifically tuned for the simulated patient. R1, R2
and C were specified for each WK3 to characterize the peripheral
circulation, while CA needed to be estimated to mimic the
compliance of the aortic model. The 0D model equations were solved with a backward-
differentiation scheme using the software 20-sim (Controllab
Products B.V., Enschede, The Netherlands). The aim of the tuning procedure was to obtain physiological
values for the mean flow rate at the outlets ð QiÞ, and target
systolic (Psys) and diastolic (Pdia) blood pressure at the inlet. (3) (3) A CFD simulation (rigid 3D aorta plus 0D WK3 models) was
run using the WK3 parameters from step 2. (3) A CFD simulation (rigid 3D aorta plus 0D WK3 models) was
run using the WK3 parameters from step 2. The systolic–diastolic pressure variation (DP, obtained
from CFD simulations) and DA* (extracted from 2D cine-
MRI sequences—equation (2.1)) were used to estimate the
distensibility of various aortic segments (aortic arch, ascend-
ing, descending and abdominal aorta) as D½Pa1 ¼ DA=DP. In the aortic segments presenting the dissection, both the (3) A CFD simulation (rigid 3D aorta plus 0D WK3 models) was
run using the WK3 parameters from step 2. Patient-specific brachial Psys and Pdia (150 and 80 mmHg,
respectively) were used as pressure target values. Target values
for Qi were set based on available PC-MRI and literature data,
as summarized in table 1. The systolic–diastolic pressure variation (DP, obtained
from CFD simulations) and DA* (extracted from 2D cine-
MRI sequences—equation (2.1)) were used to estimate the
distensibility of various aortic segments (aortic arch, ascend-
ing, descending and abdominal aorta) as D½Pa1 ¼ DA=DP. In the aortic segments presenting the dissection, both the The workflow illustrated in figure 2c was followed, as
described below. Table 1. Mean blood flow at the aortic branches used for tuning the Windkessel parameters. Table 1. Mean blood flow at the aortic branches used for tuning the Windkessel parameters. 5 5 2.3.4. Numerical simulations and post-processing (Online version in colour.) RCC
LCC
LCC
RRA
LSA
40
20
0
inlet
CT
RRA
SMA
LRA
AbAo
RSA
mean: 115 mmHg
154/76 mmHg
mean: 111 mmHg
157/74 mmHg
mean: 8.9 ml s–1
peak: 26.6 ml s–1
mean: 13.8 ml s–1
peak: 29.4 ml s–1
Q (ml s–1)
Qmean (ml s–1)
P (mmHg)
160
120
80
40
20
0
Q (ml s–1)
P (mmHg)
Pinlet (mmHg)
160
120
80
40
30
20
10
0
0
0.2
0.4
0.6
0.8
0
0.2
0.4%
0%
1.1%
0.2%
1.4%
0.7%
–1.8%
Sys
target values
CFD
RSA
RCC
LCC
LSA
CT
SMA
RRA
LRA
AbAo
Dia
150
80
0
–0.4%
–0.9% –1.6%
–1.0%
0.4
0.6
0.8
t (s)
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
t (s)
c)
(d)
(f)
(e) LCC
RRA
40
20
0
mean: 115 mmHg
154/76 mmHg
mean: 111 mmHg
157/74 mmHg
mean: 8.9 ml s–1
peak: 26.6 ml s–1
mean: 13.8 ml s–1
peak: 29.4 ml s–1
Q (ml s–1)
P (mmHg)
160
120
80
40
20
0
Q (ml s–1)
P (mmHg)
160
120
80
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
t (s)
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
t (s)
(d)
(e) 600
inlet
R
AbAo
mean: 134 ml s–1
mean: 116 mmHg
151/79 mmHg
mean: 115 mmHg
168/74 mmHg
peak: 510 ml s–1
mean: 30.9 ml s–1
peak: 157 ml s–1
400
200
Q (ml s–1)
Q (ml s–1)
P (mmHg)
P (mmHg)
0
160
120
80
200
100
0
160
120
80
0
0.2
0.4
0.6
0.8
0
0.2
0.4
t (s)
0.6
0.8
0
0.2
0.4
t (s)
0.6
0.8
0
0.2
0.4
0.6
0.8
(a)
(c
(b) 6 (a) CT
RRA
SMA
LRA
AbAo
Qmean (ml s–1)
Pinlet (mmHg)
40
30
20
10
0
0.4%
0%
1.1%
0.2%
1.4%
0.7%
–1.8%
Sys
target values
CFD
RSA
RCC
LCC
LSA
CT
SMA
RRA
LRA
AbAo
Dia
150
80
0
–0.4%
–0.9% –1.6%
–1.0%
(f) AbAo t (s) Figure 3. (a,b,d,e) Flow (Q) and pressure (P) waves obtained with CFD simulation at the inlet and selected outlets (c) of the compliant model. P is calculated as the average
pressure over the boundary area. (f) Comparison between the flow and pressure target values used for the tuning procedure of the Windkessel parameters and CFD results. 2.3.4. Numerical simulations and post-processing The relative percentage difference between CFD results and target values are reported in the figure, showing a good agreement. (Online version in colour.) pressure
velocity
1.00
0.75
0.50
0.25
0
0
1.3
2.5
3.8
>5.0
0
0.1
0.3
0.4
0.5
(m s–1)
TAWSS
(Pa)
OSI
140
130
120
110
100
(mmHg)
(a)
(b)
(c)
(d)
Figure 4. (a) Velocity magnitude at peak systole (t ¼ 0.12 s). Inset: streamlines passing through the entry tear at peak systole showing the blood flowing into the
FL. (b) Colour map of the pressure at peak systole in the ascending aorta and aortic arch. An area of increased pressure can be noted in the proximal FL where the
blood flow impinges on the vessel wall. (c) Colour map of the TAWSS. (d) Colour map of the OSI. 0
0.1
0.3
0.4
0.5
OSI
(d) (c) (a) Figure 4. (a) Velocity magnitude at peak systole (t ¼ 0.12 s). Inset: streamlines passing through the entry tear at peak systole showing the blood flowing into the
FL. (b) Colour map of the pressure at peak systole in the ascending aorta and aortic arch. An area of increased pressure can be noted in the proximal FL where the
blood flow impinges on the vessel wall. (c) Colour map of the TAWSS. (d) Colour map of the OSI. 2.3.4. Numerical simulations and post-processing Simulations were run until reaching a periodic steady state. For
the multi-scale model, this was achieved within three cardiac
cycles after appropriate initialization; the last cycle was used
for the analysis of results. Post-processing was done using 600
inlet
RCC
LCC
LCC
RRA
LSA
40
20
0
inlet
CT
RRA
SMA
LRA
AbAo
RSA
AbAo
mean: 134 ml s–1
mean: 116 mmHg
151/79 mmHg
mean: 115 mmHg
168/74 mmHg
mean: 115 mmHg
154/76 mmHg
mean: 111 mmHg
157/74 mmHg
peak: 510 ml s–1
mean: 8.9 ml s–1
peak: 26.6 ml s–1
mean: 13.8 ml s–1
peak: 29.4 ml s–1
mean: 30.9 ml s–1
peak: 157 ml s–1
400
200
Q (ml s–1)
Q (ml s–1)
Qmean (ml s–1)
Q (ml s–1)
P (mmHg)
P (mmHg)
0
160
120
80
P (mmHg)
160
120
80
40
20
0
Q (ml s–1)
P (mmHg)
Pinlet (mmHg)
160
120
80
40
30
20
10
0
200
100
0
160
120
80
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
0
0.2
0.4%
0%
1.1%
0.2%
1.4%
0.7%
–1.8%
Sys
target values
CFD
RSA
RCC
LCC
LSA
CT
SMA
RRA
LRA
AbAo
Dia
150
80
0
–0.4%
–0.9% –1.6%
–1.0%
0.4
0.6
0.8
0
0.2
0.4
t (s)
t (s)
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
t (s)
0.6
0.8
0
0.2
0.4
t (s)
0.6
0.8
0
0.2
0.4
0.6
0.8
(a)
(c)
(d)
(f)
(e)
(b)
Figure 3. (a,b,d,e) Flow (Q) and pressure (P) waves obtained with CFD simulation at the inlet and selected outlets (c) of the compliant model. P is calculated as the average
pressure over the boundary area. (f) Comparison between the flow and pressure target values used for the tuning procedure of the Windkessel parameters and CFD results. The relative percentage difference between CFD results and target values are reported in the figure, showing a good agreement. 3.3. Comparison between two-dimensional phase-
contrast MRI data and computational fluid
d
i
lt
fl
t i th di
t d in the proximal part of the FL is observed; during systole, a net
flow is drawn through the small entry tear into the FL due to
the pressure gradient between the TL and the FL (figure 4b)
and the dilation of the vessel caused by the increasing pressure. At peak systole, the flow is well organized throughout apart
from the proximal FL, where a high-velocity jet-like flow,
rolling up to form a vortex, is observed (figure 4a). y
Figure 5 compares the blood flow rate measured with PC-
MRI and obtained with the CFD model at four different
locations: the arch (distally to the LCC), two sections of the
TL in the descending aorta, and the abdominal aorta. Peak
flow rates are underestimated by the CFD model, with a rela-
tive difference between measured and computed flows of
0.1%, 215%, 222% and 29%, respectively. Overall, predicted
waveforms agree with those measured in vivo, with a good
synchronization between the two waves in the first three
locations, even if a delay is present in the AbAo. Blood flowing through the entry tear impinges on the FL
wall causing a localized pressure increase (figure 4b), poten-
tially leading to further enlargement/rupture of the already
structurally compromised FL wall. TAWSS and OSI distributions are shown in figure 4c,d. High TAWSS values (greater than 5 Pa) are observed in the
region around the entry tear, which is coherent with the
flow description presented previously. Very low TAWSS is
seen in the medial/distal part of the FL due to the almost
stagnant flow obtained here. 3.2. Aortic dissection haemodynamics (Online version in colour.) (b) (a) mean: 92.3 ml s–1; peak: 400.2 ml s–1
mean: 80.2 ml s–1; peak: 399.9 ml s–1
0.4
0.8 0 0.4 mean: 92.3 ml s–1; peak: 366.9 ml s–1
mean: 87.9 ml s–1; peak: 430.6 ml s–1
0.4
0.8 0 0.4 mean: 92.3 ml s–1; peak: 364.9 ml s–1
mean: 92.9 ml s–1; peak: 467.7 ml s–1
0.4
0.8 0 0.4 mean: 30.2 ml s–1; peak: 151.9 ml s–1
mean: 30.5 ml s–1; peak: 166.7 ml s–1 Figure 5. Comparison between computed and measured flow rates at different locations along the dissected aorta, as indicated in (a). (b) CFD results are presented
with the solid lines, while 2D PC-MRI data are shown by the dashed lines. (Online version in colour.) 3.2. Aortic dissection haemodynamics (i.e. point of lowest pressure). The PWV in the aorta was
evaluated with the foot-to-foot method (9.21 m s21), in conso-
nance with PWV findings reported by Taviani et al. [27] for
elderly people (i.e. 60–80 years). Flow characteristics at peak systole are illustrated in figure 4a,b. Figure 4a shows the velocity magnitude in the 3D domain. High blood velocities (greater than 1.0 m s21) can be seen in
the TL, where the dissection causes a reduction in cross-
sectional area, and in the coeliac trunk and renal arteries, due
to low lumen area and high blood flow demand. Velocities in
the FL are low in the medial/distal regions (less than
0.05 m s21), due to the absence of secondary communications
between the TL and FL. Nonetheless, a region of high velocity In agreement with the literature [23], there is no backflow
in the renal arteries, whereas a slight retrograde flow occurs
at the abdominal aorta at early diastole. These results suggest that the coupled 3D/0D, compliant
and finely tuned model can correctly represent the effects of
wave propagation along the vessel. CFD
PC-MRI data
400
mean: 92.3 ml s–1; peak: 400.2 ml s–1
mean: 80.2 ml s–1; peak: 399.9 ml s–1
mean: 92.3 ml s–1; peak: 366.9 ml s–1
mean: 87.9 ml s–1; peak: 430.6 ml s–1
mean: 92.3 ml s–1; peak: 364.9 ml s–1
mean: 92.9 ml s–1; peak: 467.7 ml s–1
mean: 30.2 ml s–1; peak: 151.9 ml s–1
mean: 30.5 ml s–1; peak: 166.7 ml s–1
200
0
0
0.4
0.8
0
0.4
0.8
0
0.4
0.8
0
0.4
t (s)
0.8
Q (ml s–1)
400
200
0
Q (ml s–1)
400
200
200
100
–100
0
0
Q (ml s–1)
Q (ml s–1)
prox. AbAo
medial
arch
(a)
(b)
Figure 5. Comparison between computed and measured flow rates at different locations along the dissected aorta, as indicated in (a). (b) CFD results are presented
with the solid lines, while 2D PC-MRI data are shown by the dashed lines. 3.4. Vessel wall displacement: comparison between
computational fluid dynamics results and
two-dimensional cine-MRI data Regions with moderate OSI are observed throughout the
aorta (figure 4d); although the flow is well organized in systole,
it is highly disorganized in diastole. OSI values in the aortic
branches are low because the flow is anterograde throughout
the cardiac cycle here. Instead, a very high OSI is reported in
the distal part of the FL due to the flow wave’s biphasic
nature in this region, caused by the alternating expansion
and contraction of the FL during systole and diastole (as
described in §3.6 for the compliant model). Blood pressure changes during the cardiac cycle drive aortic
dilation and contraction. At peak systole, the maximum
displacement relative to the undeformed (i.e. diastolic) con-
figuration predicted by the simulations is localized in the
ascending aorta (approx. 0.74 mm). Subsequently, the pressure
peak reaches the AbAo at t ¼ 0.23 s, with a maximum vessel displacement
1.00
20
ascending aorta
max DA%: 10.7%
max DA%: 11.0%
max DA%: 17.2%
max DA%: 16.6%
10
DA%
0
0
0.4
abdominal aorta
(s)
0.8
0
0.4
CFD
2D cine-MRI
t = 0.12 s
t = 0.23 s
t = 0.78 s
(s)
0.8
20
10
DA%
0
0.75
0.50
0.25
0
(mm)
(a)
(b)
Figure 6. (a) Displacement of the vessel wall obtained with the computational model at peak systole (t ¼ 0.12 s), systolic deceleration phase (t ¼ 0.23 s) and
end-diastole (t ¼ 0.78 s). (b) Percentage cross-sectional area variation (DA%) during a cardiac cycle at the level of the ascending and abdominal aorta: comparison
between CFD results and 2D cine-MRI measurements. DA%¼ (A – A0)/A0, where A is the cross-sectional area of the vessel lumen and A0 is the lowest cross-
sectional area of the vessel lumen throughout a cardiac cycle. (b) (a) 20
ascending aorta
max DA%: 10.7%
max DA%: 11.0%
10
DA%
0
0
0.4
(s)
0.8 max DA%: 17.2%
max DA%: 16.6%
abdominal aorta
(s)
0
0.4
(s)
0.8
20
10
DA%
0 abdominal aorta t = 0.23 s t = 0.12 s t = 0.78 s Figure 6. (a) Displacement of the vessel wall obtained with the computational model at peak systole (t ¼ 0.12 s), systolic deceleration phase (t ¼ 0.23 s) and
end-diastole (t ¼ 0.78 s). (b) Percentage cross-sectional area variation (DA%) during a cardiac cycle at the level of the ascending and abdominal aorta: comparison
between CFD results and 2D cine-MRI measurements. 3.4. Vessel wall displacement: comparison between
computational fluid dynamics results and
two-dimensional cine-MRI data DA%¼ (A – A0)/A0, where A is the cross-sectional area of the vessel lumen and A0 is the lowest cross-
sectional area of the vessel lumen throughout a cardiac cycle. variation of the transmural pressure across the IF (TMP ¼
PTL 2 PFL, where PTL and PFL represent the pressure in the
TL and FL, respectively) at proximal and distal levels is
shown in figure 7b. The TMP curves are compared with
2D-MRI sequences at the same locations showing the IF
displacement during the cardiac cycle. The TMP is positive
(i.e. pressure higher in the TL) during most of systole at
both locations, reaching a value of about 30 mmHg, with the
distal TMP curve delayed with respect to the proximal one. Instead, a negative TMP (i.e. pressure higher in the FL) is
found throughout diastole with a minimum value around
210 mmHg. Comparison of TMP curves with MRI sequences
shows that the IF displacement direction agrees well with the
computed TMP: a positive TMP corresponds with the motion
of the IF through the FL; conversely, a negative TMP relates
to the motion of the IF through the TL, indicating reliability
of the predicted pressure field while providing further
validation of the methodology presented herein. expansion ¼ 1.23 mm (figure 6a). The computed maximum
percentage cross-sectional area variation (DA%) is equal to
10.7% and 17.2% at the ascending and abdominal aorta,
respectively, and is in agreement with the values extracted
from 2D-MRI data (11.0% and 16.6%, respectively; figure 6b). Moreover, the predicted DA% waveforms compare well with
the measured ones, suggesting that the MB method can reliably
reproduce vessel expansion due to pressure changes during the
cardiac cycle. The measured DA% waveform at the ascending
aorta presents a peak at early diastole, absent in the computed
one, that may be due to the dicrotic wave, which is not
completely resolved by the simulation. The dicrotic notch in the pressure wave is caused by the
abrupt change of blood flow due to the closure of the aortic
valve. However, the flow-rate curve applied at the inlet of
the CFD model, which was extracted from PC-MRI data,
potentially affected by measurement inaccuracies, lacks
such a rapid change at the beginning of the diastole, and
thus the production of a dicrotic wave in the simulation
may be affected. 3.5. True lumen–false lumen transmural pressure and
intimal flap displacement Figure 8 compares the flow rate and pressure waves of a rigid
versus a compliant model at a medial section of the dissection. It should be noted that, even if pressure values obtained by
both models are of the same order of magnitude, the relative
pressure in the two lumina follows an opposite trend: the com-
pliant model predicts higher pressures during systole and
lower pressures during diastole in the TL than in the FL,
while the opposite is found with the rigid model. As previously
discussed, the available clinical dataset indicates that the intimal flap displacement Pressure differences between the TL and the FL can provide
insight into the actual state and probable progression of the dis-
section but cannot usually be assessed in the clinic. For
instance, high pressure in the FL can lead to further expansion,
or even rupture of the FL, while causing a narrowing of the TL
[4]. Simulated pressure distributions at peak systole (figure 7a)
show higher pressure in the TL than in the FL. The temporal pressure
30
peak systole
initial diastole
end-diastole
end-diastole
TL
IF
IF
TL
FL
0.06 s
0.20 s
0.78 s
20 mm
FL
0.10 s
0.34 s
0.78 s
10 mm
15
0
–10
30
15
0
–10
0
0.34
systolic
acceleration
systolic
deceleration
0.80
(s)
0
0.34
0.80
(s)
systole
diastole
TMP (mmHg)
TMP (mmHg)
142
133
123
113
104
(mmHg)
TMP = PTL – PFL
*
*
(a)
(b)
Figure 7. (a) Pressure colour map at peak systole (t ¼ 0.12 s). (b) Comparison between the TMP across the IF obtained with the CFD compliant model during a
cardiac cycle and the displacement of the IF observed from 2D cine-MRI data at different time instances (TMP ¼ PTL 2 PFL, where PTL and PFL are the pressures in
the TL and FL averaged over a cross-section—indicated by an asterisk in the figure—located at the same level of the respective 2D cine-MRI slice). It can be noted
that the direction of the IF displacement agrees well with the sign of the TMP: a positive TMP corresponds with the motion of the IF through the FL, whereas a
negative TMP relates to the motion of the IF through the TL. (a) (b) 30
peak systole
initial diastole
end-diastole
TL
IF
FL
0.10 s
0.34 s
0.78 s
10 mm
15
0
–10
0
0.34
0.80
( )
TMP (mmHg)
(b) TMP (mmHg) end-diastole
IF
TL
FL
0.06 s
0.20 s
0.78 s
20 mm
30
15
0
–10
systolic
acceleration
systolic
deceleration
0
0.34
0.80
(s)
systole
diastole
TMP (mmHg) diastole Figure 7. (a) Pressure colour map at peak systole (t ¼ 0.12 s). intimal flap displacement (b) Comparison between the TMP across the IF obtained with the CFD compliant model during a
cardiac cycle and the displacement of the IF observed from 2D cine-MRI data at different time instances (TMP ¼ PTL 2 PFL, where PTL and PFL are the pressures in
the TL and FL averaged over a cross-section—indicated by an asterisk in the figure—located at the same level of the respective 2D cine-MRI slice). It can be noted
that the direction of the IF displacement agrees well with the sign of the TMP: a positive TMP corresponds with the motion of the IF through the FL, whereas a
negative TMP relates to the motion of the IF through the TL. correct pressure distribution is the one computed by the
compliant model, not the rigid one. relative difference with the one computed by the compliant
model (TAWSSC). In the proximal part of the FL, TAWSSR is sig-
nificantly lower than TAWSSC. The OSI distributions obtained
with the two models also differ considerably (figure 9b). Additionally, while the cycle mean flow rate (Q) is approxi-
mately the same in both models, its distribution among the
cardiac phases is different: the compliant model predicts a
lower TL flow during systole and a higher TL flow during
diastole than the rigid model. Owing to the aortic compliance,
the vessel can store blood during systole and release it during
diastole. Following this, 28 ml of blood is accumulated in the
aorta during systole due to a pressure increase of 68 mmHg. Consequently, peak flow rates in the compliant model are less
pronounced than those obtained with the rigid model. As
expected, the flow rate in the FL computed by the rigid model
is equal to zero throughout the cardiac cycle. On the other
hand, the compliant model predicts a small but non-negligible
net flow in the FL resulting from the alternate dilation and
contraction of the FL during systole and diastole, respectively. 4. Discussion Although this approach is suit-
able for rigid models with a single outlet, it is inadequate for
multi-branched or deformable models as flow distribution
among the vascular branches strongly depends on the down-
stream vasculature. To overcome these limitations, 0D WK3s
have been used as BCs in more advanced AD simulations [2,5]. aortic system [12], may be different in pathological cases. More-
over, this imposes a constant flow diversion into each branch
that can have a major impact on wall shear stress (WSS)-
based indices, as shown by Gallo et al. [26]. Additionally,
zero-pressure conditions are often used as outflow BCs at the
abdominal aortic branches [11]. Although this approach is suit-
able for rigid models with a single outlet, it is inadequate for
multi-branched or deformable models as flow distribution
among the vascular branches strongly depends on the down-
stream vasculature. To overcome these limitations, 0D WK3s
have been used as BCs in more advanced AD simulations [2,5]. The proposed MB method allowed us to account for aortic
wall deformation and compliance in the CFD simulation. A linear, elastic relationship was assumed between wall displa-
cement and fluid forces. Although it is well known that the
stress–strain relationship of the vessel wall is generally non-
linear, experimental evidence suggests that the assumption of
a linear constitutive relation for the arterial wall is justified in
the range of physiological pressures [28]. It has been shown
that the effects of nonlinearity and viscoelasticity on pressure
and flow waves are minor in the aorta [25,29]. Also, limited
data availability on the viscoelastic properties of the arterial
wall in the literature [25] implies that using more advanced con-
stitutive relations would introduce additional complexity and
parameter uncertainties. Finally, a rigid-wall approximation is commonly adopted. However, as shown in our previous work [14] and also con-
firmed here, vessel wall motion has a significant impact
on clinically relevant haemodynamic markers for AD. FSI
methods, coupling CFD with finite-element modelling of the
aortic wall, are subject to uncertainties regarding the mechanical
properties ofthetissue, which remain unknown forthedissected
aorta and may be patient specific [13]. The high computational
cost of FSI simulations can also be prohibitive in the context of
CFD models for medical support. Thus, simpler methods to
account for wall motion in such applications are necessary. 4. Discussion The present work presents a significant advance of the cur-
rent state of the art in CFD simulations of AD for clinical
support. The vast majority of computational studies in the lit-
erature are limited by simplified assumptions about the
treatment of BCs and wall motion. For instance, despite the fact that the cardiac output is
patient specific and known to be affected by medical treatment
[10], due to the lack of in vivo data, flow waves of healthy sub-
jects examined in published studies are commonly employed as
inflow BCs [12]. Overly simplified assumptions are often made
regarding outflow BCs. A flow split is commonly adopted to
prescribe flow to the supra-aortic branches, by diverting 5%
of the total inlet flow into each branch during the entire cardiac
cycle [12]. This proportion, reported for the healthy human The dissimilar haemodynamics computed by the two
models results in different TAWSS distributions; this is particu-
larly true in the region around the entry tear, an important
indicator of disease progression. Figure 9a shows the TAWSS
distribution obtained with the rigid model (TAWSSR) and the 160
TL
FL
FL
TL
compliant model
rigid model
140
120
P (mmHg)
Q (ml s–1)
100
80
600
TL
Q: 92.3 ml s–1
Q: 0 ml s–1
Qsys: 182.8 ml s–1
Qsys: 11.8 ml s–1
Qdia: 25.5 ml s–1
Qdia: –8.7 ml s–1
FL
TL
FL
400
200
0
Q (ml s–1)
600
400
200
0
0
0.34
systole
diastole
systole
diastole
(s)
0.80
0
0.34
(s)
0.80
medial
plane
false
lumen
false lumen
true
lumen
true lumen
160
140
120
P (mmHg)
100
80
0
0.34
(s)
0.80
0
0.34
(s)
0.80
Q: 93.2 ml s–1
Qsys: 215.4 ml s–1
Qdia: 2.8 ml s–1
Q: 0 ml s–1
Qsys: 0 ml s–1
Qdia: 0 ml s–1
(a)
(b)
Figure 8. (a,b) Comparison between CFD results obtained with compliant and rigid models. TL results are presented with solid blue lines, whereas FL results are
shown by dashed red lines. The pressure results are presented as the averaged values calculated over the cross-sections shown in the schematic in (a), whereas the
flow results are calculated as the integral of the velocity over the same sections (a positive flow corresponds to fluid moving towards the abdominal aorta). Q, Qsys
and Qdia are the mean flow rates calculated over the entire cardiac cycle, the systole and diastole phase, respectively. 4. Discussion (Online version in colour.) FL
TL
rigid model
160
140
120
P (mmHg)
100
80
0
0.34
(s)
0.80 160
TL
FL
compliant model
140
120
P (mmHg)
100
80
0
0.34
(s)
0.80
(b) compliant model 10 (b) (a) medial
plane
false
lumen
false lumen
true
lumen
true lumen
(a) Q (ml s–1)
600
TL
Q: 92.3 ml s–1
Q: 0 ml s–1
Qsys: 182.8 ml s–1
Qsys: 11.8 ml s–1
Qdia: 25.5 ml s–1
Qdia: –8.7 ml s–1
FL
400
200
0
0
0.34
systole
diastole
(s)
0.80
(s) TL
FL
Q (ml s–1)
600
400
200
0
systole
diastole
0
0.34
(s)
0.80
(s)
Q: 93.2 ml s–1
Qsys: 215.4 ml s–1
Qdia: 2.8 ml s–1
Q: 0 ml s–1
Qsys: 0 ml s–1
Qdia: 0 ml s–1 Figure 8. (a,b) Comparison between CFD results obtained with compliant and rigid models. TL results are presented with solid blue lines, whereas FL results are
shown by dashed red lines. The pressure results are presented as the averaged values calculated over the cross-sections shown in the schematic in (a), whereas the
flow results are calculated as the integral of the velocity over the same sections (a positive flow corresponds to fluid moving towards the abdominal aorta). Q, Qsys
and Qdia are the mean flow rates calculated over the entire cardiac cycle, the systole and diastole phase, respectively. (Online version in colour.) MRI. The parameters of the WK3 models coupled to 3D
models of the dissected aorta were successfully set, and target
values specified on mean flows at model outlets and on blood
pressure at the inlet were met. The aortic wall distensibility/
compliance was estimated indirectly from cross-sectional
vessel area and pressure changes at different sections along
the aorta. At the level of the dissected descending aorta, a dis-
tensibility of 0.42 1025 Pa21 was found; this is comparable
to values reported by Ganten et al. [24] for a cohort of patients
with chronic type B AD. This value is significantly lower than
that for healthy patients of the same age [24]. aortic system [12], may be different in pathological cases. More-
over, this imposes a constant flow diversion into each branch
that can have a major impact on wall shear stress (WSS)-
based indices, as shown by Gallo et al. [26]. Additionally,
zero-pressure conditions are often used as outflow BCs at the
abdominal aortic branches [11]. 4. Discussion other fully rigid AD models, where differences of up to 56%
[5] and 28% [11] at peak flow rate are present. simulations. As an example, the computational time reported in
our previous work for an FSI simulation of AD was 57 h per
cycle [14]. In this context, the MB algorithm is much less compu-
tationally expensive and easier to implement, and this is critical
for clinical use. Moreover, the model can be easily tuned with
patient-specific vessel motion data obtained non-invasively in
the clinic (via 2D cine-MRI). This represents an advantage
over previous AD studies using traditional FSI methods [14],
where uncertainties related to material properties, which are
often taken from the literature, can be considerable. The results also show that the use of a rigid-wall approxi-
mation requires careful consideration. For instance, the TMP
between the two lumina predicted by a rigid model does not
agree with the magnetic resonance images. TMP is an important
physiological variable that cannot be measured in vivo non-inva-
sively and without risk for the patient, but can be predicted
instead by CFD simulations; hence, accurate prediction is essential
in order to use this parameter in the clinic. The compliant model pre-
dicted higher systolic pressure in the TL, and higher diastolic
pressure in the FL; these findings compare well with experimen-
tal results obtained by Tsai et al. [30] with a model of a chronic
typeBADlackingadistalre-entry-tear,similartotheonestudied
here. Higher diastolicFL pressure may lead to an increased aortic
wall tension and consequent risk of lumen expansion. Comparison between computed and measured blood
flows at different sections in the descending aorta showed
good agreement; modelling the compliance of the aortic
wall allowed amplitude and phase discrepancies between
measured and simulated waveforms observed in previous
rigid AD studies [5] to be reduced. Therefore, it appears clear
that introducing the compliance of the vessel wall in the CFD
model allows a better replication of the in vivo fluid dynamics
situation, and represents an improvement of this study
compared with previous rigid AD models [2,5]. TAWSS is thought to be important for AD initiation,
affecting growth and enlargement of the FL [2]. The high
TAWSS found by the compliant model in the entry-tear
region can be a predictor of further enlargement. 4. Discussion In this study, we developed a patient-specific, compliant
CFD model of a chronic type B AD with patient-specific
tuned dynamic BCs. A rich in vivo dataset comprising multiple
non-invasive imaging modalities was used to inform and vali-
date the computational model. A fast procedure for BC tuning
and a new, efficient method to model the wall motion have been
proposed to tackle some of the aforementioned challenges. The MB method allowed us to capture the essential phy-
sics of arterial deformation and wave propagation, as shown
in the Results section. The compliant model simulation took 13 h for one cardiac
cycle on a desktop computer (Intel Xeon E5, 8 cores, 32 GB
RAM) and this is a significant improvement over FSI The model was tuned using brachial blood pressure
measurements and flow and displacement data obtained via TAWSS
0
1.3
2.5
3.8
>5.0
<–50
–25
0
25
>50
0
0.1
0.3
0.4
0.5 <–50
–25
0
25
>50
TAWSS: %diff rigid-compliant
OSI
OSI: %diff rigid-compliant
(Pa)
(a)
(b)
Figure 9. Haemodynamic indices in the ascending aorta and aortic arch obtained with the rigid CFD model, and comparison with compliant model results. (a) TAWSS obtained with the rigid model and percentage difference relative to the compliant model results, calculated as: %diff ¼ (TAWSSR 2 TAWSSC)/(5
(Pa)). (b) OSI obtained with the rigid model and percentage difference relative to the compliant model results, calculated as: %diff ¼ (OSIR 2 OSIC)/0.5
(where OSIR and OSIC are the OSI values for rigid and compliant, respectively). The percentage difference figures were normalized by values representing the
range of the respective parameter distributions for better visualization. TAWSS
0
1.3
2.5
3.8
>5.0
<–50
–25
0
25
>5
TAWSS: %diff rigid-compliant
(Pa)
(a) (a) rsif.royalsocietypublishing.org >5.0
<–50 >5.0
<– Figure 9. Haemodynamic indices in the ascending aorta and aortic arch obtained with the rigid CFD model, and comparison with compliant model results. (a) TAWSS obtained with the rigid model and percentage difference relative to the compliant model results, calculated as: %diff ¼ (TAWSSR 2 TAWSSC)/(5
(Pa)). (b) OSI obtained with the rigid model and percentage difference relative to the compliant model results, calculated as: %diff ¼ (OSIR 2 OSIC)/0.5
(where OSIR and OSIC are the OSI values for rigid and compliant, respectively). The percentage difference figures were normalized by values representing the
range of the respective parameter distributions for better visualization. 4. Discussion It has
been recently reported by Doyle & Norman [4] that areas of
low TAWSS may be correlated to regions of rapid local
expansion in type B AD. In the present study, the medial
and distal parts of the FL exhibit low TAWSS and high OSI,
which may indicate elevated risk. It should be noted that there is still a difference between the
CFD results and PC-MRI, which may be attributed to factors
related to the in vivo data, such as inaccuracies in the segmenta-
tion of the lumen contours on magnetic resonance images,
noise and the spatial resolution of PC-MRI, and to the model
limitations discussed below. The under-estimation of peak
flow rates in the TL can affect the derived fluid-dynamic vari-
ables, such as TAWSS and OSI, and potentially lead to an
under-estimation of the WSS in this region. However, the dis-
crepancies reported here are smaller than those obtained in 5. Conclusion This paper presents a novel, patient-specific multi-scale model-
ling approach coupled to Windkessel BCs to study AD. The
approach accounts for wall compliance in a computationally
efficient manner not explored hitherto. Simulation results
were compared with patient-specific clinical data as part of a Acknowledgement. The authors thank Prof. D. Rodney Hose for his
useful suggestions. J. R. Soc. Interface 14: 20170632 vph-case.eu) and from the Leverhulme Trust through the Senior
Research Fellowship ’Exploring the Unknowable Using Simulation:
Structural Uncertainty in Multiscale Models’ (fellowship no. RF-
2015-482). Funding. This project has received funding from the European Union’s
Horizon 2020 research and innovation programme under the Marie
Skłodowska-Curie grant agreement no. 642612, VPH-CaSE (www. vph-case.eu) and from the Leverhulme Trust through the Senior
Research Fellowship ’Exploring the Unknowable Using Simulation:
Structural Uncertainty in Multiscale Models’ (fellowship no. RF-
2015-482). Acknowledgement. The authors thank Prof. D. Rodney Hose for his
useful suggestions. References 1. Morris PD et al. 2015 Computational fluid dynamics
modelling in cardiovascular medicine. Heart 102,
18–28. (doi:10.1136/heartjnl-2015-308044)
2. Alimohammadi M, Agu O, Balabani S, Dı´az-Zuccarini
V. 2014 Development of a patient-specific
simulation tool to analyse aortic dissections:
assessment of mixed patient-specific flow
and pressure boundary conditions. Med. Eng. Phys. 36, 275–284. (doi:10.1016/j.medengphy. 2013.11.003)
3. Elefteriades JA, Barrett PW, Kopf GS. 2007 Litigation
in nontraumatic aortic diseases—a tempest in the
malpractice maelstrom. Cardiology 109, 263–272. (doi:10.1159/000107790)
4. Doyle BJ, Norman PE. 2016 Computational
biomechanics in thoracic aortic dissection: today’s
approaches and tomorrow’s opportunities. Ann. Biomed. Eng. 44, 71–83. (doi:10.1007/s10439-015-
1366-8)
5. Dillon-Murphy D, Noorani A, Nordsletten D,
Figueroa CA. 2016 Multi-modality image-
based computational analysis of haemodynamics in
aortic dissection. Biomech. Model. Mechanobiol. 15,
857–876. (doi:10.1007/s10237-015-0729-2)
6. Menichini C, Cheng Z, Gibbs RGJ, Xu XY. 2016
Predicting false lumen thrombosis in patient-specific
models of aortic dissection. J. R. Soc. Interface 13,
20160759. (doi:10.1098/rsif.2016.0759)
7. Alimohammadi M, Bhattacharya-Ghosh B, Seshadhri
S, Penrose J, Agu O, Balabani S, Dı´az-Zuccarini V. 2014 Evaluation of the hemodynamic effectiveness
of aortic dissection treatments via virtual stenting. approaches and tomorrow’s opportunities. Ann. Biomed. Eng. 44, 71–83. (doi:10.1007/s10439-015-
1366-8)
5. Dillon-Murphy D, Noorani A, Nordsletten D,
Figueroa CA. 2016 Multi-modality image-
based computational analysis of haemodynamics in
aortic dissection. Biomech. Model. Mechanobiol. 15,
857–876. (doi:10.1007/s10237-015-0729-2)
6. Menichini C, Cheng Z, Gibbs RGJ, Xu XY. 2016
Predicting false lumen thrombosis in patient-specific
models of aortic dissection. J. R. Soc. Interface 13,
20160759. (doi:10.1098/rsif.2016.0759)
7. Alimohammadi M, Bhattacharya-Ghosh B, Seshadhri
S, Penrose J, Agu O, Balabani S, Dı´az-Zuccarini V. 2014 Evaluation of the hemodynamic effectiveness
of aortic dissection treatments via virtual stenting. 1. Morris PD et al. 2015 Computational fluid dynamics
modelling in cardiovascular medicine. Heart 102,
18–28. (doi:10.1136/heartjnl-2015-308044) Int. J. Artif. Organs 37, 753–762. (doi:10.5301/ijao. 5000310) approaches and tomorrow’s opportunities. Ann. Biomed. Eng. 44, 71–83. (doi:10.1007/s10439-015-
1366-8) approaches and tomorrow’s opportunities. Ann. Biomed. Eng. 44, 71–83. (doi:10.1007/s10439-015-
1366-8) Int. J. Artif. Organs 37, 753–762. (doi:10.5301/ijao. 5000310) 8. 8. Sun Z, Chaichana T. 2016 A systematic review of
computational fluid dynamics in type B aortic
dissection. Int. J. Cardiol. 210, 28–31. (doi:10. 1016/j.ijcard.2016.02.099) 5. 2. Alimohammadi M, Agu O, Balabani S, Dı´az-Zuccarini
V. 2014 Development of a patient-specific
simulation tool to analyse aortic dissections:
assessment of mixed patient-specific flow
and pressure boundary conditions. Med. Eng. Phys. 36, 275–284. (doi:10.1016/j.medengphy. 2013.11.003) 2. Alimohammadi M, Agu O, Balabani S, Dı´az-Zuccarini
V. J. R. Soc. Interface 14: 20170632 J. R. Soc. Interface 14: 20170632 This study assumes that the IF behaves like a rigid zero-
thickness membrane separating the two lumina asthe resolution
of CT images did not allow the IF thickness to be estimated with
reasonable accuracy. The IF thickness estimated from MRI slices
was about 2 mm, which is small compared with the diameter of
the aorta; hence, the use of this approximation was deemed
appropriate. Moreover, the IF of chronic type B AD is expected
to be stiff and not as compliant as in acute settings [30]; this
was confirmed by the cine-MRI sequences showing relatively
small displacement of the IF (approx. 3 mm maximum displace-
ment in the proximal and distal parts, and less than 1 mm in the
central segment of the flap). However, in the regions where the
IF displacement is at its maximum, the fluid flow can be signifi-
cantly affected by the flap motion, and the assumption of a rigid
and zero-thickness IF may be the cause of the discrepancy
observed between the computed and measured peak flow
rates at the locations where the TL and FL coexist. Further
work will examine the effect of IF motion on blood flow
distribution and pressure in the TL and FL. Ethics. The clinical dataset was acquired as part of an ethically
approved protocol (NHS Health Research Authority, ref: 12/YH/
0551; Leeds Teaching Hospitals NHS Trust, ref: 788/RADRES/16);
patient consent was obtained. Data accessibility. The patient-specific geometry of the dissected aorta
used in the CFD model is available in .stl format on the Digital
Repository Figshare: doi.org/10.6084/m9.figshare.5354359.v1 [15]. Additional supporting data are available as part of the electronic
supplementary material. Authors’ contributions. M.B. conceived the study, carried out model devel-
opment and numerical simulations, and drafted the manuscript; S.B. provided interpretation of results and helped to draft the manuscript;
J.P.G. collected the MRI data; S.P. collected the CT scan data and pro-
vided clinical input; S.H.-V. conceived the study and provided
clinical input; V.D.-Z. conceived the study, provided resources and
coordination, and helped to draft the manuscript. All authors revised
the manuscript and gave final approval for publication. Competing interests. We declare we have no competing interests. Funding. This project has received funding from the European Union’s
Horizon 2020 research and innovation programme under the Marie
Skłodowska-Curie grant agreement no. 642612, VPH-CaSE (www. rsif.royalsocietypublishing.org rsif.royalsocietypublishing.org 4.1. Limitations Owing to the difficulties in acquiring in vivo haemodynamic
data using non-invasive methods, some model parameters were specified using non-patient-specific information; these
include the R1-to-Rtot ratio used to calculate the WK3
parameters coupled to the aortic rigid model, and the popu-
lation-based law (equation (2.5)) used to estimate the PWV in
the aortic branches. Moreover, the flow distribution among
the abdominal branches was specified based on data taken
from the literature for healthy patients. Although this assump-
tion holds for the subject studied in this paper, in which the
visceral branches were not involved in the dissection, it may
not be valid in general. For AD cases in which the flow into
these arteries is expected to be impaired, it may be useful to
acquire additional flow data at the coeliac and infrarenal
regions to inform the model. However, for the patient studied,
these assumptions allowed us to obtain physiological pressure
and flow waves, consistent with the available clinical data. rich dataset from multiple non-invasive imaging modalities,
comprising haemodynamics and vessel motion data, and cover-
ing the entire extension of the dissection. This work, at the
interface of medicine and engineering, was developed in close
collaboration with clinicians, who have stressed that the relative
good agreement achieved between simulations and the in vivo
data demonstrates that the proposed approach can successfully
capture the haemodynamics in a chronic type B AD and can
potentially lead to a powerful decision-making tool for the clini-
cal management of AD. For instance, the model can be used to
simulate different interventional strategies (e.g. covering of the
entry tear with a stent graft, fenestration of the IF) or medical
treatments (e.g. b-blocker therapy) and analyse their effects
on the fluid dynamics in the dissection (in particular pressure
and blood flow in the FL). Further research from the authors
will include the application of the tool on a small cohort of
patients. This is work that is well under way with data already
collected for a number of patients. 12 References 2014 Development of a patient-specific
simulation tool to analyse aortic dissections:
assessment of mixed patient-specific flow
and pressure boundary conditions. Med. Eng. Phys. 36, 275–284. (doi:10.1016/j.medengphy. 2013.11.003) 5. Dillon-Murphy D, Noorani A, Nordsletten D,
Figueroa CA. 2016 Multi-modality image-
based computational analysis of haemodynamics in
aortic dissection. Biomech. Model. Mechanobiol. 15,
857–876. (doi:10.1007/s10237-015-0729-2) 9. Alastruey J, Xiao N, Fok H, Schaeffter T,
Alberto Figueroa C. 2016 On the impact of
modelling assumptions in multi-scale, subject-
specific models of aortic haemodynamics. J. R. Soc. Interface 13, 20160073. (doi:10.1098/rsif. 2016.0073) 6. Menichini C, Cheng Z, Gibbs RGJ, Xu XY. 2016
Predicting false lumen thrombosis in patient-specific
models of aortic dissection. J. R. Soc. Interface 13,
20160759. (doi:10.1098/rsif.2016.0759) 6. Menichini C, Cheng Z, Gibbs RGJ, Xu XY. 2016
Predicting false lumen thrombosis in patient-specific
models of aortic dissection. J. R. Soc. Interface 13,
20160759. (doi:10.1098/rsif.2016.0759) 3. Elefteriades JA, Barrett PW, Kopf GS. 2007 Litigation
in nontraumatic aortic diseases—a tempest in the
malpractice maelstrom. Cardiology 109, 263–272. (doi:10.1159/000107790) 7. Alimohammadi M, Bhattacharya-Ghosh B, Seshadhri
S, Penrose J, Agu O, Balabani S, Dı´az-Zuccarini V. 2014 Evaluation of the hemodynamic effectiveness
of aortic dissection treatments via virtual stenting. 7. Alimohammadi M, Bhattacharya-Ghosh B, Seshadhri
S, Penrose J, Agu O, Balabani S, Dı´az-Zuccarini V. 2014 Evaluation of the hemodynamic effectiveness
of aortic dissection treatments via virtual stenting. 10. Chen D, Mu¨ller-Eschner M, Kotelis D, Bo¨ckler D,
Ventikos Y, Von Tengg-Kobligk H. 2013 A
longitudinal study of type-B aortic dissection and
endovascular repair scenarios: computational 10. Chen D, Mu¨ller-Eschner M, Kotelis D, Bo¨ckler D,
Ventikos Y, Von Tengg-Kobligk H. 2013 A
longitudinal study of type-B aortic dissection and
endovascular repair scenarios: computational 4. Doyle BJ, Norman PE. 2016 Computational
biomechanics in thoracic aortic dissection: today’s analyses. Med. Eng. Phys. 35, 1321–1330. (doi:10. 1016/j.medengphy.2013.02.006) carotid bifurcation model. J. Biomech. 32, 601–608. (doi:10.1016/S0021-9290(99)00015-9) 24. Ganten MK et al. 2009 Motion characterization of
aortic wall and intimal flap by ECG-gated CT in
patients with chronic B-dissection. Eur. J. Radiol. 72,
146–153. (doi:10.1016/j.ejrad.2008.06.024) 13 rsif.royalsocietypublishing.org 17. Peacock J, Jones T, Tock C, Lutz R. 1998 The onset
of turbulence in physiological pulsatile flow in a
straight tube. Exp. Fluids 24, 1–9. (doi:10.1007/
s003480050144) 11. Cheng Z, Juli C, Wood NB, Gibbs RGJ, Xu XY. 2014
Predicting flow in aortic dissection: comparison of
computational model with PC-MRI velocity
measurements. Med. Eng. Phys. 36, 1176–1184. (doi:10.1016/j.medengphy.2014.07.006) 25. References Reymond P, Merenda F, Perren F, Ru D. 2009
Validation of a one-dimensional model of the
systemic arterial tree. Am. J. Physiol. Heart Circ. Physiol. 297, 208–222. (doi:10.1152/ajpheart. 00037.2009.) 18. Alastruey J, Parker KH, Peiro J, Sherwin SJ. 2008
Lumped parameter outflow models for 1-D blood flow
simulations: effect on pulse waves and parameter
estimation. Commun. Comput. Phys. 4, 317–336. 12. Tse KM, Chiu P, Lee HP, Ho P. 2011 Investigation of
hemodynamics in the development of dissecting
aneurysm within patient-specific dissecting
aneurismal aortas using computational fluid
dynamics (CFD) simulations. J. Biomech. 44,
827–836. (doi:10.1016/j.jbiomech.2010.12.014) 26. Gallo D et al. 2012 On the use of in vivo measured
flow rates as boundary conditions for image-based
hemodynamic models of the human aorta:
implications for indicators of abnormal flow. Ann. Biomed. Eng. 40, 729–741. (doi:10.1007/s10439-
011-0431-1) rg
J. R. Soc. Interface 14: 20170632 19. Westerhof N, Lankhaar JW, Westerhof BE. 2009 The
arterial Windkessel. Med. Biol. Eng. Comput. 47,
131–141. (doi:10.1007/s11517-008-0359-2) 20. ANSYS Inc. 2016 CFX-solver theory guide. Release 17. Canonsburg, PA: ANSYS Inc. 13. Sommer G, Sherifova S, Oberwalder PJ, Dapunt OE,
Ursomanno PA, DeAnda A, Griffith BE, Holzapfel GA. 2016 Mechanical strength of aneurysmatic and
dissected human thoracic aortas at different shear
loading modes. J. Biomech. 49, 2374–2382. (doi:10.1016/j.jbiomech.2016.02.042) 21. BonfantiM, Alimohammadi M, Homer-Vanniasinkam S,
Dı´az-Zuccarini V. 2016 Patient-specific simulation of the
blood flow in an aortic dissection for clinical support
including an efficient method to represent the motion
of the intimal flap and vessel wall: a case study. In
VPH2016 book of abstracts (ed. AG Hoekstra),
pp. 356–359. Amsterdam, The Netherlands: University
of Amsterdam. 27. Taviani V, Hickson SS, Hardy CJ, McEniery CM,
Patterson AJ, Gillard JH, Wilkinson IB, Graves MJ. 2011 Age-related changes of regional pulse wave
velocity in the descending aorta using Fourier
velocity encoded M-mode. Magn. Reson. Med. 65,
261–268. (doi:10.1002/mrm.22590) 14. Alimohammadi M, Sherwood JM, Karimpour M,
Agu O, Balabani S, Dı´az-Zuccarini V. 2015 Aortic
dissection simulation models for clinical support:
fluid-structure interaction vs. rigid wall models. Biomed. Eng. Online 14, 34. (doi:10.1186/s12938-
015-0032-6) 28. Zhou J, Fung YC. 1997 The degree of nonlinearity
and anisotropy of blood vessel elasticity. Proc. Natl
Acad. Sci. USA 94, 14 255–14 260. (doi:10.1073/
pnas.94.26.14255) 22. Moore JE, Ku DN. 1994 Pulsatile velocity
measurements in a model of the human abdominal
aorta under simulated exercise and postprandial
conditions. J. Biomech. Eng. 116, 107. (doi:10.1115/
1.2895692) 29. References Segers P, Stergiopulos N, Verdonck P, Verhoeven R. 1997 Assessment of distributed arterial network
models. Med. Biol. Eng. Comput. 35, 729–736. (doi:10.1007/BF02510985) 15. Bonfanti M, Balabani S, Greenwood JP, Puppala S,
Homer-Vanniasinkam S, Dı´az-Zuccarini V. 2017 Data
from: computational tools for clinical support: a
multi-scale compliant model for haemodynamic
simulations in an aortic dissection based on
multimodal imaging data. Figshare Digital
Repository. (doi:10.6084/m9.figshare.5354359.v1) 23. Les AS, Shadden SC, Figueroa CA, Park JM, Tedesco
MM, Herfkens RJ, Dalman RL, Taylor CA. 2010
Quantification of hemodynamics in abdominal aortic
aneurysms during rest and exercise using magnetic
resonance imaging and computational fluid
dynamics. Ann. Biomed. Eng. 38, 1288–1313. (doi:10.1007/s10439-010-9949-x) 30. Tsai TT, Schlicht MS, Khanafer K, Bull JL, Valassis DT,
Williams DM, Berguer R, Eagle KA. 2008 Tear size
and location impacts false lumen pressure in an ex
vivo model of chronic type B aortic dissection. J. Vasc. Surg. 47, 844–851. (doi:10.1016/j.jvs. 2007.11.059) 16. Gijsen FJH, van de Vosse FN, Janssen JD. 1999 The
influence of the non-Newtonian properties of blood
on the flow in large arteries: steady flow in a
|
https://openalex.org/W4394961049
|
https://aditum.org/images/article/1713269466Pediatrics_and_Child_Health_Issues.pdf
|
English
| null |
Biliary dyskinesia. Increasing in Incidence or Better Recognition?
|
Pediatrics and child health issues
| 2,024
|
cc-by
| 1,699
|
Biliary dyskinesia. Increasing in Incidence or Better Recognition Kennedy A Sabharwal1*, BA, Michael W Simon2, MD, PhD
1University of Kentucky College of Medicine. 2University of Kentucky Department of Pediatrics, Lexington. Abstract: Gallbladder disease has historically remained uncommon in children. Children with nausea, abdominal pain and vomiting after eating are more
likely to be diagnosed with irritable bowel syndrome. When children are
evaluated with laboratory studies, ultrasound of gallbladder and HIDA scan,
a diagnosis of biliary dyskinesia may be confirmed. Laparoscopic surgery
of the gallbladder has been shown to be effective with minimal recovery and
complications. Follow-up shows that children unlike adults generally remain
symptom-free. *Corresponding author: Kennedy Sabharwal, University
of Kentucky College of Medicine 4040 Finn Way Suite 310,
Lexington KY 40517. Key Words: biliary dyskinesia; laparoscopic cholecystectomy; gallstones;
gallbladder Citation: Kennedy A Sabharwal, Michael W Simon, (2024). “Biliary dyskinesia. Increasing in Incidence or Better
Recognition?”. Pediatrics and Child Health Issues, 5(1); DOI:
10.61148/2836-2802/JPCHI/059 Introduction: Copyright: © (2024) Kennedy Sabharwal. This is an open
access article distributed under the Creative Commons
Attribution
License,
which
permits
unrestricted
use,
distribution, and reproduction in any medium, provided the
original work is properly cited. Throughout history, gallbladder disease has remained essentially
nonexistent in pediatrics other than its association with hemolytic disease
especially hereditary spherocytosis. Recently, the incidence of gallstones in
the pediatric population has been on the rise, mostly due to the astounding
increase in rates of pediatric obesity. (1,2) Other than childhood obesity,
gallstones are also common in children who are given total parental nutrition
due to short gut syndrome because of the decrease in gastric motility. It is
much more common, however, that children with abdominal symptoms of
nausea, vomiting and abdominal distress especially after eating have been
considered to have other etiologies like irritable bowel syndrome and
chronic nonspecific abdominal pain. (2) Gallbladder disease is often
overlooked and ignored as a possible etiology, likely because there are no
specific diagnostic criteria for children; however, the incidence of
gallbladder disease is increasing in children (3). Materials And Methods Enrich your Research Enrich your Research Pediatrics and Child Health Issues Mini Review Pediatrics and Child Health Issues Materials And Methods Children presenting with sharp upper right quadrant abdominal pain, nausea
and episodic vomiting after eating were evaluated. Their history was
reviewed. If there was clinical suspicion and examination consistent with
constipation, they had a flat plate of the abdomen performed. If the
abdominal X ray was normal and they had a positive Murphy sign they had
collected
a
comprehensive
metabolic
panel
including
gamma
glutamyltransferase (GGT), complete blood count (CBC) with a manual
differential and received an ultrasound of the abdomen. If the ultrasound
showed gallstones, they were referred for surgical evaluation. If the
ultrasound and laboratory studies were normal, they received a
cholescintigraphy 99mTc hepatobiliary iminodiacetic acid (HIDA) scan
with cholecystokinin (CCK) infusion. If the HIDA scan showed an ejection Copy right © Kennedy Sabharwal 1 | P a g e
nal discomfo
normal they 1 | P a g e
nal discomfo
normal they 1
inal
s no fraction of less than 37% or infusion reproduced abdominal
discomfort, they were sent for surgical evaluation. If the HIDA
scan was normal they would be referred for gastroenterology
evaluation. puberty and the production of estrogens. Estrogens increase the
secretion of cholesterol into gallstones, which promotes the
formation of gallstones. This could easily lead to increased
frequency of cholelithiasis in teenage girls. None of the children within this data were obese. Obesity has been
identified as a significant cause of gallbladder disease in children. Results None of the children within this data were obese. Obesity has been
identified as a significant cause of gallbladder disease in children. (8) This is likely due to bile saturation with cholesterol seen in
obesity, excessive hepatic secretions, and impaired gallbladder
motility. (9) Infection with Epstein-Barr virus, streptococci,
Hepatitis A, and gram-negative infections may also cause
gallbladder disease. (10-12) Since 2015, 33 children, 26 females (12 – 24 years of age, mean 14
years) and 7 males (12 – 20 years of age, mean 16 years), were
identified with symptoms consistent with biliary dyskinesia
followed by diagnostic confirmation of gallbladder disease. All
laboratory studies were normal. One female was taking oral
contraceptives. None of these children were obese. One child had
stones visualized on ultrasound. None of the children had a thicken
gallbladder wall visualized. The HIDA scan with infusion showed
a reduced ejection fraction of less than 37% in 24 of the 26 females
and all 7 males. The only child who did not have a HIDA scan was
the subject with stones identified by ultrasound. For all the children
symptoms were reproduced by dye infusion. Indication for surgery may be either symptom-based or chronic
cholecystitis. Each child in the researcher’s data had an outpatient
laparoscopic
cholecystectomy
performed. Laparoscopic
cholecystectomy has been shown to be a safe surgical approach in
children without significant comorbidities. (13) However, the
merits and benefits of cholecystectomy as a treatment option for
biliary dyskinesia is still debated. Each child recovered
uneventfully, and their symptoms abated after the surgery. They
were seen at follow-up appointments between 1 month up to 3
years with no complications and no recurrent abdominal
symptoms. Discussion The patients reported here had a history of sharp right upper
abdominal pain, nausea, and occasional vomiting after eating. For
all the children, examination was significant for a positive Murphy
sign. These children were otherwise unremarkable for the
examination with no fever or other findings. There was no
antecedent history of illness. One female was on oral
contraceptives. She was not the patient with gallstones. If a provider is suspicious of biliary dyskinesia in a pediatric
patient, a positive Murphy's sign with consistent history may be an
indication to begin evaluation with screening laboratory studies,
ultrasound of the gallbladder and if all normal follow with a HIDA
scan with infusion. The author’s observation may reflect an
increase in the occurrence of biliary dyskinesia in children or
simply a better awareness and recognition of gallbladder disease. Only one child had stones visualized on ultrasound. None of the
children had a thicken gallbladder wall visualized. However, HIDA
scan with contrast showed a reduced ejection fraction of less than
37% in 24 of the 26 females and all 7 of the males. The only child
who did not have a HIDA scan was the subject with stones
identified by ultrasound. For all the children symptoms were
reproducible by the dye infusion. The HIDA scan is not predictive
of underlying gallbladder histopathology, but it may signal
dysfunction. A low ejection fraction during the HIDA scan or pain
triggered by sincalide biliary infusion does not predict
postoperative outcome. (4) Financial disclosures: None References 1. Rothstein, David H., and Carroll M. Harmon. “Gallbladder
disease in children.” Seminars in Pediatric Surgery, vol. 25,
no. 4, Aug. 2016, pp. 225–231. 2. Pogorelić, Zenon et al. “Gallbladder Disease in Children: A
20-year Single-center Experience.” Indian pediatrics vol. 56,5
(2019): 384-386. Symptoms of gallbladder disease in the pediatric population may
present as acute abdominal pain, nausea, vomiting, and/or fatty-
food intolerance. (5) The most common of these symptoms is
vomiting seen in 60% of children who present with biliary
dyskinesia. It is very rare to see a child with biliary dyskinesia
presenting with only abdominal pain without vomiting. (6) For
these individuals, symptoms had been present for three months up
to two years prior to diagnosis. Selected dietary items like pizza
and cheese seemed to exacerbate their symptoms, while dietary
restrictions eliminating fatty meals improved their symptoms. 3. Goldman, Deborah A. “Gallbladder, Gallstones, and Diseases
of the Gallbladder in Children.” Pediatrics in review vol. 41,12 (2020): 623-629. 4. Morris-Stiff, Gareth, et al. “The cholecystokin provocation
hida test: Recreation of symptoms is superior to ejection
fraction in predicting medium-term outcomes.” Journal of
Gastrointestinal Surgery, vol. 15, no. 2, Feb. 2011, pp. 345–
349. Within this data set, there was a gender predisposition for females
to have biliary dyskinesia. A previous longitudinal study reported
that teenage girls are four times more likely to have gallbladder
disease compared to teenage boys. (7) Oral contraceptives may
increase the risk of gallstones, but also the female gender as a
whole may have an increased susceptibility to gallstones due to 5. Al-Homaidhi, Hossam S et al. “Biliary dyskinesia in
children.” Pediatric surgery international vol. 18,5-6 (2002):
357-60. 6. Friesen, C A, and C C Roberts. “Cholelithiasis. Clinical
characteristics in children. Case analysis and literature
review.” Clinical pediatrics vol. 28,7 (1989): 294-8. Copy right © Kennedy Sabharwal 2 | P a g e 2 | P a g e 2 7. Chilimuri, Sridhar et al. “Symptomatic Gallstones in the
Young: Changing Trends of the Gallstone Disease-Related
Hospitalization in the State of New York: 1996 -
2010.” Journal of clinical medicine research vol. 9,2 (2017):
117-123. Caused
by Streptococcus
gallolyticus
subspecies
pasteurianus: A Case Report.” Microorganisms vol. 10,10
1929. 28 Sep. 2022. Caused
by Streptococcus
gallolyticus
subspecies
pasteurianus: A Case Report.” Microorganisms vol. 10,10
1929. 28 Sep. 2022. 12. Ariobimo, Bonfilio Neltio; et al. References Acute “Acalculous
Cholecystitis Associated with Hepatitis A Viral Infection.”
Biomolecular and Health Science Journal Vol 6,1: 74-77. 8. Zdanowicz, Katarzyna et al. “The Etiology of Cholelithiasis
in
Children
and
Adolescents-A
Literature
Review.” International journal of molecular sciences vol. 23,21 13376. 2 Nov. 2022. 13. Mattson, Anja, et al. “Laparoscopic cholecystectomy in
children: A systematic review and meta-analysis.” The
Surgeon, vol. 21, no. 3, June 2023. 9. Bonfrate, Leonilde et al. “Obesity and the risk and prognosis
of gallstone disease and pancreatitis.” Best practice &
research. Clinical gastroenterology vol. 28,4 (2014): 623-35. 14. Santucci, Neha, et al. "Biliary Dyskinesia in Children: A
Systematic Review". Journal of Pediatric Gastroenterology
and Nutrition, vol. 64, no. 2, February 2017: 186-193. 10. Yi, Dae Yong et al. “Ultrasonographic Gallbladder
Abnormality of Primary Epstein-Barr Virus Infection in
Children
and
Its
Influence
on
Clinical
Outcome.” Medicine vol. 94,27 (2015): e1120. 15. Ozden, Nuri, and John K DiBaise. “Gallbladder ejection
fraction and symptom outcome in patients with acalculous
biliary-like pain.” Digestive diseases and sciences vol. 48,5
(2003): 890-7. 11. Shigemori, Tsunehiko et al. “Acute Calculous Cholecystitis Copy right © Kennedy Sabharwal 3 | P a g e 3 | P a g e 3
|
https://openalex.org/W2782785506
|
https://angeo.copernicus.org/articles/36/641/2018/angeo-36-641-2018.pdf
|
English
| null |
On application of asymmetric Kan-like exact equilibria to the Earth magnetotail modeling
|
Annales geophysicae
| 2,018
|
cc-by
| 12,759
|
Daniil B. Korovinskiy1, Darya I. Kubyshkina1, Vladimir S. Semenov2, Marina V. Kubyshkina2, Nikolai V. Erkaev3,4,
and Stefan A Kiehas1 Daniil B. Korovinskiy1, Darya I. Kubyshkina1, Vladimir S. Semenov2, Marina V. Kubyshkin
and Stefan A Kiehas1 vinskiy1, Darya I. Kubyshkina1, Vladimir S. Semenov2, Marina V. Kubyshkina2, Nikolai V. E
Ki h
1 1Space Research Institute, Austrian Academy of Sciences, Graz, Austria
2Earth’s Physics Department, Saint Petersburg State University, St. Petersburg, Russia
3Institute of Computational Modelling, FRC “Krasnoyarsk Science Center” SBRAS, Krasnoyarsk, Russia
4The Applied Mechanics Department, Siberian Federal University, Krasnoyarsk, Russia 1Space Research Institute, Austrian Academy of Sciences, Graz, Austria
2Earth’s Physics Department, Saint Petersburg State University, St. Petersburg, Russia
3Institute of Computational Modelling, FRC “Krasnoyarsk Science Center” SBRAS, Krasnoyarsk, Russia
4The Applied Mechanics Department, Siberian Federal University, Krasnoyarsk, Russia Correspondence: Daniil B. Korovinskiy (daniil.korovinskiy@oeaw.ac.at) Correspondence: Daniil B. Korovinskiy (daniil.korovinskiy@oeaw.ac.at) Correspondence: Daniil B. Korovinskiy (daniil.korovinskiy@oeaw.ac.at) Received: 2 January 2018 – Discussion started: 17 January 2018
Accepted: 22 March 2018 – Published: 19 April 2018 Received: 2 January 2018 – Discussion started: 17 January 2018
Accepted: 22 March 2018 – Published: 19 April 2018 ground magnetoplasma configuration. In applications to col-
lisionless plasma, the background equilibrium is to be de-
rived from a solution of the kinetic Vlasov–Maxwell equa-
tions. A number of such solutions are derived both numer-
ically (e.g., Burkhart et al., 1992; Pritchett and Coroniti,
1992; Cargill et al., 1994, and others) and analytically (e.g.,
Schindler and Birn, 2002; Yoon and Lui, 2005; Sitnov and
Merkin, 2016; Vinogradov et al., 2016). All these solutions
describe symmetric planar current sheets; the only approx-
imate equilibrium solution for bent CS was introduced in
the paper of Panov et al. (2012), where the authors present
an analysis of direct THEMIS and GOES observations of
plasma sheet evolution near substorm onset. Panov et al. (2012) have found the CS bending to be a source of the tail-
ward growing normal magnetic field component Bz (in the
present paper we use the reference system with x axis point-
ing tailward, y axis pointing dawnward and z axis pointing
north). Hence, bending of the current sheet turns out to be an
important parameter for the sheet stability, controlled by the
sign of the derivative ∂Bz/∂x (e.g., Hau et al., 1989; Erkaev
et al., 2007, 2009; Pritchett and Coroniti, 2010) in many in-
stances. Abstract. A specific class of solutions of the Vlasov–
Maxwell equations, developed by means of generalization of
the well-known Harris–Fadeev–Kan–Manankova family of
exact two-dimensional equilibria, is studied. Daniil B. Korovinskiy1, Darya I. Kubyshkina1, Vladimir S. Semenov2, Marina V. Kubyshkina2, Nikolai V. Erkaev3,4,
and Stefan A Kiehas1 The examined
model reproduces the current sheet bending and shifting in
the vertical plane, arising from the Earth dipole tilting and the
solar wind nonradial propagation. The generalized model al-
lows magnetic configurations with equatorial magnetic fields
decreasing in a tailward direction as slow as 1/x, contrary
to the original Kan model (1/x3); magnetic configurations
with a single X point are also available. The analytical solu-
tion is compared with the empirical T96 model in terms of
the magnetic flux tube volume. It is found that parameters
of the analytical model may be adjusted to fit a wide range
of averaged magnetotail configurations. The best agreement
between analytical and empirical models is obtained for the
midtail at distances beyond 10–15 RE at high levels of mag-
netospheric activity. The essential model parameters (current
sheet scale, current density) are compared to Cluster data of
magnetotail crossings. The best match of parameters is found
for single-peaked current sheets with medium values of num-
ber density, proton temperature and drift velocity. This result is in line with previous findings revealing that
the configuration asymmetry can be an important factor of
magnetosphere dynamics. Particularly, Kivelson and Hughes
(1990) have first suggested that the CS bending may drop
down the reconnection onset threshold. This idea was con-
firmed later, when Partamies et al. (2009) noticed the sea-
sonal variations in the number of substorm events with maxi- 1
Introduction Studies of magnetosphere dynamics, including substorm
events, require a relevant current sheet (CS) stability anal-
ysis. This in turn requires a proper choice of the back- Ann. Geophys., 36, 641–653, 2018
https://doi.org/10.5194/angeo-36-641-2018
© Author(s) 2018. This work is distributed under
the Creative Commons Attribution 4.0 License. Ann. Geophys., 36, 641–653, 2018
https://doi.org/10.5194/angeo-36-641-2018
© Author(s) 2018. This work is distributed under
the Creative Commons Attribution 4.0 License. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets mums in winter and summer periods, when dipole tilt angle is
bigger (the known geomagnetic activity maximums, e.g., in
the Kp index, are registered contrary around the equinoxes). The paper is organized as follows. In Sect. 2 we describe
the analytical solution for bent CS. In Sect. 3 we compare
analytical and empirical T96 solutions. In Sect. 4 we present
the further generalization of the analytical model, providing
more realistic profiles of Bz in the equatorial plane. Then, the
model-typical scales are compared with in situ data. Discus-
sion and conclusions finalize the paper in Sect. 5. p
g
y
q
Later, this effect was investigated in detail in the paper
of Kubyshkina et al. (2015), where it was shown that the
substorm probability is higher for about 10–25 % during the
periods with tilt angle > 15◦, compared to the periods with
smaller tilt angles. The direction of the solar wind (SW) flow
also affects the substorm probability: it grows for 10–20 %
when SW flow direction forces the CS tilt to increase. The
statistical analysis has shown that the average substorm in-
tensity (defined by AL value during the event) is lower for
larger effective tilts (dipole tilt angle plus solar wind flow in-
clination). In other words, a large number of weak substorms
occur in those time intervals in which effective tilt angles
are high, and a smaller number of more intense substorms
is observed when tilt angles are small. This also agrees with
the results of Nowada et al. (2009), where both AL and AU
indices were analyzed for the intervals of negative interplan-
etary magnetic field Bz. 2
Analytical solution For two-component (proton + electron) isothermal plasma
with Maxwellian distribution functions and constant current
velocity the system of Vlasov–Maxwell equations can be re-
duced to the 2-D Grad–Shafranov equation (see Schindler,
1972; Yoon and Lui, 2005) for the dimensionless magnetic
potential 9 = (0, 9, 0), ∂29
∂x2 + ∂29
∂z2 = e−29. (1) (1) The quantity 9 is normalized for (−B0L), where L =
2cTi/(eB0Vi) is the typical scale of CS in the normal di-
rection, B0 = √8πn0(Te + Ti) is the lobe magnetic field,
n0 = n0e = n0i is the typical number density, Te,i are the
electron and ion temperatures, respectively, and Ve,i are the
corresponding drift velocities, fulfilling the condition In Kubyshkina et al. (2015), the dependence of magneto-
tail lobe magnetic field (as a proxy of the magnetic flux) on
the dipole tilt angle was studied by means of empirical mod-
eling. The average lobe field was found to be smaller for all
radial distances in a case of nonzero tilt angles. The decrease
reached 10–20 % for maximum tilt angle. This result is rea-
sonable under the assumption that substorm onsets require
a lower energy input during the periods of increased dipole
tilt. Next, in the paper of Semenov et al. (2015) it was found
that there is a clear dependence of the substorm probability
on the jumps of the z component of the SW velocity (asym-
metric factor), while the jumps of number density or plasma
pressure (symmetric factor) turn out to be noneffective. Fi-
nally, we should note that the Earth’s dipole tilt angle un-
dergoes daily and seasonal variations in the interval of about
±35◦, so that it is equal to zero twice a day within about
4 months a year, and during the other 8 months it is never
zero. In addition, the solar wind flow direction varies for
about ±6◦. These variations produce CS inclination, bending
and shift from the ecliptic plane. Therefore, the simplest so-
lar wind–magnetosphere configuration (vertical dipole, pla-
nar CS, radial solar wind) adopted by the majority of models,
is rather untypical and the development of the relevant bent
CS models is in high demand. Vi/Ti + Ve/Te = 0. (2) (2) Vi/Ti + Ve/Te = 0. Equation (2) expresses the condition of the zero electro-
static potential. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 642 2
Analytical solution The model of an ion-dominated CS, where
|Vi/Ve| > Ti/Te, is considered in the paper of Yoon and
Lui (2004). In the case of Maxwellian distribution func-
tions condition (2) can be satisfied by means of the proper
choice of the reference system, while in the general case of
non-Maxwellian distribution functions it cannot be fulfilled
(Schindler and Birn, 2002). Equation (2) expresses the condition of the zero electro-
static potential. The model of an ion-dominated CS, where
|Vi/Ve| > Ti/Te, is considered in the paper of Yoon and
Lui (2004). In the case of Maxwellian distribution func-
tions condition (2) can be satisfied by means of the proper
choice of the reference system, while in the general case of
non-Maxwellian distribution functions it cannot be fulfilled
(Schindler and Birn, 2002). A series of analytical solutions of Eq. (1) was found by
Walker (1915), who showed that the solution may be ex-
pressed via an arbitrary generating function g of the complex
variable ζ = x + iz, e−29 =
4|g′|2
(1 + |g|2)2 ,
g′ = dg(ζ)
dζ
. (3) (3) The first exact solution for two-dimensional (2-D) equi-
librium bent CS with nonzero dipole tilt was presented in
short notes of Semenov et al. (2015). This solution general-
izes the well-known Harris–Fadeev–Kan–Manankova equi-
libria family (see Yoon and Lui, 2005). In the present paper
we investigate the obtained solution to estimate its relevance
for the magnetotail CS modeling and stability analysis. For
this end, we compare the analytical solution with the empir-
ical Tsyganenko (1995) T96 model and define the analytical
model parameters, providing the best agreement. With the solution (3), the equilibrium magnetoplasma con-
figuration takes the form 9 = ln
1 + |g|2
2|g′|
,
(4)
n = exp(−29),
p = 0.5exp(−29),
(5) (4) (5) where p is the plasma pressure. By definition, the dimen-
sionless magnetic field components are Bx = −∂9/∂z and
Bz = +∂9/∂x. www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets Namely, one should
set (f = 0, a ̸= 0, b ̸= 0) for the Kan solution; (f ̸= 0, a =
b = 0) for the Fadeev solution, and (f = a = b = 0) for the
Harris solution. PHI = 30 The solution for a bent CS is developed in the paper of
Semenov et al. (2015) by substituting the complex parame-
ters a →ia and b →b0eiϕ in Eq. (6). The complex param-
eter a controls the shift of the CS in the z direction and ϕ
controls the dipole tilt angle. For the case of bent CS with-
out plasmoids (Kan-like model, f = 0) the solution (6) takes
relatively simple form, 0
5
10
15
20
, (−B0L)
−15
−10
−5
0
5
10
15
z, L
−10
0
10
20
30
40
50
x, L
(c)
PHI = 60
(−B L)
(d) PHI = 60 9 = ln
coshZ∗
√
W
,
(7)
Z∗= z −b0x sin(ϕ) −b0(z −a)cos(ϕ)
R2
,
(8)
W =
1 + b2
0 + 2b0(x2 −(z −a)2)cos(ϕ) + 4b0x(z −a)sin(ϕ)
R4
,
(9) (7) (8) 0
5
10
15
20
, (−B0L)
−15
−10
−5
0
5
10
15
z, L
−10
0
10
20
30
40
50
x, L
(d)
PHI = 120 1 + b2
0 + 2b0(x2 −(z −a)2)cos(ϕ) + 4b0x(z −a)sin(ϕ)
R4
,
(9) (9) where R2 = x2 + (z −a)2. Configurations of this type pos-
sess a dipole singularity at (x, z) = (0, a) and two additional
singularities at (x, z) = (±√b0 sin(ϕ/2), a∓√b0 cos(ϕ/2)),
rotating twice as slow as a dipole does. Hence, the effective
dipole tilt is equal to ϕ/2. For positive tilt angles the CS is
bent and uplifted over the ecliptic plane, and for negative tilts
the CS is shifted down. The set of magnetic configurations for dipole tilt angle
PHI = {0, 30, 60, 120} degrees clockwise (PHI = −ϕ/2) is
shown in Fig. 1. The two first cases (0 and 30◦) can be
observed in the Earth’s magnetosphere, and other cases are
shown here to illustrate the model behavior. White asterisks
in Fig. 1c and d mark the X points (Bx = Bz = 0), being
an attribute of the Kan-like solution. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 643 , (−B0L)
−10
0
10
20
30
40
50
x, L
−15
−10
−5
0
5
10
15
z, L
−4
−2
0
2
4
6
8
10
12
14
(a)
PHI = 0
0
5
10
15
20
, (−B0L)
−15
−10
−5
0
5
10
15
z, L
−10
0
10
20
30
40
50
x, L
(b)
PHI = 30
(
) , (−B0L)
−10
0
10
20
30
40
50
x, L
−15
−10
−5
0
5
10
15
z, L
−4
−2
0
2
4
6
8
10
12
14
(a)
PHI = 0
0
5
10
15
20
, (−B0L)
−15
−10
−5
0
5
10
15
z, L
−10
0
10
20
30
40
50
x, L
(b)
PHI = 30
0
5
10
15
20
, (−B0L)
−15
−10
−5
0
5
10
15
z, L
−10
0
10
20
30
40
50
x, L
(c)
PHI = 60
(−B L)
(d) , (−B0L)
−10
0
10
20
30
40
50
x, L
−15
−10
−5
0
5
10
15
z, L
−4
−2
0
2
4
6
8
10
12
14
(a)
PHI = 0
The particular choice of the generating function g specifies
the particular CS model. In the current paper we consider the
family of Harris-like models, including the classical Harris
(1962) current sheet, the Fadeev et al. (1965) solution (Har-
ris sheet complemented by an infinite chain of magnetic is-
lands along the neutral plane), the Kan (1973) solution (Har-
ris sheet with quasi-dipole), and the Manankova et al. (2000)
solution, representing the combination of all previous mod-
els. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets The last one is specified by the generating function , (
0 )
−10
0
10
20
30
40
50
x, L
−15
−10
−5
0
5
10
15
z, L
−4
−2
0
2
4
6
8
10
12
14
(a)
PHI = 0
0
5
10
15
20
, (−B0L)
−15
−10
−5
0
5
10
15
z, L
−10
0
10
20
30
40
50
x, L
(b)
PHI = 30
0
5
10
15
20
, (−B0L)
−15
−10
−5
0
5
10
15
z, L
−10
0
10
20
30
40
50
x, L
(c)
PHI = 60
0
5
10
15
20
, (−B0L)
−15
−10
−5
0
5
10
15
z, L
−10
0
10
20
30
40
50
x, L
(d)
PHI = 120
Figure 1. Magnetic potential 9(x,z), calculated from the asymmet-
ric Kan model (Eqs. 7–9) with parameters a = 0 and b0 = 8. Solu-
tions with dipole tilt angles PHI = {0◦, 30◦, 60◦,120◦} clockwise
are plotted on panels (a)–(d), respectively. PHI = −ϕ/2 of the ana-
lytical model. Spatial units are normalized for typical CS width L. Magnetic potential is normalized for (−B0L). X points are marked
white. PHI = 0 g(ζ) = f +
q
1 + f 2 exp
i
ζ −
b
ζ −a
. (6) (6) Solution (6) contains three real parameters a, b and f , where
a specifies the shift along the x axis, b controls the field line
elongation, and f defines the current density in the mag-
netic islands. Generating functions for other listed models
are the special cases of the function (6). Namely, one should
set (f = 0, a ̸= 0, b ̸= 0) for the Kan solution; (f ̸= 0, a =
b = 0) for the Fadeev solution, and (f = a = b = 0) for the
Harris solution. Solution (6) contains three real parameters a, b and f , where
a specifies the shift along the x axis, b controls the field line
elongation, and f defines the current density in the mag-
netic islands. Generating functions for other listed models
are the special cases of the function (6). D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets In the symmetric Kan
model the X point is located at infinity, but in bent sheets it
starts to approach the dipole with increasing tilt angle. This
X point is not produced by magnetic reconnection, and it
does not break a steady state equilibrium of the CS. However, Figure 1. Magnetic potential 9(x,z), calculated from the asymmet-
ric Kan model (Eqs. 7–9) with parameters a = 0 and b0 = 8. Solu-
tions with dipole tilt angles PHI = {0◦, 30◦, 60◦,120◦} clockwise
are plotted on panels (a)–(d), respectively. PHI = −ϕ/2 of the ana-
lytical model. Spatial units are normalized for typical CS width L. Magnetic potential is normalized for (−B0L). X points are marked
white. the appearance of the X point can be considered as a mani-
festation of potentially unstable configuration. In such a case, the appearance of the X point can be considered as a mani-
festation of potentially unstable configuration. In such a case, www.ann-geophys.net/36/641/2018/ 3
Comparison with the T96 model (2009) and veri-
fied by in situ data analysis (Sergeev et al., 2014), any bursty
bulk flow (BBF), produced by reconnection in the magneto-
tail and moving toward the Earth, stops near that particular
point where the entropy of the ambient plasma is equal to
that inside the BBF. The distribution of entropy along the
magnetotail is also an important factor for the stability anal-
ysis (Birn et al., 2009) and for the study of wave (oscillation)
generation and dissipation (Panov et al., 2016). Figure 3. Flux tube volume
R (x,z)
(30,−2.4)(dS/B) normalized to the full
FTV
R (5,1.5)
(30,−2.4)(dS/B), in percent, calculated by T96 model (blue)
and by the Kan model (red) for the quiet conditions with tilt an-
gle PHI = 30◦clockwise (model parameter ϕ = −60). Other model
parameters are given in the legend of Fig. 4b. The Earth is on the
left. Figure 3. Flux tube volume
R (x,z)
(30,−2.4)(dS/B) normalized to the full
(5 1 5) FTV
R (5,1.5)
(30,−2.4)(dS/B), in percent, calculated by T96 model (blue)
and by the Kan model (red) for the quiet conditions with tilt an-
gle PHI = 30◦clockwise (model parameter ϕ = −60). Other model
parameters are given in the legend of Fig. 4b. The Earth is on the
left. The FTV is determined in the same way for both analyt-
ical and empirical models: we integrate dS/B along mag-
netic field lines, where dS is the field line length element and
B =
q
B2x + B2z . In the T96 model the location of the flux
tube is computed by means of field line tracing; in the an-
alytical model this is a curve of constant 9. As one of the
first steps, the values of FTV of a single flux tube are com-
pared. The model parameters correspond to the quiet magne-
tospheric conditions with tilt angle of 30◦clockwise (see the
legend of Fig. 4b). FTVs are calculated along the magnetic
field line with a node at (x, z) = (30, −2.4). To eliminate
singularities, we excluded the near-Earth region x < 5RE, so
that the total FTVs are calculated as
R (5,1.5)
(30,−2.4)dS/B. The re-
sults are shown in Fig. 3 by the blue curve for T96 and by
the red curve for the Kan model. The values of FTV, normal-
ized for total FTV, are plotted as a function of x. 3
Comparison with the T96 model Topologically, magnetic configurations plotted in Fig. 1 are
very similar to that of the Earth’s magnetosphere. However,
to estimate the relevance of the analytical solution one should
compare some important numerical characteristics of the CS
model with the corresponding values registered in real ob-
servations. This can be done by utilizing empirical magnetic
field models, providing realistic averaged magnetospheric
configurations at various levels of magnetospheric activity. Of course, we should keep in mind that the real magneto-
sphere is an essentially three-dimensional structure. Follow-
ing the dipole tilt (and solar wind flow direction) variations,
the magnetotail CS bends and shifts from the equatorial plane
in the z direction (at most ∼3RE for maximum tilt) and also
warps in the y direction. These effects are well pronounced
in empirical magnetospheric models, but the 2-D analytical
model is evidently unable to reproduce all these complex
deformations. Therefore, we restrict our study to the noon–
midnight plane y = 0, and the two main effects manifested
in that plane: CS bending and shifting in z direction. Figure 2. X-point location vs. the effective tilt angle ϕ/2 in the
asymmetric Kan solution (Eqs. 7–9) with a = 0 and parameters
b0 = 40 (red), b0 = 15 (blue), and b0 = 9 (green). X, RE
30
25
20
15
10
5
FTV, %
0
20
40
60
80
100
Normalized ∫(dS/B)
T96
Kan
90 %
50 % X, RE
30
25
20
15
10
5
FTV, %
0
20
40
60
80
100
Normalized ∫(dS/B)
T96
Kan
90 %
50 %
Figure 3. Flux tube volume
R (x,z)
(30,−2.4)(dS/B) normalized to the full
FTV
R (5,1.5)
(30,−2.4)(dS/B), in percent, calculated by T96 model (blue)
and by the Kan model (red) for the quiet conditions with tilt an-
gle PHI = 30◦clockwise (model parameter ϕ = −60). Other model
parameters are given in the legend of Fig. 4b. The Earth is on the
left. To explore the appropriateness of the here presented ana-
lytical solution for bent CS, we compare the predicted mag-
netic flux tube volume (a proxy for the entropy) with that
calculated from the empirical model of Tsyganenko (1995)
T96. We consider the flux tube volume (FTV) instead of
the entropy, since the analytical solution is isothermal. This
quantity is chosen due to its importance for the magnetotail
dynamics. As was claimed by Birn et al. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 644 φ/2, o
45
50
55
60
65
70
75
80
85
90
0
20
40
60
80
100
Distance to the X point versus tilt angle
b0 = 40
b0 = 15
b0 = 9
x, L
Figure 2. X-point location vs. the effective tilt angle ϕ/2 in the
asymmetric Kan solution (Eqs. 7–9) with a = 0 and parameters
b0 = 40 (red), b0 = 15 (blue), and b0 = 9 (green). φ/2, o
45
50
55
60
65
70
75
80
85
90
0
20
40
60
80
100
Distance to the X point versus tilt angle
b0 = 40
b0 = 15
b0 = 9
x, L www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets (a)
25
5
PHI=0
0
FTV, R /nT
E
X, RE
0
30
20
15
10
25
5
6
4
2
pdyn = 2 nPa
Dst = -10nT
B = 2nT
z
b = 8.8
a = -0.05
d = 7.4%
max
d = 5.3%
med (b)
PHI=30
0
FTV, R /nT
E
X, RE
0
30
20
15
10
25
5
8
6
4
2
pdyn = 2 nPa
Dst = -10nT
B = 2nT
z
b = 8.8
a = -2.35
d = 10.6%
max
d = 7.7%
med (b)
(d)
4
3
2
1
PHI=30
0
FTV, R /nT
E
FTV, R /nT
E
X, RE
X, RE
0
30
20
15
10
25
5
0
30
20
15
10
25
5
8
6
4
2
4
3
2
1
pdyn = 2 nPa
pdyn = 3 nPa
Dst = -10nT
Dst = -50nT
B = 2nT
z
B = -3nT
z
b = 8.8
b = 14
a = -2.35
a = -2
d = 10.6%
max
d = 7.2%
max
d = 7.7%
med
d = 4.4%
med PHI=30
0 (b)
(d)
(f)
4
3
2
1
PHI 30
FTV, R /nT
E
FTV, R /nT
E
FTV, R /nT
E
X, RE
X, RE
X, RE
X, RE
0
30
20
15
10
25
5
0
30
20
15
10
25
5
0
30
20
15
10
25
5
8
6
4
2
4
3
2
1
1.6
1.2
0.8
0.4
pdyn = 2 nPa
pdyn = 3 nPa
pdyn = 6 nPa
Dst = -10nT
Dst = -50nT
Dst = -150nT
B = 2nT
z
B = -3nT
z
B = -7nT
z
b = 8.8
b = 14
b = 41.5
a = -2.35
a = -2
d = 10.6%
max
d = 7.2%
max
d = 3.7%
max
d = 7.7%
med
d = 4.4%
med
d = 1.9%
med
a = -1.84 (b)
(d)
(f)
4
3
2
1
FTV, R /nT
E
FTV, R /nT
E
FTV, R /nT
E
X, RE
X, RE
X, RE
X, RE
0
30
20
15
10
25
5
0
30
20
15
10
25
5
0
30
20
15
10
25
5
8
6
4
2
4
3
2
1
1.6
1.2
0.8
0.4
pdyn = 2 nPa
pdyn = 3 nPa
pdyn = 6 nPa
Dst = -10nT
Dst = -50nT
Dst = -150nT
B = 2nT
z
B = -3nT
z
B = -7nT
z
b = 8.8
b = 14
b = 41.5
a = -2.35
a = -2
d = 10.6%
max
d = 7.2%
max
d = 3.7%
max
d = 7.7%
med
d = 4.4%
med
d = 1.9%
med
a = -1.84 (b) (a) (d)
(f)
4
3
2
1
FTV, R /nT
E
FTV, R /nT
E
X, RE
X, RE
X, RE
X, RE
0
30
20
15
10
25
5
0
30
20
15
10
25
5
4
3
2
1
1.6
1.2
0.8
0.4
pdyn = 3 nPa
pdyn = 6 nPa
Dst = -50nT
Dst = -150nT
B = -3nT
z
B = -7nT
z
b = 14
b = 41.5
a = -2
d = 7.2%
max
d = 3.7%
max
d = 4.4%
med
d = 1.9%
med
a = -1.84 (c) (d) (e) Figure 4. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets Flux tube volumes: analytical solution (red curves) and T96 (black curves) for quiet (a, b), substorm (c, d) and storm (e, f)
conditions are plotted for tilt angles 0◦(a, c, e) and PHI = 30◦clockwise (b, d, f). Input parameters for the T96 model (black text), for the
Kan-like model (red text), and SD normalized for average FTV (blue text) are given in legends. The Earth is on the left. 90% of FTV are provided by the farther half of the tube,
x ∈[15, 30]RE, and 50% of FTV are concentrated in the
most distant interval within 3–4 RE in the x direction from
the tube node. sets of parameters are taken to specify the quiet magneto-
tail {Dst = −10, pdyn = 2 nPa, Bsw
z
= 2 nT}, substorm con-
ditions {Dst = −50, pdyn = 3 nPa, Bsw
z
= −3 nT} and storm
{Dst = −150, pdyn = 6 nPa, Bsw
z
= −7 nT}. Magnetic field
component Bsw
y
was set equal to zero. Parameters a and b0
of the analytical solution (7–9) are found numerically to min-
imize the SD between two models. The results (FTV vs. x co-
ordinate of the flux tube node) are presented in Fig. 4, where
red lines plot analytical solutions and black ones plot the T96 Then, FTVs, calculated by means of analytical and em-
pirical models, are compared at different levels of magne-
tospheric activity, characterized by input parameters of the
T96 model (Dst index, the SW dynamical pressure, pdyn,
and SW magnetic field components Bsw
y
and Bsw
z ). Three D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 645 y
q
y
(a)
(b)
(c)
(d)
(e)
(f)
25
5
4
3
2
1
PHI=0
0
PHI=30
0
FTV, R /nT
E
FTV, R /nT
E
FTV, R /nT
E
FTV, R /nT
E
FTV, R /nT
E
FTV, R /nT
E
X, RE
X, RE
X, RE
X, RE
X, RE
X, RE
X, RE
0
30
20
15
10
25
5
0
30
20
15
10
25
5
0
30
20
15
10
25
5
0
30
20
15
10
25
5
0
30
20
15
10
25
5
0
30
20
15
10
25
5
6
4
2
8
6
4
2
3
2
1
4
3
2
1
1.6
1.2
0.8
0.4
1.6
1.2
0.8
0.4
pdyn = 2 nPa
pdyn = 2 nPa
pdyn = 3 nPa
pdyn = 3 nPa
pdyn = 6 nPa
pdyn = 6 nPa
Dst = -10nT
Dst = -10nT
Dst = -50nT
Dst = -50nT
Dst = -150nT
Dst = -150nT
B = 2nT
z
B = 2nT
z
B = -3nT
z
B = -3nT
z
B = -7nT
z
B = -7nT
z
b = 8.8
b = 8.8
b = 22.13
b = 14
b = 51
b = 41.5
a = -2.35
a = -0.05
a = -2
a = -0.03
a = -0.02
d = 7.4%
max
d = 10.6%
max
d = 7.2%
max
d = 4.2%
max
d = 3.6%
max
d = 3.7%
max
d = 5.3%
med
d = 7.7%
med
d = 2.9%
med
d = 4.4%
med
d = 1.9%
med
d = 1.9%
med
a = -1.84
Figure 4. Flux tube volumes: analytical solution (red curves) and T96 (black curves) for quiet (a, b), substorm (c, d) and storm (e, f)
conditions are plotted for tilt angles 0◦(a, c, e) and PHI = 30◦clockwise (b, d, f). Input parameters for the T96 model (black text), for the
Kan-like model (red text), and SD normalized for average FTV (blue text) are given in legends. The Earth is on the left. 3
Comparison with the T96 model It is seen
that two models demonstrate rather close results. A total of the X-point motion towards the dipole with increasing tilt an-
gle could mean that CS evolves toward an unstable state. Ac-
cording to the solution (Eqs. 7–9), the X-point location also
depends on the CS width L and model parameter b0. The X-
point position as a function of ϕ is plotted in Fig. 2 for three
values of b0, corresponding to three different levels of geo-
magnetic activity (see Fig. 4, right column). It is seen that
for tilt angles |ϕ/2| < 45◦the X point stays very far beyond
60RE for any realistic value of L and b0, e.g., for b0 = 8 (the
value, corresponding to quiet magnetotail) and |ϕ/2| = 45◦
the X point stays as far as ≈340 L. For |ϕ/2| = 60◦(almost
2 times more than the Earth maximal dipole tilt) an approach
to 8.5 L is achieved. www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets
645 rovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 646 σ / <FTV>
(a) σ / <FTV>
σ / <FTV>
(a)
(b)
Figure 5. Comparison of analytical and empirical models for symmetric (dipole tilt 0◦, a) and bent (30◦clockwise, b) current sheets. Standard deviations, σ, normalized for average FTV, are shown as functions of x0, where x0 is the center of the region under consideration
[x0 −2.5, x0 + 2.5], for quiet (black curves), substorm (blue curves) and storm (red curves) conditions. The Earth is on the left. σ / <FTV>
(b) Figure 5. Comparison of analytical and empirical models for symmetric (dipole tilt 0◦, a) and bent (30◦clockwise, b) current sheets. Standard deviations, σ, normalized for average FTV, are shown as functions of x0, where x0 is the center of the region under consideration
[x0 −2.5, x0 + 2.5], for quiet (black curves), substorm (blue curves) and storm (red curves) conditions. The Earth is on the left. results. The left column shows the symmetrical case (zero
dipole tilt), and the right column corresponds to the dipole
tilt angle of 30◦clockwise. ations are bigger for the more quiet magnetosphere environ-
ment, and (3) deviations are smaller for a tilt angle of 30◦. Compared to results of the large interval analysis (Fig. 4),
dependence on the activity level is the same, and dependence
on the tilt angle demonstrates opposite behavior. Overall, an-
alytical and empirical models show good agreement beyond
15RE, improving with growing activity. One can see that the agreement between two models is
quite good, with the maximal SDs varying within 2–11 %. The values of dmax = σmax/⟨FTV⟩are given in legends of
Fig. 4, where σ is the SD and ⟨FTV⟩is the average FTV. The
better agreement is achieved for disturbed magnetospheric
conditions, i.e., the analytical model describes the stretched
CSs even better than the thicker ones. It is found that minimal
difference between two models is obtained when parameter a
is very close to the medium neutral sheet position determined
from the empirical model. The best-fit value of the parameter
b0, controlling the field lines stretching and the CS thinning,
depends on the level of activity and the dipole tilt angle. It
grows from 8.8 for the quiet magnetosphere to 51 for storm
conditions. At any fixed distance, the stretching of field lines
makes the FTV decrease with growing magnetospheric activ-
ity. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets For example, at the distance of x = 30RE it changes from
≈5RE/nT for “quiet” conditions to ≈3RE/nT for “sub-
storm” conditions and to the ≈1.6RE/nT for “storm-time”
conditions. On the contrary, the asymmetric deformation of
CS (dipole tilt angle) forces the FTV to increase. www.ann-geophys.net/36/641/2018/ www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 647 2
4
6
8
10
12
14
−500
0
500
1000
1500
x, L
Beq
−1
1/Bz, B0
−1
b=1.0, k=1.0, n=1.00
b=1.0, k=2.0, n=0.99
b=1.0, k=0.1, n=0.99
b=1.0, k=1.0, n=0.99
b=0.5, k=1.0, n=0.98
b=1.0, k=1.0, n=0.98
b=2.0, k=1.0, n=0.98
b=1.0, k=1.0, n=1.10
Figure 7. Profiles 1/Bz(x,0) for symmetric Kan-like CS, calcu-
lated from an analytical model (Eqs. 11–16). Parameters {a1 =
0, a2 = 0, ϕ = 0, f = 0} are the same. Other parameters are {b0 =
1, k = 1, n = 1} (red), {b0 = 1, k = 2, n = 0.99} (dark-green dash-
dotted), {b0 = 1, k = 0.1, n = 0.99} (dark-green dashed), {b0 =
1, k = 1, n = 0.99} (dark-green solid), {b0 = 0.5, k = 1, n = 0.98}
(violet dash-dotted), {b0 = 1, k = 1, n = 0.98} (violet solid), {b0 =
2, k = 1, n = 0.98} (violet dashed), and {b0 = 1, k = 1, n = 1.1}
(cyan). Red curve shows the original Kan solution. Units are nor-
malized for CS typical width L and for B−1
0 . 2
4
6
8
10
12
14
−500
0
500
1000
1500
x, L
Beq
−1
1/Bz, B0
−1
b=1.0, k=1.0, n=1.00
b=1.0, k=2.0, n=0.99
b=1.0, k=0.1, n=0.99
b=1.0, k=1.0, n=0.99
b=0.5, k=1.0, n=0.98
b=1.0, k=1.0, n=0.98
b=2.0, k=1.0, n=0.98
b=1.0, k=1.0, n=1.10 z, L
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
(a)
0L)
(-B
x, L
2
3
4
5
6
7
8
9
10
n = 0.95
−0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
(b)
z, L
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
x, L
2
3
4
5
6
7
8
9
10
0L)
(-B
n = 1.00
−0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
(c)
z, L
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
x, L
2
3
4
5
6
7
8
9
10
0L)
(-B
n = 1.05
x, L
2
3
4
5
6
7
8
9
10
−0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
(d)
z, L
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
0L)
(-B
n = 1.10
Figure 6. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets It is seen that the Kan solution
(n = 1) is the only degenerated case when the first term of W
turns to 1 and its derivative to zero; hence, in the distant tail
Bz ∼(1/x2+k) due to the rightmost term of expression (14). For any n ̸= 1 we have (∂W/∂x)/W →O(1/x). For symmetric Kan-like CS without plasmoids (a = 0, f =
0, ϕ = 0), the quantity Bz at the x axis takes the simple form
Bz(x,0) = −(∂W/∂x)/(2W). It is seen that the Kan solution
(n = 1) is the only degenerated case when the first term of W
turns to 1 and its derivative to zero; hence, in the distant tail
Bz ∼(1/x2+k) due to the rightmost term of expression (14). For any n ̸= 1 we have (∂W/∂x)/W →O(1/x). Figure 6. The values of magnetic potential 9(x,z), calculated
from analytical model (Eqs. 11–16), are shown by color for
model parameter n = {0.95, 1, 1.05, 1.1} on panels (a)–(d), respec-
tively. Other parameters {a = 0, b0 = 1, ϕ = 0, f = 0, k = 1} are
the same. Magnetic field lines are plotted by white curves. Panel (b)
shows the original Kan solution. Parameter n controls flaring of magnetic field lines; values
of n > 1 force strong convergence of the CS field lines to-
ward the x axis, and hence the location of the X-line is dras-
tically dependent on n. This feature is illustrated in Fig. 6,
where four symmetric magnetic configurations with (a =
0, ϕ = 0, f = 0, b0 = 1, k = 1) and n = {0.95, 1, 1.05, 1.1}
are plotted. In Fig. 7 reverse values of the equatorial magnetic
field, B−1
z (x,0), are plotted for several sets of the model pa-
rameters. The set of green curves illustrates contribution of
the parameter k. The set of violet curves shows the effect of
the parameter b0 variation. The set of solid curves demon-
strates the parameters n impact. It is seen that (a) all curves
except the red one (original Kan solution, n = 1) tend to Assuming {f, n, k} to be real values, a = a1 + ia2, and b =
b0 exp(iϕ), we derive 9 = ln
f cosX∗+
p
1 + f 2 coshZ∗
√
W
! D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets The values of magnetic potential 9(x,z), calculat
from analytical model (Eqs. 11–16), are shown by color f
model parameter n = {0.95, 1, 1.05, 1.1} on panels (a)–(d), respe
tively. Other parameters {a = 0, b0 = 1, ϕ = 0, f = 0, k = 1} a
the same. Magnetic field lines are plotted by white curves. Panel (
shows the original Kan solution. z, L
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
(a)
0L)
(-B
x, L
2
3
4
5
6
7
8
9
10
n = 0.95
L)
( B x, L
−0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
(b)
z, L
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
x, L
2
3
4
5
6
7
8
9
10
0L)
(-B
n = 1.00
(c)
0L)
(-B Figure 7. Profiles 1/Bz(x,0) for symmetric Kan-like CS, calcu- Figure 7. Profiles 1/Bz(x,0) for symmetric Kan-like CS, calcu-
lated from an analytical model (Eqs. 11–16). Parameters {a1 =
0, a2 = 0, ϕ = 0, f = 0} are the same. Other parameters are {b0 =
1, k = 1, n = 1} (red), {b0 = 1, k = 2, n = 0.99} (dark-green dash-
dotted), {b0 = 1, k = 0.1, n = 0.99} (dark-green dashed), {b0 =
1, k = 1, n = 0.99} (dark-green solid), {b0 = 0.5, k = 1, n = 0.98}
(violet dash-dotted), {b0 = 1, k = 1, n = 0.98} (violet solid), {b0 =
2, k = 1, n = 0.98} (violet dashed), and {b0 = 1, k = 1, n = 1.1}
(cyan). Red curve shows the original Kan solution. Units are nor-
malized for CS typical width L and for B−1
0 . n = 1.05 Z∗= rn sin(nϑ) + b0
Rk sin(k2 −ϕ),
(13)
W = n2r2(n−1) +
b2
0k2
R2(k+1) + 2nkb0
rn−1
Rk+1 cos[(n −1)ϑ
+ (k + 1)2 −ϕ],
(14)
r =
p
x2 + z2,
ϑ = arctan
z
x
,
(15)
R =
p
(x −a1)2 + (z −a2)2,
θ = arctan
z −a2
x −a1
. (16) (13) For symmetric Kan-like CS without plasmoids (a = 0, f =
0, ϕ = 0), the quantity Bz at the x axis takes the simple form
Bz(x,0) = −(∂W/∂x)/(2W). 4
Normal magnetic component and current density The results of the previous section show that parameters of
the asymmetric Kan-like model may be adapted to provide
rather good agreement with the magnetotail CS, especially in
a distant tail beyond 15–20 RE, and especially for bent cur-
rent sheets. However, until now the practical usage of this
model encountered a substantial obstacle, related to the be-
havior of the normal magnetic field component. It can be
easily checked that in the distant tail the Kan model yields
Bz ∼1/x3, while in reality Bz decreases as 1/x or even
slower (e.g., Behannon and Ness, 1966; Mihalov et al., 1968;
Behannon, 1970; Wang and Lyons, 2004; Yue et al., 2013). For plane and axially symmetric current sheets the solution
with Bz ∼1/xα with arbitrary α is found in Vasko et al. (2013). For Kan-like models considered in the current paper
the Bz problem may be solved by introducing one more pa-
rameter in the generating function g(ζ). With the additional
parameter n, general asymmetric model takes the form (com-
pare to Eq. 16 of Yoon and Lui, 2005) Figure 4 shows a comparison of two models within the
large interval x ∈[5, 30]. To detect the best-matching region
we performed the same analysis for eight short overlapping
intervals x ∈[7.5, 12.5] + 2.5n, where n = 0, 1,...,8. The
normalized SD as a function of x0n, where x0n is the cen-
ter of corresponding interval, is shown in Fig. 5, where three
features are observed: (1) SD grows toward the Earth and
exceeds 10% for x < 15RE for all activity levels, (2) devi- g(ζ) = f +
q
1 + f 2 exp
i
ζ n −
b
(ζ −a)k
. (10) (10) www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets
647 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets −1.2
−1
−0.8
−0.6
−0.4
−0.2
20
30
40
50
60
70
80
x, L
10
b)
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
Jy, J0
Jy J0
) 20
30
40
50
60
70
80
x, L
10
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
a)
−1.2
−1
−0.8
−0.6
−0.4
−0.2
−1.4
Jy, J0
Jy J
) 20
30
40
50
60
70
80
x, L
10
c)
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
−1.2
−1
−0.8
−0.6
−0.4
−0.2
−1.4
Jy, J0
) 20
30
40
50
60
70
80
x, L
10
d)
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
−1.2
−1
−0.8
−0.6
−0.4
−0.2
Jy, J0
) ,
20
30
40
50
60
70
80
x, L
10
f)
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
−1.2
−1
−0.8
−0.6
−0.4
−0.2
−1.4
Jy, J0
) Figure 8. Current density Jy(x,z) by an analytical model (Eqs. 11–16) for {a1 = 0, a2 = −0.03, b = 22.13, f = 0, k = 1} is shown for
three values of the parameter n for plane sheets (a, c, e, ϕ = 0) and curved sheets (b, d, f, ϕ = 60). In panels (a, b), n = 0.995. In (c, d)
n = 1. In (e, f), n = 1.005. Panel (c) corresponds to the plane substorm sheet (see Fig. 4c). Units are normalized for CS typical width L and
J0 = cB0/(4πL). O(x), and (b) numerical values of Bz are highly variable de-
pending on different combinations of parameters {b0, k, n}. tion of the CS width and 20% growth of the peaking current
density. In two dimensions, contributions of parameters n and ϕ are
shown in the next two plots. Figures 8 and 9 present Jy(x,z)
and Bz(x,z), respectively, for six sets of the model param-
eters, where parameters a1 = 0, a2 = −0.03, f = 0, b0 =
22.13, k = 1 are the same. Panels (a) show the solutions for
(n = 0.995, ϕ = 0). Panels (b) show the solutions for the
bent sheet (n = 0.995, ϕ/2 = 30). On panels (c) solutions
for a plane substorm CS model (see Fig. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets ,
(11)
X∗= rn cos(nϑ) −b0
Rk cos(k2 −ϕ),
(12) (11) (12) www.ann-geophys.net/36/641/2018/ www.ann-geophys.net/36/641/2018/
Ann. Geophys., 36, 641–653, 2018 www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 www.ann-geophys.net/36/641/2018/ D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 648 20
30
40
50
60
70
80
x, L
10
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
a)
−1.2
−1
−0.8
−0.6
−0.4
−0.2
−1.4
Jy, J0
−1.2
−1
−0.8
−0.6
−0.4
−0.2
20
30
40
50
60
70
80
x, L
10
b)
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
Jy, J0
20
30
40
50
60
70
80
x, L
10
c)
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
−1.2
−1
−0.8
−0.6
−0.4
−0.2
−1.4
Jy, J0
20
30
40
50
60
70
80
x, L
10
d)
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
−1.2
−1
−0.8
−0.6
−0.4
−0.2
Jy, J0
20
30
40
50
60
70
80
x, L
10
e)e)
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
−1.2
−1
−0.8
−0.6
−0.4
−0.2
−1.4
Jy, J0
20
30
40
50
60
70
80
x, L
10
f)
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
−1.2
−1
−0.8
−0.6
−0.4
−0.2
−1.4
Jy, J0
)
)
)
)
)
)
Figure 8. Current density Jy(x,z) by an analytical model (Eqs. 11–16) for {a1 = 0, a2 = −0.03, b = 22.13, f = 0, k = 1} is shown for
three values of the parameter n for plane sheets (a, c, e, ϕ = 0) and curved sheets (b, d, f, ϕ = 60). In panels (a, b), n = 0.995. In (c, d)
n = 1. In (e, f), n = 1.005. Panel (c) corresponds to the plane substorm sheet (see Fig. 4c). Units are normalized for CS typical width L and
J0 = cB0/(4πL). D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets −2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
−0.05
−0.04
−0.03
−0.02
−0.01
0
0.01
0.02
0.03
20
30
40
50
60
70
80
x, L
b)
b)
Bz, B0
) 20
30
40
50
60
70
80
x, L
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
−5
−4
−3
−2
−1
0
1
2
3
4
5
x 10−3
a)a)
Bz, B0 x, L
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
20
30
40
50
60
70
80
x, L
−5
−4
−3
−2
−1
0
1
2
3
4
x 10−3
c)c)
Bz, B0
L
) ,
−3
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
20
30
40
50
60
70
80
x, L
−0.05
−0.04
−0.03
−0.02
−0.01
0
0.01
0.02
0.03
d)
d)
Bz, B0
) x, L
−3
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
20
30
40
50
60
70
80
x, L
−0.04
−0.03
−0.02
−0.01
0
0.01
0.02
0.03
f)f)
Bz, B0
) Figure 9. Magnetic field component Bz(x,z) by an analytical model (Eqs. 11–16) for {a1 = 0, a2 = −0.03, b = 22.13, f = 0, k = 1} is
shown for three values of the parameter n for plane sheets (a, c, e, ϕ = 0) and curved sheets (b, d, f, ϕ = 60). In panels (a, b), n = 0.995. In (c, d), n = 1. In (e, f) n = 1.005. Panel (c) corresponds to the plane substorm sheet (see Fig. 4c). Units are normalized for CS typical width
L and B0. sented in Table 1 of Runov et al. (2006). Assuming Vi =
q
V 2x + V 2y + V 2z and B0 = BL, the quantities L and J0 are
calculated. The plot of J0(L) is shown in Fig. 10. Most of
the points, which we call “regular”, lie within the interval
of L ∈[2, 8]103 km and J0 ∈[3, 15]nAm−2 (red asterisks). Other points represent extremely small values of CS param-
eters, such as very low ion temperature (Ti < 2keV, blue
crosses), drift velocity (Vi < 35kms−1, blue diamonds) and
number density (ni < 0.2cm−3, blue asterisks). A single case
an of extremely high value of Vi = 659kms−1 is marked by
a magenta circle. which are mostly single-peaked current sheets. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 4c) with n = 1 and
ϕ = 0 are shown; the bent sheet (n = 1, ϕ/2 = 30) quantities
are plotted on panels (d). On panels (e) parameter n = 1.005
and ϕ = 0, and on panels (f) n = 1.005 and ϕ/2 = 30. Figure 9 shows that even so weak a variation of parame-
ter n affects the distribution of Bz, mostly near to the sheet
center. The range of appropriate values of n is restricted from
above by the solution geometry (X-point location). Say, for
current model parameters and with n = 1.01, the X point is
located at x ≈65 in a plane sheet, and it approaches x ≈50
for ϕ/2 = 30. As expected, the increase of tilt angle ϕ en-
hances the value of Bz, so that for ϕ/2 = 30◦Bz is growing
10 times. The solution (Eqs. 11–16) is written in normalized units,
where the magnetic field is normalized for the lobe value B0,
and normalization constants for the length scale and current
density are Figure 8 demonstrates that the CS width is almost uniform
on x and is not affected by tilt angle, controlling only the
sheet location (vertical shift may be recouped by the proper
choice of parameter a2). With increasing parameter n, the
sheet is thinning and, correspondingly, the peaking current
density is growing. The same effect is produced by enhanced
geomagnetic activity. Comparison of current densities for
quiet and storm conditions (not shown) reveal 20% reduc- L = 2cTi
eB0Vi
= 2 · 103 ·
Ti [keV]
B0 [nT]Vi [kms−1], 103 km,
(17)
J0 = cB0
4πL = 0.8 ·
B0 [nT]
L[103 km]
, nAm−2. (18) (17) (18) (18) To estimate the relevance of this scaling, we make
use of Cluster data of magnetotail CS crossings, pre- Ann. Geophys., 36, 641–653, 2018 www.ann-geophys.net/36/641/2018/ rovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets The analyti-
cal model (Eqs. 11–16) preserves basic features of the initial
Harris solution, and hence it is unable to resolve the com-
plex CS structure, such as bifurcated or embedded current
sheets (see, e.g., Hoshino et al., 1996; Nakamura et al., 2006;
Runov et al., 2006; Artemyev et al., 2009; Petrukovich et al.,
2015). It means that the cross-sheet profiles of current den-
sity in our model (not shown) resemble the Harris profiles,
shown in Figs. 2 and 3 of Runov et al. (2006). Hence, analyt-
ical estimates of the CS width usually exceed the real values. However, in some cases (e.g., cases 3, 27 and 28) the Harris
profiles may be more or less relevant to real current sheets. Figure 11 shows the model normalization constant J0 vs. peaking observed perpendicular current density (blue curve
in Fig. 2 of Runov et al., 2006). It is seen that analytical es-
timates and measured values of J0 mismatch in all extreme
cases of Fig. 10. In other cases (“regular” points, red aster-
isks) the model estimate agrees with observed values with
an accuracy up to a coefficient k ∈[0.5, 2] (except for the
cases 4 and 23 of Runov et al., 2006, when the discrep-
ancy increases by 2.5 times). Thus, the best match of current
densities is found for cases {1–3, 5–7, 11, 15–18, 22, 25–28}, D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 649 20
30
40
50
60
70
80
x, L
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
−5
−4
−3
−2
−1
0
1
2
3
4
5
x 10−3
a)a)
Bz, B0
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
−0.05
−0.04
−0.03
−0.02
−0.01
0
0.01
0.02
0.03
20
30
40
50
60
70
80
x, L
b)
b)
Bz, B0
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
20
30
40
50
60
70
80
x, L
−5
−4
−3
−2
−1
0
1
2
3
4
x 10−3
c)c)
Bz, B0
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
20
30
40
50
60
70
80
x, L
−0.05
−0.04
−0.03
−0.02
−0.01
0
0.01
0.02
0.03
d)
d)
Bz, B0
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
20
30
40
50
60
70
80
x, L
−5
−4
−3
−2
−1
0
1
2
3
4
x 10−3
e)e)
Bz, B0
−2
−1.5
−1
−0.5
0
0.5
1
1.5
2
z, L
20
30
40
50
60
70
80
x, L
−0.04
−0.03
−0.02
−0.01
0
0.01
0.02
0.03
f)f)
Bz, B0
)
)
)
)
)
Figure 9. Magnetic field component Bz(x,z) by an analytical model (Eqs. 11–16) for {a1 = 0, a2 = −0.03, b = 22.13, f = 0, k = 1} is
shown for three values of the parameter n for plane sheets (a, c, e, ϕ = 0) and curved sheets (b, d, f, ϕ = 60). In panels (a, b), n = 0.995. In (c, d), n = 1. In (e, f) n = 1.005. Panel (c) corresponds to the plane substorm sheet (see Fig. 4c). Units are normalized for CS typical width
L and B0. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets If two plasma components
give zero contribution in the current velocity, Vi2 −Ve2 =
0, Eq. (1) stays valid for nonuniform plasma tempera-
ture (Voronina and Kan, 1993). The four-component-plasma
model could be probably appropriate for magnetotail stud-
ies at high levels of geomagnetic activity. Indeed, in the
quiet magnetotail the population of ions {O+, O++, He++},
penetrating from the ionosphere, is less than 1% (Lennarts-
son et al., 1986), and hence the approximation of “pro-
ton + electron” plasma is relevant. With the growth of ge-
omagnetic activity, the O+ contribution becomes essential
during the main and recovery phases of intensive storm
events. However, practical application of the nonisothermal
model requires thorough studies, going beyond the scope of
the present paper. Figure 10. Analytical model units: current density J0 vs. spa-
tial scale L from Eqs. (17) and (18) for Cluster data of cur-
rent sheet crossings, presented in Table 1 of Runov et al. (2006). Blue crosses show cases of the lowest ion temperature, Ti <
2keV (8, 9, 12, 13, 14 in Table 1 of Runov et al., 2006); blue dia-
monds show cases of the lowest ion drift velocity, Vi < 35kms−1
(8, 9, 10, 19, 21, 29, 30); and blue asterisks show cases (20, 24) of
the lowest ion number density, ni < 0.2cm−3. Magenta circle
shows the case 20 of extremely high velocity, Vi = 659kms−1. All
other “regular” cases are shown by red asterisks. The red line plots
the fitting curve y = 1/(0.045x −0.01). plasma, CS stability analysis and numerical simulations of
magnetotail dynamics. So far these studies were restricted
by purely symmetric background equilibria. In this paper we
present the extension of the well-known family of exact ki-
netic Harris–Fadeev–Kan–Manankova solutions to the 2-D
bent CS. This extension is really important, since the Earth
dipole is tilted most of the time. The constancy of the proton temperature is not reflected
in observations (e.g., Kissinger et al., 2012; Wang et al.,
2012), and hence the isothermal model may be considered
as a first approximation only, although for some local anal-
ysis it seems to be rather suitable due to the small (∼10–
20 %) cross-cut variations of proton temperature, detected
in observations of central-peaked current sheets (see Fig. 5
in Runov et al., 2006). D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 650 0
5
10
15
20
0
5
10
15
20
25
30
35
40
L, 1000 km
J , nA m
0
-2
Regular
Ti < 2 keV
Vi < 35 km/s
V = 659 km s
i
ni < 0.2 cm−3
The curve y = 1 / (0.045x − 0.01)
−1
Figure 10. Analytical model units: current density J0 vs. spa-
tial scale L from Eqs. (17) and (18) for Cluster data of cur-
rent sheet crossings, presented in Table 1 of Runov et al. (2006). Blue crosses show cases of the lowest ion temperature, Ti <
2keV (8, 9, 12, 13, 14 in Table 1 of Runov et al., 2006); blue dia-
monds show cases of the lowest ion drift velocity, Vi < 35kms−1
(8, 9, 10, 19, 21, 29, 30); and blue asterisks show cases (20, 24) of
the lowest ion number density, ni < 0.2cm−3. Magenta circle
shows the case 20 of extremely high velocity, Vi = 659kms−1. All
other “regular” cases are shown by red asterisks. The red line plots
the fitting curve y = 1/(0.045x −0.01). and parameter ϕ specifies the CS bending. For further stud-
ies the more general model (Eqs. 11–16) can be considered,
where additional parameters n and k provide the more accu-
rate adjustment of the magnetoplasma quantities. Moreover,
for sub-Alfvénic plasma, i.e., for the low-activity periods, all
model parameters may be treated as time-dependent quanti-
ties (Wolf, 1983; Semenov et al., 2015). The time-dependent
approach in such a modeling approach is not appropriate for
the periods of explosive activity, such as storms and sub-
storms, when BBFs with Alfvénic speed are produced. 0
5
10
15
20
0
5
10
15
20
25
30
35
40
L, 1000 km
J , nA m
0
-2
Regular
Ti < 2 keV
Vi < 35 km/s
V = 659 km s
i
ni < 0.2 cm−3
The curve y = 1 / (0.045x − 0.01)
−1 Of course, the suggested analytical model is still far from
universality. One significant limitation of this model is re-
lated to the isothermal constraint. This constraint may be
released for four-component (two positive + two negative)
plasma with bi-Maxwellian distribution functions for each
particle species (Kan, 1973; Voronina and Kan, 1993). In
such a case the condition (2) takes the form Vik/Tik +
Vek/Tek = 0, where k = {1, 2}. 5
Discussion and conclusions In empirical models (T89, T96, T01, TS05, etc.) magnetic
field configurations with any plasma populations are not
force-balanced since ∇× [j × B] ̸= 0, or there is no ∇P
to balance Ampere’s force (Zaharia et al., 2003). That is
why we crucially need kinetic force-balanced CS models for
many magnetospheric studies, such as wave generation in www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets In such sheets, inaccuracy of the
constant-temperature estimate does not exceed the model in-
accuracy in current density or CS width. To validate the obtained analytic solution for bent CS
we performed a comparison with the T96 model, used as
a proxy of realistic averaged magnetospheric configuration. It is shown that the proposed model provides a reasonable ap-
proximation for the magnetotail CS in a wide range of dipole
tilt angles and geomagnetic activity levels. Particularly, the
parameters of the analytical model can always be adjusted
to fit the behavior of the magnetic FTV with an accuracy of
about 10% for all distances from 5 to 30 RE tailward. For
short segments (5RE) of the CS, located beyond 15RE, the
agreement may be improved up to 5% (except the case of
the bent CS at quiet magnetospheric conditions). The agree-
ment between analytical and empirical models is found to be
better for the stretched magnetic configuration, i.e., for the
pre-substorm conditions. Other model limitations are the two-dimensionality and
isotropy of the plasma pressure. Even with these limitations,
the model stays appropriate for a wide class of problems,
mentioned in the beginning of the current section. Particu-
larly, we lay hopes that application of the presented model
can stimulate investigations on the magnetotail CS stability
to resolve the questions suggested by Kivelson and Hughes
(1990): why can symmetric CS accumulate magnetic flux en-
ergy more effectively, and does the threshold of substorm-
initiating instability depend on degree of the CS bending? Notably, such a good agreement is obtained for the sim-
plest three-parametric Kan-like model (Eqs. 7–9), where pa-
rameter a controls the CS displacement from the equatorial
plane, parameter b0 controls magnetic field lines stretching, Our findings are summarized as follows: www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 rovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 651 0
5
10
15
20
25
0
5
10
15
20
25
30
35
40
J , nA m , Runov et al. (2006)
0
-2
J , nA m , model
0
-2
Regular
Ti < 2 keV
V < 35 km s
i
V = 659 km s
i
ni < 0.2 cm−3
The curve y = x
4
23
−1
Figure 11. Typical current density J0 of the analytical model vs. peaking perpendicular current density from Fig. Competing interests. The authors declare that they have no conflict
of interest. Competing interests. The authors declare that they have no conflict
of interest. – Parameters of the asymmetric model may be adjusted to
reproduce the realistic distribution of the magnetic flux
tube volume at any level of geomagnetic activity; with
enhancing activity the model relevance improves. The
model-typical scales for CS width and current density
match the corresponding parameters of the in situ regis-
tered single-peaked current sheets with medium values
of number density, proton temperature and drift veloc-
ity; disagreement does not exceed a factor of 2. – Parameters of the asymmetric model may be adjusted to
reproduce the realistic distribution of the magnetic flux
tube volume at any level of geomagnetic activity; with
enhancing activity the model relevance improves. The
model-typical scales for CS width and current density
match the corresponding parameters of the in situ regis-
tered single-peaked current sheets with medium values
of number density, proton temperature and drift veloc-
ity; disagreement does not exceed a factor of 2. Acknowledgements. This study has been supported by the Austrian
Science Fund (FWF), P 27012-N27 and I 3506-N27, and by Rus-
sian Science Foundation (RSF) grant no. 18-47-05001. The authors
thank Anna V. Egorova for her help with preparation of the images,
and reviewers for their help in improving the paper. The topical editor, Elias Roussos, thanks two anonymous refer-
ees for help in evaluating this paper. – The asymmetric solution does not contain any limitation
for the tilt angle values, and hence the model is appro-
priate for any Earth-like magnetosphere with arbitrary
dipole inclination. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 2 of Runov et al. (2006). Blue crosses show cases of the lowest ion temperature, Ti < 2keV (8, 9, 12, 13, 14 in Table 1 of Runov et al. (2006)); blue diamonds show
cases of the lowest ion drift velocity, Vi < 35kms−1 (8, 9, 10, 19, 21, 29, 30); and blue asterisks show cases (20, 24) of the lowest ion number
density, ni < 0.2cm−3. Magenta circle shows the case 20 of extremely high velocity, Vi = 659kms−1. All other “regular” cases are shown
by red asterisks. The red line plots y = x. Black arrows mark cases 4 and 23, demonstrating the largest (amongst red points) discrepancy of
the observed and model values. 0
5
10
15
20
25
0
5
10
15
20
25
30
35
40
J , nA m , Runov et al. (2006)
0
-2
J , nA m , model
0
-2
Regular
Ti < 2 keV
V < 35 km s
i
V = 659 km s
i
ni < 0.2 cm−3
The curve y = x
4
23
−1 Figure 11. Typical current density J0 of the analytical model vs. peaking perpendicular current density from Fig. 2 of Runov et al. (2006). Blue crosses show cases of the lowest ion temperature, Ti < 2keV (8, 9, 12, 13, 14 in Table 1 of Runov et al. (2006)); blue diamonds show
cases of the lowest ion drift velocity, Vi < 35kms−1 (8, 9, 10, 19, 21, 29, 30); and blue asterisks show cases (20, 24) of the lowest ion number
density, ni < 0.2cm−3. Magenta circle shows the case 20 of extremely high velocity, Vi = 659kms−1. All other “regular” cases are shown
by red asterisks. The red line plots y = x. Black arrows mark cases 4 and 23, demonstrating the largest (amongst red points) discrepancy of
the observed and model values. of the X point is much more effectively controlled by
the new parameter n of the generalized model (Eqs. 11–
16). – An exact 2-D bent CS equilibrium, built by means of
generalization of the Harris–Fadeev–Kan–Manankova
family of symmetric solutions of the Vlasov–Maxwell
equations, is considered. The examined model repro-
duces the effects, related to the Earth dipole tilt and CS
bending. The further generalization releases degeneracy
of the original model, which caused of the normal mag-
netic component to decrease too rapidly. Data availability. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets No data sets were used in this article. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets J., Reeves, G. D., and
Thomsen, M. F.: Different magnetospheric modes: solar wind
driving and coupling efficiency, Ann. Geophys., 27, 4281–4291,
https://doi.org/10.5194/angeo-27-4281-2009, 2009. Harris, E. G.: On a plasma sheath separating regions of oppo-
sitely directed magnetic field, Nuovo Cimento, 23, 115–121,
https://doi.org/10.1007/BF02733547, 1962. Petrukovich,
A.,
Artemyev,
A.,
Vasko,
I.,
Nakamura,
R.,
and Zelenyi, L.: Current Sheets in the Earth Magneto-
tail:
Plasma
and
Magnetic
Field
Structure
with
Cluster
Project Observations, Space Sci. Rev., 188(1–4), 311–337,
https://doi.org/10.1007/s11214-014-0126-7, 2015. Hau, L. N., Wolf, R. A., Voigt, G. H., and Wu, C. C.:
Steady
state
magnetic
field
configurations
for
the
Earth’s
magnetotail,
J. Geophys. Res. 94,
1303–1316,
https://doi.org/10.1029/JA094iA02p01303, 1989. Hoshino, M., Nishida, A., Mukai, T., Saito, Y., and Yamamoto, T.:
Structure of plasma sheet in magnetotail: Double-peaked
electric current sheet, J. Geophys. Res., 101, 24775–24786,
https://doi.org/10.1029/96JA02313, 1996. Pritchett, P. L. and Coroniti, F. V.: Formation and stability of the
self-consistent one-dimensional current sheet, J. Geophys. Res.,
97, 16773–16787, https://doi.org/10.1029/92JA01550, 1992. Pritchett, P. L. and Coroniti, F. V.: A kinetic ballooning/interchange
instability in the magnetotail, J. Geophys. Res., 115, A06301,
https://doi.org/10.1029/2009JA014752, 2010. Kan,
J. R.:
On
the
structure
of
the
magnetotail
cur-
rent
sheet,
J. Geophys. Res.,
78,
3773–3781,
https://doi.org/10.1029/JA078i019p03773, 1973. Runov, A., Sergeev, V. A., Nakamura, R., Baumjohann, W., Ap-
atenkov, S., Asano, Y., Takada, T., Volwerk, M., Vörös, Z.,
Zhang, T. L., Sauvaud, J.-A., Réme, H., and Balogh, A.: Local
structure of the magnetotail current sheet: 2001 Cluster observa-
tions, Ann. Geophys., 24, 247–262, 2006. Kissinger, J., McPherron, R. L., Hsu, T.-S., and Angelopoulos, V.:
Diversion of plasma due to high pressure in the inner magneto-
sphere during steady magnetospheric convection, J. Geophys. Res.,
117,
A05206,
https://doi.org/10.1029/2012JA017579,
2012. Schindler, K.: A self-consistent theory of the tail of the magneto-
sphere, in: Earth’s Magnetospheric Processes, vol. 32, edited by:
McComas, B. M., D. Reidel, Dordrecht, pp. 200–209, 1972. Kivelson,
M. G. and
Hughes,
W. J.:
On
the
threshold
for triggering substorms, Planet. Space Sci., 38, 211–220,
https://doi.org/10.1016/0032-0633(90)90085-5, 1990. Schindler, K. and Birn, J.: Models of two-dimensional embedded
thin current sheets from Vlasov theory, J. Geophys. Res., 107,
1193, https://doi.org/10.1029/2001JA000304, 2002. Kubyshkina, M., Tsyganenko, N., Semenov, V., Kubyshkina, D.,
Partamies, N., and Gordeev, E.: Further evidence for the role
of magnetotail current shape in substorm initiation, Earth Plan-
ets Space, 67, 139, https://doi.org/10.1186/s40623-015-0304-1,
2015. Semenov,
V. S.,
Kubyshkina,
D. I.,
Kubyshkina,
M. V.,
Kubyshkin,
I. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 652 Behannon, K. W.: Geometry of the geomagnetic tail, J. Geophys. Res., 75, 743–753, https://doi.org/10.1029/JA075i004p00743,
1970. Manankova, A. V., Pudovkin, M. I., and Runov, A. V.: Stationary
configurations of the two-dimensional current-carrying plasma
sheet: Exact solutions, Geomagn. Aeronomy+, 40, 430–438,
2000. Behannon, K. W., and Ness, N. F.: Magnetic storms in the
earth’s magnetic tail, J. Geophys. Res., 71, 2327–2351,
https://doi.org/10.1029/JZ071i009p02327, 1966. Mihalov, J. D., Colburn, D. S., Currie, R. G., and Sonett, C. P.: Con-
figuration and reconnection of the geomagnetic tail, J. Geophys. Res., 73, 943–959, https://doi.org/10.1029/JA073i003p00943,
1968. Birn, J., Hesse, M., Schindler, K., and Zaharia, S.: Role of en-
tropy in magnetotail dynamics, J. Geophys. Res., 114, A00D03,
https://doi.org/10.1029/2008JA014015, 2009. Nakamura, R., Baumjohann, W., Runov, A., and Asano, Y.: Thin
Current Sheets in the Magnetotail Observed by Cluster, Space
Sci. Rev., 122, 29–38, https://doi.org/10.1007/s11214-006-6219-
1, 2006. Burkhart,
G. R.,
Drake,
J. F.,
Dusenbery,
P. B.,
and
Speiser,
T. W.:
A
particle
model
for
magnetotail
neu-
tral sheet equilibria, J. Geophys. Res., 97, 13799–13815,
https://doi.org/10.1029/92JA00495, 1992. Nowada, M., Shue, J.-H., and Russel, C. T.: Effects of dipole tilt an-
gle on geomagnetic activity, Planet. Space Sci., 57, 1254–1259,
https://doi.org/10.1016/j.pss.2009.04.007, 2009. Cargill, P. G., Chen, J., and Holland, J. B.: One-dimensional hybrid
simulations of current sheets in the quiet magnetotail, Geophys. Res. Lett., 21, 2251–2254, https://doi.org/10.1029/94GL01693,
1994. Panov, E. V., Nakamura, R., Baumjohann, W., Kubyshkina, M. G.,
Artemyev, A. V., Sergeev, V. A., Petrukovich, A. A., An-
gelopoulos, V., Glassmeier, K.-H., McFadden, J. P., and
Larson,
D.:
Kinetic
ballooning/interchange
instability
in a bent plasma sheet, J. Geophys. Res., 117, A06228,
https://doi.org/10.1029/2011JA017496, 2012. Erkaev, N. V., Semenov, V. S., and Biernat, H. K.: Mag-
netic
double-gradient
instability
and
flapping
waves
in
a
current
sheet,
Phys. Rev. Lett.,
99,
235003,
https://doi.org/10.1103/PhysRevLett.99.235003, 2007. Erkaev, N. V., Semenov, V. S., Kubyshkin, I. V., Kubyshkina, M. V., and Biernat, H. K.: MHD aspect of current sheet oscillations
related to magnetic field gradients, Ann. Geophys., 27, 417–425,
https://doi.org/10.5194/angeo-27-417-2009, 2009. Panov, E. V., Baumjohann, W., Wolf, R. A., Nakamura, R., An-
gelopoulos, V., Weygand, J. M., and Kubyshkina, M. V.: Magne-
totail energy dissipation during an auroral substorm, Nat. Phys.,
12, 1158–1163, https://doi.org/10.1038/nphys3879, 2016. Fadeev, V. M., Kvartskhava, I. F., and Komarov, N. N.: Self-
focusing of the local plasma currents, Nucl. Fusion, 5, 202–209,
https://doi.org/10.1088/0029-5515/5/3/003, 1965 (in Russian). Partamies, N., Pulkkinen, T. I., McPherron, R. L., McWilliams, K.,
Bryant, C. R., Tanskanen, E., Singer, H. References Artemyev, A. V., Petrukovich, A. A., Zelenyi, L. M., Naka-
mura, R., Malova, H. V., and Popov, V. Y.: Thin embed-
ded current sheets: Cluster observations of ion kinetic struc-
ture and analytical models, Ann. Geophys., 27, 4075–4087,
https://doi.org/10.5194/angeo-27-4075-2009, 2009. – The obtained bent CS solution contains the X point,
moving from infinity toward the dipole with the dipole
tilt increase, staying still far beyond the lunar orbit for
the Earth magnetotail realistic tilt angles. The location www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets V.,
and
Partamies,
N.:
On
the
correla-
tion between the fast solar wind flow changes and sub-
storm
occurrence,
Geophys. Res. Lett.,
42,
5117–5124,
https://doi.org/10.1002/2015GL064806, 2015. Lennartsson, W. and Shelley, E. G.: Survey of 0.1- to 16-keV/e
plasma sheet ion composition, J. Geophys. Res., 91, 3061–3076,
https://doi.org/10.1029/JA091iA03p03061, 1986. Sergeev, V. A., Chernyaev, I. A., Angelopoulos, V., Runov, A. V.,
and Nakamura, R.: Stopping flow bursts and their role in the gen- Ann. Geophys., 36, 641–653, 2018 www.ann-geophys.net/36/641/2018/ D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 653 eration of the substorm current wedge, Geophys. Res. Lett., 41,
1106–1112, https://doi.org/10.1002/2014GL059309, 2014. Wang, C.-P., Gkioulidou, M., Lyons, L. R., and Angelopoulos, V.:
Spatial distributions of the ion to electron temperature ratio in
the magnetosheath and plasma sheet, J. Geophys. Res., 117,
A08215, https://doi.org/10.1029/2012JA017658, 2012. eration of the substorm current wedge, Geophys. Res. Lett., 41,
1106–1112, https://doi.org/10.1002/2014GL059309, 2014. Sitnov, M. I. and Merkin, V. G.: Generalized magnetotail equilib-
ria: Effects of the dipole field, thin current sheets, and mag-
netic flux accumulation, J. Geophys. Res., 121, 7664–7683,
https://doi.org/10.1002/2016JA023001, 2016. Wolf, R. A.: The quasi-static (slow-flow) region of the mag-
netosphere, in: Solar-Terrestrial Physics, vol. 104, edited by:
Carovillano, R. L and Forbes, J. M., Springer, Dordrecht,
https://doi.org/10.1007/978-94-009-7194-3_14, 303–368, 1983. Tsyganenko, N. A.: Modeling the Earth’s magnetospheric magnetic
field confined within a realistic magnetopause, J. Geophys. Res.,
100, 5599–5612, https://doi.org/10.1029/94JA03193, 1995. Yoon, P. H. and Lui, A. T. Y.: Lower-hybrid-drift and modified-
two-stream instabilities in current sheet equilibrium, J. Geophys. Res.,
110,
A02210,
https://doi.org/10.1029/2003JA010180,
2004. Vasko, I. Y., Artemyev, A. V., Popov, V. Y., and Malova, H. V.:
Kinetic models of two-dimensional plane and axially symmet-
ric current sheets: Group theory approach, Phys. Plasmas, 20,
022110, https://doi.org/10.1063/1.4792263, 2013. Yoon, P. H. and Lui, A. T. Y.: A class of exact two-dimensional
kinetic current sheet equilibria, J. Geophys. Res., 110, A01202,
https://doi.org/10.1029/2003JA010308, 2005. Vinogradov, A. A., Vasko, I. Y., Artemyev, A. V., Yushkov, E. V.,
Petrukovich, A. A., and Zelenyi, L. M.: Kinetic models of mag-
netic flux ropes observed in the earth magnetosphere, Phys. Plas-
mas, 23, 072901, https://doi.org/10.1063/1.4958319, 2016. Yue, C., Wang, C.-P., Zaharia, S. G., Xing, X., and Lyons, L.:
Empirical modeling of plasma sheet pressure and three-
dimensional force-balanced magnetospheric magnetic field
structure: 2. Modeling, J. Geophys. Res., 118, 6166–6175,
https://doi.org/10.1002/2013JA018943, 2013. Voronina, V. A. and Kan, J. R.: A kinetic model of the plasma sheet:
isotropic nonuniform plasma temperature, J. Geophys. Res., 98,
13395–13402, https://doi.org/10.1029/93JA00959, 1993. Walker,
G. W.:
Some
problems
illustrating
the
forms
of
nebulae,
P. Roy. Soc. Lond. A
Mat.,
91,
410–420,
https://doi.org/10.1098/rspa.1915.0032, 1915. Zaharia, S. and Cheng, C. Z.: Can an isotropic plasma pressure dis-
tribution be in force balance with the T96 model field?, J. Geo-
phys. Res., 108, 1412, https://doi.org/10.1029/2002JA009501,
2003. Wang,
C.-P. and
Lyons,
L. R.:
Midnight
radial
pro-
files
of
the
quiet
and
growth-phase
plasma
sheet:
The
Geotail
observations,
J. Geophys. Res.,
109,
A12201,
https://doi.org/10.1029/2004JA010590, 2004. Ann. Ann. Geophys., 36, 641–653, 2018 www.ann-geophys.net/36/641/2018/ D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets Geophys., 36, 641–653, 2018
|
https://openalex.org/W4292981881
|
https://zenodo.org/records/7006565/files/Death_of_thermosensitive_genic_male_sterile_seedlings_in_Malaysian_rice_fields.pdf
|
English
| null |
Death of thermosensitive genic male sterile seedlings in Malaysian rice fields
|
Zenodo (CERN European Organization for Nuclear Research)
| 1,997
|
cc-by
| 702
|
Breeding methods Dead seedlings in Malaysian ricefield, 3 wk
after transplanting. fertile and sterile. The same pheno-
menon was observed in the H93-106
family line. Thirteen out of 18 family
lines showed the same phenomenon. All five lines of the H92-150 family line
showed more seedling deaths than the
lines previously mentioned. TG-43
and TG-44 had a few sterile plants. All plants were fertile in the next
generation of TG-43 and TG-44. Two
out of 18 family lines showed the same
phenomenon. Death of thermosensitive genic
male sterile seedlings in
Malaysian ricefields H. Kato and M. Sobri, Japan International
Research Center for Agricultural Sciences,
Ohwashi 1-2, Tsukuba, Ibaraki 305, Japan; and
Guok Hup Ping, Malaysian Agricultural
Research and Development Institute (MARDI),
13200 Kepala Batas, Seberang Perai, Penang,
Malaysia One hundred and fifty-four thermo-
sensitive genic male sterile (TGMS)
lines were introduced in Malaysia from
the National Agricultural Research
Center in Japan. The lines were bred
from indica and japonica crosses and
comprised 31 family lines, ranging
from B 1 F 9 to F 4 . These TGMS lines were
transplanted in ricefields at MARDI
during the 1994-95 dry season (Nov-
Feb) and the 1995 wet season (Apr-
Aug). Some of the plants in 18 family
lines died during the dry season
(see figure). This type of seedling death rate has
not been observed in nonTGMS lines
introduced from Japan, and the phe-
nomenon has never occurred in Japan. The 30-yr average temperature during
the seedling growth month for Tokyo is
18.4 °C and that for Kuala Lumpur is
25.9 °C. Dead seedlings in Malaysian ricefield, 3 wk
after transplanting. the next generation of TG-3, which
originated from the fertile plants of the
TG-3 line, plants were segregated into
fertile and sterile. This trait came from
PL12, which contains a single and
recessive TGMS gene. The segregation
ratio of the TGMS plants, however, was
less than the expected ratio (1/4). Three
out of five lines of the H92-62 family
line also had seedling deaths. TG-22,
TG-23, TG-24, and TG-25 had some
sterile plants. In the next generation of
TG-23, plants were segregated into Seedling death was observed only
during high temperatures. Germina-
tion of TGMS seeds was also poor. We suspect that the high temperature
caused the TGMS seedlings to die. can be explained as follows: all the
TGMS seedlings died from high tem-
perature, so no TGMS plants lived to
flower in the hot dry season. But the
TGMS gene in heterozygous plants was
expressed and both fertile and sterile
plants were produced in the next gener-
ation. The phenomenon of H92-62 was
different from that of H91-4. Perhaps
only a few of the TGMS seedlings died
from high temperature. For H92-150,
we believe most of the TGMS seedlings
and the fertile plants, including the
TGMS heterozygous plants, died from
the high temperature. Death of thermosensitive genic
male sterile seedlings in
Malaysian ricefields The phenomenon observed in H91-4 Seedlings died in four of the five
lines of the H91-4 family line, and no
sterile plants were found (see table). In Dead seedlings in four TGMS family lines. MARDI, Penang, Malaysia. 1994-95. Line
Family line
Combination
Generation
Dead/total Sterile plants
Next generation
seedlings
(no.)
TG-1
H91-4
H89-1//H89-1/
B 1 F 9
1/20
0
TG-2
Mangetsumochi
0/20
0
TG-3
3/20
0
Fertlle; sterile
TG-4
1/20
0
segregation
TG-5
2/20
0
TG-21
H92-62
H89-1/X88
F 10
0/20
0
TG-22
6/20
2/14
TG-23
3/10
2/7
Fertile; sterile
TG-24
12/20
2/8
segregation
TG-25
4/20
6/16
TG-41
H92-150
H89-1/Mansoek//H87-35
F 9
4/20
0
TG-42
8/20
0
TG-43
11/20
1/9
All fertile plants
TG-44
10/20
1/10
All fertile plants
TG-45
4/20
0
TG-56
H93-106
PL12/90SL495
F 7
7/20
3/13
Fertile, sterile
TG-57
5/20
3/15
segregation
TG-58
2/20
0
TG-59
5/20
0
TG-60
2/20
1/18 Dead seedlings in four TGMS family lines. MARDI, Penang, Malaysia. 1994-95. The conclusions drawn from this
study are speculative and need further
confirmation through studies con-
ducted with controlled temperature. Family lines with dead seedlings, how-
ever, cannot be used to produce hybrid
seed. Further selection has to be done to
eliminate this trait. Thirteen out of 31
family lines did not show this pheno-
menon and can therefore be used as
parents for hybrids.
|
https://openalex.org/W4385760204
|
https://vti.diva-portal.org/smash/get/diva2:1792152/FULLTEXT01
|
English
| null |
Exploring interdependencies, vulnerabilities, gaps and bridges in care transitions of patients with complex care needs using the Functional Resonance Analysis Method
|
BMC health services research
| 2,023
|
cc-by
| 20,321
|
© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Exploring interdependencies, vulnerabilities,
gaps and bridges in care transitions of patients
with complex care needs using the Functional
Resonance Analysis Method Ann‑Therese Hedqvist1,2* , Gesa Praetorius3,4 and Mirjam Ekstedt1,5 Hedqvist et al. BMC Health Services Research (2023) 23:851
https://doi.org/10.1186/s12913-023-09832-7 Hedqvist et al. BMC Health Services Research (2023) 23:851
https://doi.org/10.1186/s12913-023-09832-7 BMC Health Services Research Open Access Abstract Background Hospital discharge is a complex process encompassing multiple interactions and requiring coordi‑
nation. To identify potential improvement measures in care transitions for people with complex care needs, intra-
and inter-organisational everyday work needs to be properly understood, including its interdependencies, vulnerabili‑
ties and gaps. The aims of this study were to 1) map coordination and team collaboration across healthcare and social
care organisations, 2) describe interdependencies and system variability in the discharge process for older people
with complex care needs, and 3) evaluate the alignment between discharge planning and the needs in the home. Methods Data were collected through participant observations, interviews, and document review in a region
of southern Sweden. The Functional Resonance Analysis Method (FRAM) was used to model the discharge process
and visualise and analyse coordination of care across healthcare and social care organisations. Results Hospital discharge is a time-sensitive process with numerous couplings and interdependencies
where healthcare professionals’ performance is constrained by system design and organisational boundaries. The
greatest vulnerability can be found when the patient arrives at home, as maladaptation earlier in the care chain can
lead to an accumulation of issues for the municipal personnel in health and social care working closest to the patient. The possibilities for the personnel to adapt are limited, especially at certain times of day, pushing them to make
trade-offs to ensure patient safety. Flexibility and appropriate resources enable for handling variability and responding
to uncertainties in care after discharge. Conclusions Mapping hospital discharge using the FRAM reveals couplings and interdependencies between vari‑
ous individuals, teams, and organisations and the most vulnerable point, when the patient arrives at home. Resilient
performance in responding to unexpected events and variations during the first days after the return home requires
a system allowing flexibility and facilitating successful adaptation of discharge planning. Keywords Complexity, Care transitions, Hospital discharge, Patient safety, Resilience, Functional Resonance Analysis
Method (FRAM) *Correspondence:
Ann‑Therese Hedqvist
anntherese.hedqvist@lnu.se
Full list of author information is available at the end of the article *Correspondence:
Ann‑Therese Hedqvist
anntherese.hedqvist@lnu.se
Full list of author information is available at the end of the article Background Healthcare can be classified as a complex adaptive
system (CAS), as its components have multiple interde-
pendencies and work in nonlinear ways [8, 9]. Complex
systems have fuzzy boundaries and co-evolve with other
systems [10]. A CAS has intrinsic laws or principles, such
as self-organisation, emergent behaviour and the capac-
ity to learn, making it difficult to predict how its compo-
nents will interact temporally in a given situation [11]. This implies that the view of the horizon for each person
in the system is quite limited – and the further away it is,
the more unpredictable the consequences of any actions
taken become. Safety problems are thus not necessarily
a direct result of lacking knowledge or limited effort on
the part of the healthcare professionals – rather, they are
usually a result of the complexity of the system. f
Poor healthcare quality is often related to the inher-
ent complexity of the healthcare system, characterised
by silos, multiple stakeholder interactions and a signifi-
cant degree of performance variability within and across
system levels [19–21]. Care transitions, especially hos-
pital discharges, are complex [15, 22] and require exten-
sive collaboration and information exchange. Previous
research indicates the hazards associated with hospital
discharge and the need of interventions to improve care
transitions for people with complex care needs [15, 23–
25]. The vulnerable exchange points in care transitions
may increase both the need of healthcare services and
healthcare costs [26]. Previous studies on care transitions
highlight the importance of applying a systems thinking
perspective instead of linear thinking to investigate hos-
pital discharge as a process [27–30]. Understanding and
improving care transitions is considered to be of major
importance [31] and is an international healthcare prior-
ity [26, 32], as these are error-prone situations [33]. For-
mer studies describing the discharge planning process,
using the FRAM, have mapped the variability and WAD
up to the discharge [27–30]. One common failure identi-
fied [30] was a lack of teamwork collaboration across hos-
pital and community care, something the current study
has focused on. One recent study [28] evaluated both
pre-discharge processes, follow-up, and readmission. An illustrative example of complexity in healthcare is
the multidisciplinary teamwork in coordinated discharge
planning [12] and the coordination of care for older peo-
ple with complex care needs. Background and favour teamwork over professional and organisa-
tional boundaries. Advancements in modern medicine and technology have
led to new medical interventions and promote high-
quality care, creating possibilities to effectively treat
acute diseases and leading to a decline in mortality and
increased life expectancy [1]. The advancements also lead
to increased specialisation and differentiation which may
create gaps in communication and knowledge transfer
between different healthcare professions and care pro-
viders. Patients with chronic illnesses and complex care
needs, requiring care from both healthcare and social
care providers [2, 3], are especially vulnerable to frag-
mentation of care as they will meet different profession-
als working for several care providers, whether seeking
help for chronic or acute conditions [4, 5]. Coordinating
care between different care providers is recognised as dif-
ficult [6] and is complicated by the diversity of actors and
organisations involved. Due to the burden of increased
chronic illness in the aging population, healthcare sys-
tems worldwide experience challenges in providing safe
and efficient care to older people with complex care
needs [3, 7]. As outlined above, care transitions can be under-
stood as processes in a CAS. To reflect this, care transi-
tions can be analysed using systemic safety models that
promote a focus not only on the individuals, but on the
interactions between the people, the technology and the
organisations involved. Systemic models aid the identifi-
cation and understanding of potential vulnerabilities at
the system level instead of focusing on individuals as the
root causes of any failure (or ‘scapegoats’) [16]. However,
there is often a mismatch between how work is presumed
to be performed and how it is actually performed. This
mismatch is also referred to as the discrepancy between
work-as-imagined (WAI) and work-as-done (WAD) (16,
17). When the concepts of WAI and WAD are related
to the framework of WAx [17], work may also be rep-
resented by the perspectives of work-as-prescribed (in
procedures and guidelines), work-as-normative (in rules
or laws), work-as-observed (by the analyst), and work-
as-disclosed (by the sharp-end or blunt-end operator). Depending on the dimension being investigated, as well
as the perspective used for the analysis, work may thus
be represented differently [18]. Regardless of this, when
performance of tasks is adapted to changing operational
conditions across organisations, such as constraints in
time or resources, variability will occur due to trade-offs. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 2 of 28 The Functional Resonance Analysis Method (FRAM)hf The Functional Resonance Analysis Method (FRAM)
The FRAM [36, 39] offers an approach to map and visual-
ise variability and interdependencies in complex systems
and examine relationships between individual processes
and elements. The FRAM has been used in numerous
studies within healthcare [40], providing deeper under-
standing on how different components interact and
drive non-linear series of actions in complex healthcare
processes such as transitional care [15, 28] and hospital
discharge [27, 29]. In the FRAM, the discharge process
can be modelled as consisting of activities that occur
daily within and between organisations. The purpose of a
FRAM analysis is to describe how a system should work
to achieve the intended goals and to understand how
potential variability can prevent this from happening or
enhance functionality [36]. It is first necessary to map
and construct a model of the system (as in the current
study) and then instantiate the model [41] by analysing
patient scenarios from WAD (to be presented in a sepa-
rate study). The aims were to 1) map coordination and team collab-
oration across healthcare and social care organisations,
2) describe interdependencies and system variability in
the discharge process for older people with complex care
needs, and 3) evaluate the alignment between discharge
planning and the needs at home. Background Achieving safe care transi-
tions from hospital to home for patients with complex
care needs is a complicated process requiring success-
ful completion of a number of tasks, e.g., coordinating
care with healthcare personnel outside hospital [13]. The
structural gaps between services and settings involved in
these care transitions create safety gaps [14] and a health-
care system design with highly specialised care units may
raise walls that obscure a patient’s care pathway and hide
safety risks [15]. In a CAS, the known and the unpredict-
able exist side by side. During discharge planning, this
creates a need for adaptability and flexibility, where each
system unit may need to relinquish some of its autonomy Hedqvist et al. BMC Health Services Research (2023) 23:851 Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 3 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Fig. 1 Study design with multiple convergent data collection
methods However, that study focused on the frail older patients’
health status between admission and potential readmis-
sion to identify challenges they faced during the tran-
sition process. The current study differs from former
studies in that it incorporates multi-professional and
cross-organisational team collaboration and coordination
processes from all care providers involved (hospital, pri-
mary care, municipality), related to both pre- and post-
discharge processes, to evaluate how discharge planning
matches the needs arising when the patient returns home. This study sought to provide a detailed understanding of
how coordination of care at hospital discharge is organ-
ised, including inherent interdependencies and potential
system variability where avoidable adverse events may
occur. Furthermore, we hoped to find how discharge
planning in cross-organisational collaboration aligns with
the needs that arise when the patient is enrolled in or
returned to home healthcare and social care. Therefore,
it was considered to be valuable to map the critical situa-
tions where alignments between demands and resources
occur and where there were misalignments between how
was described in procedures and intended to be done,
and how it was done in practice.h Fig. 1 Study design with multiple convergent data collection
methods Design and study setting We conducted a qualitative study using multiple con-
vergent data collection methods [34, 35] to gather data
on care transitions and then analysed the data with the
Functional Resonance Analysis Method (FRAM) [36]
(Fig. 1). Qualitative methods comprising document
review, observations and interviews with healthcare and
social care professionals (HSCPs) were used for data
triangulation [34]. This study focused on couplings and
interdependencies in care transitions, delimited to hospi-
tal admission, in-patient care, hospital discharge and the
first 72 h at home. In the FRAM, a function refers to the means, acts or
activities that are necessary to achieve a goal or pro-
duce a certain result [36, 42]. However, a function can
also refer to something the organisation does, such as
‘treat patients’, or something that a technical system does
automatically or after manual input. Each function is
described by six aspects – Input, Output, Precondition,
Resource, Control and Time [43] – as illustrated in Fig. 2. An example of how this can be realised is presented in
Table 1. i
The study is part of a research project performed in col-
laboration with a region and municipalities in southern
Sweden. The region encompasses three small to medium-
sized hospitals, 40 primary care centres and twelve
municipalities. Sweden has a decentralised healthcare
system managed and run by regions and municipalities. The regions are responsible for primary, secondary and
specialist care, while the municipalities provide health-
care and social care in nursing homes and home care ser-
vices [37, 38]. The output of one function interacts or ‘couples’ with
other functions. In a visual representation of a FRAM
model, this is illustrated by lines or ‘couplings’ connect-
ing the functions. Functional upstream–downstream
couplings create the basis for functional resonance
[41], as they mean that the variability of a function may Page 4 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Fig. 2 The six aspects of a function in the FRAM model [41] information. They in turn informed their personnel. All
participants were informed prior to observations and
interviews and gave informed consent to participate in
the study. The sample consisted of 60 HSCPs from in-hospital
care, ambulance care, primary care and municipal care. The participants had a range of professions and roles:
registered nurses, physicians, care coordinators, occu-
pational therapists, physiotherapists, social service
officers, assistant nurses, ambulance nurses, as well as
first-line managers. Data collection and procedureh The data collection was performed from June 2020 to
October 2021, as an iterative and reflexive process, con-
verging data from observations, interviews, and docu-
ment review. Our use of multiple data sources allowed
for convergence, to achieve comprehensive data and to
expand and enrich the understanding of the complex care
transition processes [34]. The data collection encom-
passed a total of 45 interviews, eleven participant obser-
vations and five meeting observations (Table 2). be amplified or dampened by other system functions. Internal variability relates to the nature of a function,
for example bias in decision-making or assessment or
the quality and effectiveness of communication in an
organisation. External variability emerges from the vari-
ability of the conditions or work environment in which
the function is performed, affected by unspoken norms
and expectations or by organisational factors such as
regulations and legal constraints. By creating a visual of
the intra- and inter-organisational complexity and vari-
ability of the care transition process, the FRAM may aid
discovery of potential interdependencies, vulnerabilities
and gaps where discontinuities occur, thereby potentially
contributing to improved patient safety. Design and study setting This provided a multidisciplinary and
cross-stakeholder perspective on collaboration in care
transitions. Fig. 2 The six aspects of a function in the FRAM model [41 Document review First, to develop observation protocols and questions for
interviews, written materials were collected by search-
ing and accessing available documents on government
websites as well as digital and printed versions used
in everyday work at the units being observed. Docu-
ments included in the review were national laws [45–48],
regional guidelines (n = 3) and local routines from in-
hospital care (n = 2) and municipal care (n = 2) describ-
ing the discharge process. The documents were analysed
using document review, mapping the discharge process
according to regulations and routines (i.e., WAI [41]). Sampling and participants A purposive sampling strategy was adopted [44] to
recruit participants from various professions and care
providers involved in care transitions from hospital to
home, to maximise data variation [44]. The extensive
sampling enabled capture of the intra- and inter-organ-
isational perspectives of stakeholders involved in the
delivery of healthcare and social care in such transitions. The operation managers from each organisation received
an introduction to the project through written and verbal Participants a total of 86 h, with personnel shadowed in their daily
work. Various forms of meetings were observed, includ-
ing coordination meetings, care planning meetings and
discharge conversations between patients and physicians. Fieldnotes taken in association with the observations
were rewritten later the same day. background) was present and recognisable to the par-
ticipants, but not an active participant with a role in the
social context, enabling observation and occasional inter-
actions leading to a high level of involvement while main-
taining detachment [35]. Before meeting observations, all
participants were informed and gave consent. To protect
the privacy of all parties, as well as to avoid gathering sen-
sitive information like patient data, none of the meetings
were recorded. Instead, the observer wrote fieldnotes, in
line with participant observation methods [35]. Observations To gain an understanding of how the everyday work
was carried out [36], participant observations were per-
formed [35] in multiple contexts. The researcher (nurse Table 1 Example of aspects of a function (‘Initiate planning of coordinated care’) in the FRAM model
Aspect
Description
Example
Input
That which starts the function. Link to previous/upstream functions
What a function uses to produce the output
Need of coordinated care
Outputs
The result of a function. Constitutes links to subsequent/downstream functions
Initiated coordination
Precondition
Conditions that should be satisfied for a function to be executed
Consent from patient
Resource
What is needed or consumed to execute a function
Care contact
Control
What regulates or monitors a function
Policies for coordinated care
Time
Temporal constraints and requirements affecting the function
Within 24 h Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 5 of 28 Table 2 Data collection and participants
2a)
Data collection
n
Interviews
45
Participant observations
11
Meeting observations:
5
Coordination meeting
2
Care planning meeting
2
Discharge conversation
1
2b)
Occurrences in
Participants
n (60)
Interviews (45)
Participant observations (11)
Meeting observations (24)
Medical and geriatric hospital wards
(12)
(7)
(6)
(12)
Registered nurse
5
2
4
3
Care coordinator
4
4
2
4
Physician
3
2
1
Ambulance care
(5)
(5)
Ambulance nurse
5
5
Primary care
(8)
(3)
(2)
(2)
Care coordinator
6
3
2
4
Physician
2
2
Municipal care
(35)
(27)
(3)
(14)
Occupational therapist
5
4
2
Physiotherapist
6
4
3
Registered nurse
8
7
2
Social service officer
5
2
4
Care coordinator
5
4
3
3
Assistant nurse
4
4
First-line manager
2
2 Table 2 Data collection and participants 2b) Occurrences in
Participants
n (60)
Interviews (45)
Participant observations (11)
Meeting observations (24)
Medical and geriatric hospital wards
(12)
(7)
(6)
(12)
Registered nurse
5
2
4
3
Care coordinator
4
4
2
4
Physician
3
2
1
Ambulance care
(5)
(5)
Ambulance nurse
5
5
Primary care
(8)
(3)
(2)
(2)
Care coordinator
6
3
2
4
Physician
2
2
Municipal care
(35)
(27)
(3)
(14)
Occupational therapist
5
4
2
Physiotherapist
6
4
3
Registered nurse
8
7
2
Social service officer
5
2
4
Care coordinator
5
4
3
3
Assistant nurse
4
4
First-line manager
2
2 Interviewsh The knowledge gleaned from the document review was
expanded through interviews with first-line managers. These interviews related to the work performed at the
management level and what tasks were supposed to be
performed in the discharge process, contributing to the
understanding of WAI. The interviews were recorded and
transcribed verbatim. A semi-structured observation guide was used, build-
ing on the understanding of WAI gained from the docu-
ment review. It encompassed themes relevant to care
transitions and hospital discharge, such as coordination,
communication, and information exchange. Observa-
tions were complemented with contextual inquiries [35]
to understand what was happening (for example ‘Could
you tell me about what you are doing there?’). These
observations were performed in hospital wards (n = 6),
primary care (n = 2) and community settings (n = 3) for To create a more in-depth view of the research prob-
lem, and to study different ways of understanding the
work processes in care transitions, the observations
were complemented with semi-structured interviews
with personnel from throughout the care trajectory. Page 6 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Hedqvist et al. BMC Health Services Research (2023) 23:851 The interviews were conducted on site, via telephone
or via Skype/Zoom and lasted 32–75 min (mean
57.8, SD 21.2). An interview guide was developed
and revised during the process, reflecting the analyti-
cal process and addressing the gaps in understanding
remaining after the observations. The interviews thus
offered a deeper, clarifying perspective on actions
already observed. The interviews were recorded and
then transcribed verbatim. how the process could be performed. Then, a preliminary
chronology was drafted to position the functions in order
based on their internal couplings. The functions were
revised until final consensus was reached. i
Next, functions that – at an abstract level – made up
the discharge process were identified. These functions
were stepwise extended into a comprehensive model
of everyday work in care transitions from hospital to
home, visualised using the ‘FRAM Model Visualizer’
[42]. The model was validated through expert audits
by managers and HSCPs involved in discharge process
[15] with recognition and affirmation of the process,
confirming the relevance of the results and the man-
agement of contradictory views. Hence, no revision of
the model was needed. Interviewsh Then, the potential variability
of function output was assessed [41] based on what
can reasonably be expected to happen in the clinical
setting, based on observational and interview data. The variability determined the quality of the output,
which in turn influenced aspects of other functions in
the system. The FRAM model was thus used to under-
stand how variability and subsequent adjustments can
affect other functions and thus the discharge process as
a whole. Based on different phenotypes such as timing,
precision, and duration, previously suggested as vari-
ability manifestations [41], two analytical clusters (tim-
ing and precision) emerged in this process. The clusters
characterised how the interdependence of functions
could potentially lead to unexpected results, depending
on how the outputs of each function could vary in tim-
ing (too early, on time, too late, not at all) and precision
(precise, acceptable, imprecise), from the perspective of
the needs of downstream functions. This step was fol-
lowed by identification of functional dependencies and
potential for functional resonance that could affect the
system’s ability to perform the discharge process suc-
cessfully. The couplings made it possible to analyse and
describe how variability of the output from one func-
tion could affect other functions, without any claims
of a cause-effect perspective. Lastly, the couplings and
dependencies between functions informed propos-
als on ways to manage possible occurrences of uncon-
trolled performance emerging from implementation
of changes that either dampened negative effects and
absorbed variability or reinforced positive effects and
amplified variability [51]. Data analysis First, materials from the document review and inter-
views with managers were analysed using manifest con-
tent analysis [49], mapping WAI. The materials were
coded and then combined by comparing and contrast-
ing them, in order to further abstract the codes into cat-
egories describing the different steps in the discharge
process [50].h The four-step approach described by Hollnagel [41]
was applied for the analysis of WAD, preceded by a prep-
aration step (Step 0), as follows: Step 0: Define the purpose of the FRAM analysis. i
Step 1: Identify vital functions that are required for
everyday work to succeed. Step 2: Determine and describe the system’s potential
for variability. Step 3: Identify functional dependencies and poten-
tial for functional resonance that may affect the sys-
tem. Step 4: Propose ways to manage possible occurrences
of uncontrolled performance variability. Data analysis and creation of the FRAM model of
WAD were performed through an iterative and reflexive
process of identifying patterns in the transcribed data
and fieldnotes from observations, to find a foundation
in which existing work and communication processes
could be recognised as functions. The datasets were
combined by comparing and contrasting the data, with
contextual factors investigated and taken into considera-
tion. Differences and similarities in the process descrip-
tion were identified and discussed by the researchers
to allow emerging contradictions to promote a deeper
understanding and breadth of perspectives from the par-
ticipants, which further illuminated the complexity of the
phenomenon.hi Results
Th The functions identified were then listed in a Microsoft
Excel spreadsheet. For each function, a detailed descrip-
tion including as many aspects (time, control, input,
output, resources, and preconditions) as possible was
identified and described, to create an understanding of The results of this study are presented in five subsec-
tions. The first subsection relates to the discharge pro-
cess as described in regulations and policy documents
(WAI), while the three subsequent subsections present Page 7 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 in-hospital stay subsequently leading to a hospital dis-
charge. During this initial phase, the patient is treated
at the hospital ward and the potential need of coordi-
nated care after discharge is assessed and identified,
constituting the function ‘Identify the need of coordi-
nated care’. If appropriate and the patient consents, the
care coordination process is initiated by the function
‘Initiate planning of coordinated care’. During the ‘Plan
and Agree’ phase, the future needs of home care, social
support, and care aids are planned for and agreed upon,
leading to the function ‘Co-create plan of coordinated
care’. A coordinated individual planning meeting is per-
formed (‘Execute discharge planning’) or scheduled to
be held at the patient’s home after discharge. Next, the
care transition is facilitated by transferring informa-
tion between stakeholders (‘Transfer information from
in-hospital care’) and the list of medicines is updated. To facilitate for home care personnel to administer
new or changed medicines to the patient in the first
days after their return home, the medicines in question
are sent along with the patient by the hospital nurse. The next phase is ‘Transfer’, where the patient is dis-
charged and returns home. Here, the function ‘Execute
care transition’ marks the point in the process where a
transition in both the physical location and caregiver
responsibility is performed. There is thus a transfer of
responsibility where primary care (re-)assumes medi-
cal responsibility for the patient, guided by any refer-
rals from the treating physician at hospital. During
the ‘Implementation’ phase, the patient is received at
home, initiating ‘Execute coordinated care’ where care
aids are installed and care needs are handled with avail-
able resources. After the return home, during the ‘Re-
evaluate’ phase, coordinated care is re-evaluated to
consider new or altered care needs or exacerbations in
pre-existing illnesses. Results
Th If possible, actions may be taken
to respond to the altered needs with available resources
through the functions ‘Adapt home care’, ‘Adapt social
care’, or ‘Adapt care aids’. If this adaptation is insufficient WAD, using the FRAM steps to outline potential gaps
within the care transition process. Based on the results of
the analysis (steps 1–3), measures to manage variability,
risks, and gaps are suggested in the final step. The discharge process as described in regulations
and policy documentsh The review of laws, regulations, and policy documents
showed that the discharge process was intended to be
achieved through eight overarching steps involving mul-
tiple stakeholders from several professions, as well as the
patient and family. The steps, listed in Table 3, represent
WAI and serve to achieve the goal of safe and secure dis-
charge from in-patient care.hii The final step of re-evaluating also includes identifi-
cation of increased or altered needs for coordination,
which may lead to the beginning of a new cycle of care
transition. Vital functions required for everyday work to succeed The next step is then to discharge the patient so they can go home. If there
is a need for coordinated care and the patient needs new or altered care efforts at home, the physician may delay discharge
until preparations for the efforts are completed Function (i.e., event)
Description d
A patient is assessed as needing inpatient care. If there are hospital beds available, the patient is admitted to the ward
and treated and cared for by healthcare professionals Perceived need of prehospital assessment and care
Need of inpatient care
Emerging needs
Inadequate possibility of care in home
Adequate care efforts in home
Individual plan of care
Medical plan at exacerbation
Medical assessment or dialogue Vital functions required for everyday work to succeed Vital functions required for everyday work to succeed
Hospital discharge as performed in practice was mod-
elled as a complex multi-stakeholder process (Table 4),
describing relevant coordination and activities that
occurred daily within and between different parts of the
organisations, constituting WAD. The comprehensive model encompassed a sizeable
number of functions (n = 52) required for the discharge
process to succeed. In the visual presentation (Fig. 3), the
functions have been clustered to follow the patient and
their location through the different phases that constitute
the discharge process (below the FRAM model). Addi-
tionally, WAD is illustrated in relation to the phases of
the transition process in WAI (above the FRAM model),
which is described by eight steps (Table 3). Furthermore,
different colours show the range of actors involved in
each function. The model begins with the function ‘Admit the
patient to the ward’ located in the ‘Identify and Initi-
ate’ phase, as this function indicates the start of an Table 3 The eight steps describing the hospital discharge process as work-as-imagined
Step in process
Description of action
Identify
Identify patients in need of coordinated care
Initiate
Initiate coordination process
Plan
Plan and prepare for discharge
Agree
Jointly determine and agree that the patient is fit for discharge
Facilitate
Facilitate the care transition from hospital by supplying medicines, lists and care aids
Transfer
Transfer information and care responsibility from one care provider to another
Implement
Implement and perform the coordinated efforts in the home
Re-evaluate
Re-evaluate and reconsider the coordinated efforts in light of perceived needs
after initiating the planned efforts Table 3 The eight steps describing the hospital discharge process as work-as-imagined
Step in process
Description of action
Identify
Identify patients in need of coordinated care
Initiate
Initiate coordination process
Plan
Plan and prepare for discharge
Agree
Jointly determine and agree that the patient is fit for discharge
Facilitate
Facilitate the care transition from hospital by supplying medicines, lists and care aids
Transfer
Transfer information and care responsibility from one care provider to another
Implement
Implement and perform the coordinated efforts in the home
Re-evaluate
Re-evaluate and reconsider the coordinated efforts in light of perceived needs
after initiating the planned efforts Page 8 of 28 Hedqvist et al. Vital functions required for everyday work to succeed BMC Health Services Research (2023) 23:851 Table 4 Work-as-done in the hospital discharge process as modelled by the FRAM
Function (i.e., event)
Description
Admit patient to ward
A patient is assessed as needing inpatient care. If there are hospital beds available, the patient is admitted to the ward
and treated and cared for by healthcare professionals
Identified aspects:
Input
Patient to hospital
Output
Patient to inpatient care
During inpatient care
Precondition
Need of inpatient care
Resource
Availability to beds on ward
Control
Decision on admission
Decision to re-admit patient
Adapt aids at home
When there is an altered need of aids, the aids supplied may be altered, depending on availability
Identified aspects:
Input
Altered need of aids
Output
Adequate aids in home
Adapt home care
When medical or care needs are altered, the home care efforts may be adapted to better suit the patient’s needs
Identified aspects:
Input
Altered need of home care
Output
Adequate home care efforts
Adapt social care
at home
When there is an altered need of social care, the social care efforts may be altered to better suit the patient’s needs
Identified aspects:
Input
Altered need of social care
Output
Adequate social care efforts in home
Assess ADL
and mobility
If the patient needs coordinated care after discharge, an assessment of ADL and mobility is conducted to get an under‑
standing of what support and aids the patient may need after discharge. This is done by rehab personnel such as physical
therapists or occupational therapists at hospital, depending on availability. This assessment is otherwise performed by nurses
on the ward
Identified aspects:
Input
Initiated care coordination process
Output
Rehab assessment
Resource
Availability to rehab personnel
Assess patient in pre‑
hospital setting
If there is a perceived need of prehospital assessments and care, the municipal staff, family or patient may call emergency
services. The ambulance personnel then conduct an assessment in the prehospital setting
Identified aspects:
Input
Perceived need of prehospital assessment and care
Output
Need of inpatient care
Emerging needs
Inadequate possibility of care in home
Resource
Adequate care efforts in home
Control
Individual plan of care
Medical plan at exacerbation
Medical assessment or dialogue
Assess patient ready
for discharge
When the medical treatment is completed, the patient is assessed as medically fit for discharge and the physician can assess
whether the patient is ready for discharge. Assess patient ready
for discharge Table 4 (continued)
Function (i.e., event)
Description Primary care designates a person as a care contact for the patient. This person can call for a CIP (Coordinated Individual Plan‑
ning) Care contact Care contact e
The permanent care contact in primary care calls for a CIP at home no later than three days after the discharge notice
Identified aspects: Need for CIP in home Call for CIP Patient ready for discharge Care contact Plan for evaluation Within 3 days The physician checks if there are beds available on the ward in question. If the patient has a great need of inpatient care
and there are no beds available, they might be placed on a different ward Resource
Control Assess patient ready
for discharge When the medical treatment is completed, the patient is assessed as medically fit for discharge and the physician can assess
whether the patient is ready for discharge. The next step is then to discharge the patient so they can go home. If there
is a need for coordinated care and the patient needs new or altered care efforts at home, the physician may delay discharge
until preparations for the efforts are completed Identified aspects:
Input
Output Identified aspects:
Input
Output Before discharge Page 9 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 4 (continued)
Function (i.e., event)
Description
Assign care contact
Primary care designates a person as a care contact for the patient. This person can call for a CIP (Coordinated Individual Plan‑
ning)
Identified aspects:
Output
Care contact
Call for a CIP in home
The permanent care contact in primary care calls for a CIP at home no later than three days after the discharge notice
Identified aspects:
Input
Need for CIP in home
Output
Call for CIP
Precondition
Patient ready for discharge
Resource
Care contact
Control
Plan for evaluation
Time
Within 3 days
Check bed availability
The physician checks if there are beds available on the ward in question. If the patient has a great need of inpatient care
and there are no beds available, they might be placed on a different ward
Identified aspects:
Output
Availability to beds on ward
Co-create plan
of coordinated care
There is an ongoing dialogue between the various units involved in the patient’s case. Regular status updates are com‑
municated from healthcare professionals in inpatient care. Questions are asked between the units to plan and coordinate
efforts and aids before the patient returns home. Time Collaborate
within and between
organizations A lot of planning and dialogue takes place within and between the various healthcare organizations, though the patient
and family do not appear to be represented Resource
Control A preliminary plan for the care efforts is written after dialogue between all
the parties involved
Identified aspects:
Input
Initiated care coordination process
Output
Prepared plan for care in home
Plan for evaluation
Plan for aid tools in home
Possibility of dialogue
Precondition
Consent from patient
Rehab assessment
Need of coordinated care
Resource
Knowledge of patient
Knowledge of family
Interprofessional collaboration
Digital communication for collaboration
Control
Needs of patient
Needs of family
Time
During inpatient care
Collaborate
within and between
organizations
A lot of planning and dialogue takes place within and between the various healthcare organizations, though the patient
and family do not appear to be represented
Identified aspects:
Output
Interprofessional collaboration
Conduct admis‑
sion conversation
with patient
When admitting a patient, the physician and the registered nurse on the ward conduct an admission conversation
with the patient to acquire knowledge of their situation, symptoms and needs. At this juncture, family can also be involved
or contacted. The patient’s requests for care are listened to. Patients and family are offered the chance to share their views
on assumed needs when returning home and whether there is a need for coordination of care
Identified aspects:
Input
Patient in inpatient care
Output
Requests from patient or family
Needs of patient Collaborate
within and between
organizations Co-create plan
of coordinated care Output Co-create plan
of coordinated care There is an ongoing dialogue between the various units involved in the patient’s case. Regular status updates are com‑
municated from healthcare professionals in inpatient care. Questions are asked between the units to plan and coordinate
efforts and aids before the patient returns home. A preliminary plan for the care efforts is written after dialogue between all
the parties involved There is an ongoing dialogue between the various units involved in the patient’s case. Regular status updates are com‑
municated from healthcare professionals in inpatient care. Questions are asked between the units to plan and coordinate
efforts and aids before the patient returns home. A preliminary plan for the care efforts is written after dialogue between all
the parties involved
Identified aspects: Output When admitting a patient, the physician and the registered nurse on the ward conduct an admission conversation
with the patient to acquire knowledge of their situation, symptoms and needs. At this juncture, family can also be involved
or contacted. The patient’s requests for care are listened to. Patients and family are offered the chance to share their views
on assumed needs when returning home and whether there is a need for coordination of care Identified aspects:
Input
Output Patient in inpatient care
Requests from patient or family
Needs of patient Patient in inpatient care
Requests from patient or family
Needs of patient Needs of patient Page 10 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 4 (continued)
Function (i.e., event)
Description
Consult physician
in primary care
When there is a need for medical assessment of the patient, for example in case of exacerbation of a chronic illness, a dia‑
logue with the primary care physician responsible for patient care is requested
Identified aspects:
Input
Need of medical assessment or dialogue
Output
Decision to re-admit patient
Medical assessment or dialogue
Resource
Individual plan of care
Decide on admission
The physician decides whether to admit the patient to the ward
Identified aspects:
Output
Decision on admission
Precondition
Need of inpatient care
Resource
Availability to beds on ward
Decide upon level
of care
An increased need of medical care leads to a decision about what may be the most appropriate level of care. To inform this
decision, the medical plan and agreed plan for care at home are used. Resource
Control Updated list of medicines
Resource
Patient ready to go home
Control
Agreed upon time for homecoming
Enclose medicines
at discharge
New medicines that have been added to the medication list are enclosed for the first few days at home. This is to ensure
that the patient gets the medicines even if the pharmacy is closed, for example during discharge on the weekend
Identified aspects:
Input
Patient goes home
Output
Updated list of medicines
Escalate care
If the situation at home is untenable despite care efforts or when the patient needs inpatient care, the patient is readmitted
to hospital
Identified aspects:
Input
Inadequate possibility of care in home
Output
Decision to re-admit patient
Patient to hospital
Get consent to share
information
Healthcare professionals contact the patient (or family if the patient does not have the ability to consent) to get consent
to share information across caregiver boundaries. Without such consent, the coordination process cannot proceed
Identified aspects:
Output
Consent from patient Output The patient should preferably receive adapted care
efforts at home, but if this is not possible, an escalation of care may be needed, where the patient is admitted to hospital
Identified aspects:
Input
Increased need of medical care
Output
Inadequate possibility of care in home
Need of medical assessment or dialogue
Perceived need of prehospital assessment and care
Resource
Adequate care efforts in home
Control
Medical plan at exacerbation
Decision on medical care in home
Agreed upon plan for care in home
Individual plan of care
Discharge patient
The attending physician discharges the patient from inpatient care, thus terminating their inpatient trajectory in this case
Identified aspects:
Input
Patient ready for discharge
Output
Patient discharged
Continuous medical plan in primary care
Discharge letter to patient
Updated list of medicines
Resource
Patient ready to go home
Control
Agreed upon time for homecoming
Enclose medicines
at discharge
New medicines that have been added to the medication list are enclosed for the first few days at home. This is to ensure
that the patient gets the medicines even if the pharmacy is closed, for example during discharge on the weekend
Identified aspects:
Input
Patient goes home
Output
Updated list of medicines
Escalate care
If the situation at home is untenable despite care efforts or when the patient needs inpatient care, the patient is readmitted
to hospital When there is a need for medical assessment of the patient, for example in case of exacerbation of a chronic illness, a dia‑
logue with the primary care physician responsible for patient care is requested Identified aspects: Need of medical assessment or dialogue
Decision to re-admit patient
Medical assessment or dialogue
Individual plan of care Decision on admission
Need of inpatient care
Availability to beds on ward An increased need of medical care leads to a decision about what may be the most appropriate level of care. To inform this
decision, the medical plan and agreed plan for care at home are used. The patient should preferably receive adapted care
efforts at home, but if this is not possible, an escalation of care may be needed, where the patient is admitted to hospital Identify need of coor‑
dinated care Resource f the situation at home is untenable despite care efforts or when the patient needs inpatient care, the patient is readmitted
to hospital Consent from patient Page 11 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 4 (continued)
Function (i.e., event)
Description
Handle needs
with available
resources
When new or altered needs emerge, they may be handled by family or through self-care, if their abilities are sufficient
to achieve adequate care
Identified aspects:
Input
Emerging needs
Output
Adequate care efforts in home
Resource
Support from family
Self-care
Have capacity for care
and social support
The capacity and ability of the family to provide or aid in care and social support of the patient
Identified aspects:
Output
Ability to provide care and social support
Have capacity for self-
care
The patient’s knowledge, experience and ability for self-care
Identified aspects:
Output
Ability in self-care
Identify altered care
needs
During the continued care at home, the patient, family and healthcare professionals pay attention to any changes that may
indicate altered care needs and act accordingly
Identified aspects:
Input
Monitoring of care efforts
Attention to patient health status
Assessment of efforts in home according to plan
Output
Altered need of social care
Al
d
d f h Table 4 (continued) Function (i.e., event)
Description When new or altered needs emerge, they may be handled by family or through self-care, if their abilities are sufficient
to achieve adequate care Emerging needs Adequate care efforts in home Support from family Self-care Ability to provide care and social support During the continued care at home, the patient, family and healthcare professionals pay attention to any changes that m
indicate altered care needs and act accordinglyi Monitoring of care efforts
Attention to patient health status
Assessment of efforts in home according to plan
Altered need of social care
Altered need of home care
Altered need of aid tools
Emerging needs
Increased need of medical care Output Emerging needs Increased need of medical care An admission conversation is conducted with the patient where the physician or other healthcare professional determine
the need for coordinated care after returning home. Admission to hospital may have been preceded by a referral from pri‑
mary care which may clearly state that there is a need for coordinated care. Alternatively, the patient may have been assessed
by a registered nurse in home care before hospitalization, who has made clear that there is a need for coordinated care. The patient or family can also communicate their wishes or needs for coordination of care to the healthcare professionals. A prerequisite to considering coordination of care is that the patient consents to information sharing between healthcare
providers Identified aspects:
Input
Output
Precondition
Resource Identified aspects:
Input
Patient in inpatient care
Output
Need of coordinated care
Within 24 h
Precondition
Consent from patient
Resource
Requests from patient or family
Referral from municipal or primary care
Knowledge of patient
Knowledge of family
Control
Needs of patient
Needs of family
Inform family of dis‑
charge
No later than the day the patient is returning home, the family of the patient is contacted by the ward and informed of this
Identified aspects:
Input
Patient ready to go home
Output
Family ready to receive patient at home
Agreed upon time for homecoming
Time
Before discharge Table 4 (continued) Table 4 (continued) Monitor and evaluate
care efforts at home Function (i.e., event)
Description Inpatient healthcare professionals make an admission announcement in the digital system Cosmic Link, where the estimated
date of discharge is presented. Further, healthcare professionals assess and announce the need for efforts and describe
the status of the patient. A coordination case is created and units with ongoing efforts for the patient are linked to the case Initiate planning
of coordinated care of coordinated care
date of discharge is presented. Further, healthcare professionals assess and announce the need for efforts and describe
the status of the patient. A coordination case is created and units with ongoing efforts for the patient are linked to the case
Identified aspects:
Input
Need of coordinated care
Output
Initiated care coordination process
Precondition
Consent from patient
Resource
Care contact
Control
Policies for coordinated care
Time
Within 24 h
Install aids at home
Rehab personnel meet with the patient upon their return home. The staff test and install the necessary aids. Some aids
and adaptations may take longer than others to implement and therefore the patient may be at home for several days
before they are used
Identified aspects:
Input
Patient goes home
Output
Aid tools in home
Precondition
Rehab assessment
Aid tools available
Control
Plan for aid tools in home
Time
Patient goes home
Invite patient
and family in dialogue
When care efforts for the return home are planned, patients and family are invited to participate in this dialogue, for instance
in discharge planning meetings
Identified aspects:
Output
Needs of family
Needs of patient
Knowledge of patient Invite patient
and family in dialogue Time Within 24 h Install aids at home me
Rehab personnel meet with the patient upon their return home. The staff test and install the necessary aids. Some aids
and adaptations may take longer than others to implement and therefore the patient may be at home for several days
before they are used Identified aspects:
Input
Output
Precondition Patient goes home Aid tools in home Rehab assessment Aid tools available Install aids at home Notify receiving units
of pending discharge Control Plan for aid tools in home Patient goes home Invite patient
and family in dialogue When care efforts for the return home are planned, patients and family are invited to participate in this dialogue, for instance
in discharge planning meetings Needs of family Needs of patient Knowledge of patient Knowledge of family Knowledge of family
Monitor and evaluate
care efforts at home
An evaluation of the planned and performed care efforts is made in the patient’s home within a couple of days, together
with the patient, family and relevant healthcare professionals. Based on what has worked well and less well, adjustments can
be made
Identified aspects:
Input
Patient goes home
Output
Monitoring of care efforts
Assessment of efforts in home according to plan
Precondition
Execution of efforts in home according to plan
Resources
Call for CIP
Control
Individual plan of care
Plan for evaluation
Time
Days after homecoming
Notify receiving units
of pending discharge
As soon as possible after the decision has been made that the patient is ready for discharge, the receiving units are notified
of this, so they can start preparing to receive the patient
Identified aspects:
Input
Patient ready for discharge
Output
Units informed of discharge
Within 3 days
Need for CIP in home
Time
As soon as possible An evaluation of the planned and performed care efforts is made in the patient’s home within a couple of days, together
with the patient, family and relevant healthcare professionals. Based on what has worked well and less well, adjustments can
be made Identified aspects:
Input
Output Input Control Policies for coordinated care Control Before discharge Page 12 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Identified aspects: Consent from patient Care contact Time As soon as possible after the decision has been made that the patient is ready for discharge, the receiving units are notified
of this, so they can start preparing to receive the patient Identified aspects:
Input
Output Input Output Within 3 days Need for CIP in home As soon as possible Page 13 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 4 (continued) Function (i.e., event)
Description Resource Perform CIP Perform CIP Self-care and care efforts are carried out at home by patients, family and social care along with registered nurses
from the municipality. The intended care is based on the medical plan from primary care and the agreed plan devised dur‑
ing the care planning before discharge. When returning home from hospital, the patient receives information both verbally
and in writing from the hospital regarding their care and treatment, such as medicine changes Function (i.e., event)
Description The care transition from hospital to home takes place in connection with the patient being discharged by the attending
physician. Healthcare professionals on the ward ensure that the patient comes home to their accommodations and any
transport needed is ordered Perform care transi‑
tion Precondition Prepared municipal home care efforts
Resource
Information from inpatient care
Discharge letter to patient
Perform CIP
A coordinated individual plan is made, usually within the first few days of homecoming. Several professionals collaborate
in this meeting together with patient and family, to make sure the care efforts and aids are appropriate for the current needs
Identified aspects:
Input
Call for CIP
Output
Individual plan of care
Time
Days after homecoming
Perform coordinated
care at home
Self-care and care efforts are carried out at home by patients, family and social care along with registered nurses
from the municipality. The intended care is based on the medical plan from primary care and the agreed plan devised dur‑
ing the care planning before discharge. When returning home from hospital, the patient receives information both verbally
and in writing from the hospital regarding their care and treatment, such as medicine changes
Identified aspects:
Input
Patient goes home
Output
Execution of efforts in home according to plan
Attention to patient health status
Precondition
Medical plan and treatment in primary care
Adequate aid tools in home
Adequate social care efforts in home
Adequate home care efforts
Consent from patient
Updated list of medicines
Medicines for homecoming
Adequate care efforts in home
Patient met in home
Resource
Information from inpatient care
Booked social care personnel
Aid tools in home
Summary of medical care
Care contact
Self-care
Support from family
Control
Agreed upon plan for care in home
Decision on medical care in home Identified aspects: Input
Patient discharged
Output
Patient goes home
Days after homecoming
Precondition
Patient ready to go home
Municipal efforts ready
Family ready to receive patient at home
Updated list of medicines
Medicines for homecoming
Prepared municipal home care efforts
Resource
Information from inpatient care
Discharge letter to patient
A coordinated individual plan is made, usually within the first few days of homecoming. Several professionals collaborate
in this meeting together with patient and family, to make sure the care efforts and aids are appropriate for the current needs
Identified aspects: Resource Control Page 14 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 4 (continued) Identified aspects:
Output
Resource Self-care Ability in self-care Information from inpatient care Rehab personnel plan what aids may be needed at home after discharge. This depends on the assessment of the patient’s
ADL and mobility. The rehab personnel order the aids needed and make sure they are available when the patient returns
home Identified aspects:
Input
Output
Precondition Aid tools available Rehab assessment Agreed upon plan for care in home When a decision is made regarding when it is believed the patient will be discharged and go home, the care coordinator
may plan for home visits and schedule this for the social care personnel. The social care personnel are then also informed
that the patient is on their way home and what interventions will be needed. The earlier this is done, the greater the chances
of bringing in enough staff to perform all these tasks while still having time for other assignments already planned Function (i.e., event)
Description Before the patient is discharged, a discharge planning is performed. This is often performed as a hybrid meeting
where the patient (and if possible, a member of the family) and the nurse from the ward or a designated care coordinator
attend physically at the hospital, while professionals from primary care and municipal care attend via video. At this meeting,
the patient is informed of the prepared plan for care in home and this is agreed upon in dialogue with the family Perform discharge
planning Control Policies for coordinated care
Time
Before discharge
Perform self-care
The patient performs self-care at home depending on their own ability and physical condition. The self-care is guided
by the information from inpatient care
Identified aspects:
Output
Self-care
Resource
Ability in self-care
Control
Information from inpatient care
Plan and order aids
Rehab personnel plan what aids may be needed at home after discharge. This depends on the assessment of the patient’s
ADL and mobility. The rehab personnel order the aids needed and make sure they are available when the patient returns
home
Identified aspects:
Input
Patient ready for discharge
Output
Aid tools available
Precondition
Rehab assessment
Agreed upon plan for care in home
Plan and schedule
social care personnel
When a decision is made regarding when it is believed the patient will be discharged and go home, the care coordinator
may plan for home visits and schedule this for the social care personnel. The social care personnel are then also informed
that the patient is on their way home and what interventions will be needed. The earlier this is done, the greater the chances
of bringing in enough staff to perform all these tasks while still having time for other assignments already planned
Identified aspects:
Input
Units informed of discharge
Output
Booked social care personnel
Informed social care personnel
Precondition
Agreed upon plan for care in home
Plan municipal home
care
The municipal home care nurses plan for what will be needed when the patient returns home. Examples could include
wound dressing or distribution and administration of medicines
Identified aspects:
Input
Patient ready for discharge
Output
Prepared municipal home care efforts
Precondition
Agreed upon plan for care in home Time Before discharge The patient performs self-care at home depending on their own ability and physical condition. The self-care is guided
by the information from inpatient carei Plan and schedule
social care personnel Table 4 (continued) Table 4 (continued) Table 4 (continued) Function (i.e., event)
Description Care units, patients and families are prepared for the discharge and the patient to return home. A discharge planning meet‑
ing is held where the patient and family talk to healthcare professionals in the care units that are discharging and receiving
the patient at home. Aids are ordered and planned to be installed and provided in the patient’s home upon their return
home. Social care personnel are notified that the patient is coming home, what time they should meet them and what meas‑
ures are to be taken. Registered nurses in home care set up care plans and medical records for the patient and plan visits,
medicine administration and home care efforts for when the patient comes home Prepare care transition
to home Identified aspects: Identified aspects:
Input i
Input
Patient ready for discharge
Units informed of discharge
Output
Patient ready to go home
Latest the same day as discharge
Municipal efforts ready
Before discharge
Medicines for homecoming
Precondition
Consent from patient
Informed patient and family
Resource
Plan for aid tools in home
Informed social care personnel
Control
Agreed upon plan for care in home
Home care personnel or rehab personnel meet the patient upon their arrival at home after discharge. This requires
that the municipal efforts are ready and that social care personnel are both scheduled and informed about the patient’s
status and needsi Resource Control
Agreed upon plan for care in home
Home care personnel or rehab personnel meet the patient upon their arrival at home after discharge. This requires
that the municipal efforts are ready and that social care personnel are both scheduled and informed about the patient’s
status and needs Receive patient
in home Control Identified aspects:
Input
Output
Precondition Prepare care transition
to home Precondition The municipal home care nurses plan for what will be needed when the patient returns home. Examples could include
wound dressing or distribution and administration of medicines Patient ready for discharge
Prepared municipal home care efforts
Agreed upon plan for care in home Input Output Precondition Page 15 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Policies for coordinated care When the medical care is concluded in inpatient care, the physician writes a medical summary that is made available digi‑
tally, but is also printed and handed to the patient to take home Patient discharged
Summary of medical care
Medical plan at exacerbation Summary of medical care Take part of infor‑
mation from other
caregivers Resource Physicians at the hospital can contact primary care or read medical records to get information, see plans, etc. about the patient’s long-term care. Admission to a hospital can also be initiated after assessment and a referral by a regis‑
tered nurse in home care or a physician in primary care Function (i.e., event)
Description Function (i.e., event)
Description
Support care at home
In addition to the self-care performed by the patient, the family may also support care at home, depending on their abilities. This support can be used as a dampening factor if care is not functioning or coordinated, with family stepping in instead
Identified aspects:
Output
Support from family
Precondition
Information from inpatient care
Resource
Ability to provide care and social support
Take part of infor‑
mation from other
caregivers
Physicians at the hospital can contact primary care or read medical records to get information, see plans, etc. about the patient’s long-term care. Admission to a hospital can also be initiated after assessment and a referral by a regis‑
tered nurse in home care or a physician in primary care
Identified aspects:
Output
Referral from municipal or primary care
Transfer information
from inpatient care
Information is transferred between inpatient and primary care, municipal health and social care, and to patients and family. The information is provided both verbally and in writing
Identified aspects:
Input
Patient ready to go home
Output
Information from inpatient care
Time
Latest the same day as discharge
Treat patient in hos‑
pital
The medically responsible physician on the ward treats the patient. It is possible to review documentation and medical
records from the patient’s previous and ongoing care and treatment in primary care. The physician at the hospital can
also contact the physician responsible for the patient in primary care, to discuss the focus of care, treatment and plans
Identified aspects:
Output
Medically treated patient
Precondition
Patient in inpatient care
Resource
Documentation from primary care
Update list of medi‑
cines
At discharge, the current list of medicines needs to be updated by the attending physician. This list is then printed
and handed to the patient to take home. Time Digital systems are used for healthcare and social care professionals to communicate in writing and via video meetings
to update each other on the status of the patient and the presumed needs at home There are explicit guidelines and laws regarding discharge from inpatient care and what steps should be performed
and by whom. There are also criteria regarding when, where and how coordination should take place Output Information from inpatient care Function (i.e., event)
Description The list is also made available digitally, to be accessed by primary care and munici‑
pal home care nurses
Identified aspects:
Input
Patient ready for discharge
Output
Updated list of medicines
Use digital systems
for collaboration
Digital systems are used for healthcare and social care professionals to communicate in writing and via video meetings
to update each other on the status of the patient and the presumed needs at home
Identified aspects:
Output
Digital communication for collaboration
Use policies on coor‑
dination of care
There are explicit guidelines and laws regarding discharge from inpatient care and what steps should be performed
and by whom. There are also criteria regarding when, where and how coordination should take place
Identified aspects:
Output
Policies for coordinated care
Write medical sum‑
mary
When the medical care is concluded in inpatient care, the physician writes a medical summary that is made available digi‑
tally, but is also printed and handed to the patient to take home
Identified aspects:
Input
Patient discharged
Output
Summary of medical care
Medical plan at exacerbation Support care at home
In addition to the self-care performed by the patient, the family may also support care at home, depending on their abilities. This support can be used as a dampening factor if care is not functioning or coordinated, with family stepping in instead
Identified aspects: Treat patient in hos‑
pital Identified aspects: Support from family Output Information is transferred between inpatient and primary care, municipal health and social care, and to patients and fam
The information is provided both verbally and in writing Update list of medi‑
cines Function (i.e., event)
Description Function (i.e., event)
Description Availability to rehab personnel The physician in primary care receives a referral from the treating physician in inpatient care and thereby gets a medical
assignment for follow-up or further medical measures to be performed after inpatient care. This can also determine parts
of the care performed at home Referral to primary care Page 16 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 4 (continued)
Function (i.e., event)
Description Potential variability in the system in relation to the patient’s needs, the physician in pri-
mary care may be consulted or an ambulance called
to provide prehospital assessment, care, and transport
back to hospital if required. The model of the discharge process reveals normal eve-
ryday changeability in the functional output, reflect-
ing potential variability. Variability with the potential
to cause impacts on other parts of the process is most Page 17 of 28 Page 17 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 s-done in relation to the discharge process and the status of the patient Fig. 3 Work-as-done in relation to the discharge process and the status of the patient Page 18 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 5 Examples of potential for variability in the hospital discharge process Table 5 Examples of potential for variability in the hospital discharge process
Function
Output
Potential for variability
Expressions of variability
Timing
Precision
Initiate planning of coordinated care
Initiated care coordination process
Too early
Input possibly missed, risk of lacking
information for correct decision
On time
No effect or possible damping of fur‑
ther variability
Too late / Omission
Delayed or non-initiation of coordi‑
nation may mean delaying the case
or other healthcare providers being
given a very short time to communi‑
cate and plan
Precise
Possible damping of further variability
Acceptable
No effect
Imprecise
Misjudging the need for coordi‑
nated care (the patient is excluded
from the subsequent process and dis‑
charged without intervention)
Co-create plan of coordinated care
Agreed upon plan for care at home
Too early
A care plan created too early
in the patient’s hospital care
risk becoming imprecise due
to the dynamic conditions of recov‑
ery. Prepare care transition to home Monitor and evaluate care efforts at
home Assessment of efforts in home
according to plan Potential variability in the system However,
when a patient is discharged prema‑
turely, the patient may not actually
be ready for discharge increasing
the workload for the home care per‑
sonnel with the risk of readmissions
if the situation becomes untenable
in the home
On time
Possible damping of variability
Too late
A discharge late in the afternoon
presses the home care personnel
to receive the patient with less person‑
nel on duty and limited availability
to contact managers in need of added
resources
Omission
May contribute to resource issues
in the hospital ward if the patient
could have been discharged success‑
fully but is now occupying a hospital
bed
Precise
Possible damping of variability
Acceptable
No effect
Imprecise
Increased variability in downstream
functions to compensate for missed
aspects
Execute coordinated care at home
Execution of efforts in home accord‑
ing to plan
Too early
-
On time
Dampening of variability
Too late
Delayed activity affecting the care
of the patient, ad hoc solutions
Omission
Ad hoc solutions or potential omitted
care of the patient
Precise
Possible dampening of variability
Acceptable
No effect
Imprecise
If the preparation of care efforts does
not correspond to the actual needs,
it may mean a lack of care and social
support for the patient and "firefight‑
ing" by the various actors to solve this
Monitor and evaluate care efforts at
home
Assessment of efforts in home
according to plan
Too early
If the follow-up occurs too early,
the patient has not had time to set‑
tle at home or to know what works
and what does not
On time
Dampening of variability, promot‑
ing a safe and secure care adapted
to the patient’s care needs
Too late / Omission
If it takes too long or the evalua‑
tion is omitted, there may emerge
urgent needs or problems due
to the care measures not being
adapted to the current care needs
and resources The discharge of a patient frees
up a hospital bed, making it avail‑
able to a new patient. Potential variability in the system Depending on the status
of the patient, the care plan may need
to be re-created
On time
Possible damping of further variability
Too late / omission
Delayed or non-initiation of coordi‑
nated care plan may mean delaying
the case or other healthcare providers
being given a very short time to com‑
municate and plan
Precise
Possible damping of variability
as a precise care plan will aid in situa‑
tions that may arise downstream
Acceptable
No effect or increased variability due
to ambiguity
Imprecise
Incorrect depiction of current care
efforts and aid tools at home may
misrepresent the support available
to the patient when discharged
Prepare care transition to home
Patient ready to go home
Too early
Increased variability as the patient may
not be ready for discharge or the prep‑
arations made will not be relevant,
the function may need to be repeated
On time
Possible damping of further variability
Too late / omission
Ad hoc solutions are used to prepare
the patient with limited time
Precise
Possible damping of variability
Acceptable
Increases variability downstream
to compensate for flaws in preparation
Imprecise
In lack of accurate preparation,
the correct medications, adapted aid
tools or care efforts will not be avail‑
able when the patient comes home. The municipal staff will then need
to devote considerable time to try
to solve this, taking time from other
patients and assignments Co-create plan of coordinated care
Agreed upon plan for care at home
Too early Co-create plan of coordinated care
Agreed upon plan for care at home
Too early On time
Too late / omission Page 19 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 5 (continued)
Function
Output
Potential for variability
Expressions of variability
Timing
Precision
Execute care transition
Patient goes home
Too early
The discharge of a patient frees
up a hospital bed, making it avail‑
able to a new patient. Execution of efforts in home accord‑
ing to plan Potential variability in the system apparent in functions related to the planning and prepa-
ration for the care transition (during the ‘Plan and Agree’
and ‘Facilitate’ phases) and the evaluation at home (the
‘Re-evaluate’ phase) based on timing and precision of
performance. The potential for internal variability relates
to bias in assessments of what care aids a patient may
need on arrival at home (originating from the func-
tions ‘Assess ADL and mobility’ and ‘Plan and order
care aids’), or whether a patient is truly ready for dis-
charge (‘Assess patient ready for discharge’). The quality
of communication via digital systems can also be a fac-
tor complicating the assessment of care needs. External
variability is exemplified by the legal constraints limiting
the transfer and sharing of patient information between
the stakeholders involved in a care transition or by the
limited personnel resources available when a patient is
discharged late in the afternoon. Examples of potential
variability related to timing and precision in a selection
of functions are presented in Table 5. One of the functions with the most couplings
upstream and downstream is ‘Execute coordinated care
at home’. The quality of the output is affected by numer-
ous upstream functions that will amplify or dampen the
variability, depending on the timing and precision of
their performance. Consequently, when healthcare per-
sonnel perform coordinated care in a patient’s home,
the quality of the outcome depends on the previous
performance of functions in the planning and prepara-
tion for hospital discharge and care transition. When
necessary, adjustments are made by municipal person-
nel to get the work done. If the care needs of a patient
are more extensive than the assessment and planning
had prepared for, the personnel will be unable to fol-
low protocol. Instead, they must resort to using ad hoc
solutions or workarounds to get the work done, which
may trigger functional resonance, as the variability in
output of the functions creates a suboptimal basis for
downstream functions. The functional dependencies and potential for func-
tional resonance are here clustered into the perspec-
tives of timing and precision. Functional dependencies and potential for functional
resonanceh The complexity of the system becomes apparent as
functional interdependencies that embody the multidis-
ciplinary teamwork in care transitions. For example, the
variability in the function ‘Execute care transition’ as
performed by hospital personnel can affect the circum-
stances for municipal personnel to execute downstream
functions, creating the basis for functional resonance. Potential variability in the system However,
when a patient is discharged prema‑
turely, the patient may not actually
be ready for discharge increasing
the workload for the home care per‑
sonnel with the risk of readmissions
if the situation becomes untenable
in the home Execution of efforts in home accord‑
ing to plan
Too early
On time
Too late
Omission Execute coordinated care at home Monitor and evaluate care efforts at
home Monitor and evaluate care efforts at
home Page 20 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Function
Output
Potential for variability
Expressions of variability
Timing
Precision
Handle needs with available resources
Adequate care efforts in home
Too early
Increased variability, possibility of miss‑
ing actual care needs
On time
Dampening of variability, promot‑
ing a safe and secure care adapted
to the patient’s care needs
Too late
Increased variability
Omission
Increased variability
Precise
Dampening of variability
Acceptable
No effect
Imprecise
Timing of and variability in upstream
functions determines the avail‑
ability of resources for the patient. Depending on the day of the week
and the time of day, the resulting
needs can be handled either at short
notice, with a large amount of effort
or not at all. May result in a readmis‑
sion in hospital when all options are
exhausted at home apparent in functions related to the planning and prepa-
ration for the care transition (during the ‘Plan and Agree’
and ‘Facilitate’ phases) and the evaluation at home (the
‘Re-evaluate’ phase) based on timing and precision of
performance. The potential for internal variability relates
to bias in assessments of what care aids a patient may
need on arrival at home (originating from the func-
tions ‘Assess ADL and mobility’ and ‘Plan and order
care aids’), or whether a patient is truly ready for dis-
charge (‘Assess patient ready for discharge’). The quality
of communication via digital systems can also be a fac-
tor complicating the assessment of care needs. External
variability is exemplified by the legal constraints limiting
the transfer and sharing of patient information between
the stakeholders involved in a care transition or by the
limited personnel resources available when a patient is
discharged late in the afternoon. Examples of potential
variability related to timing and precision in a selection
of functions are presented in Table 5. Timing Timing is essential within the WAD process as it may
impact on both the prerequisites for and the outcomes
of a care transition. If a patient is discharged from hos-
pital while still in recovery, the course of the next few Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 21 of 28 days will be dynamic. If care planning is performed too
early, the patient’s status may change and no longer
match the decisions made, resulting in inadequate care
or a need to review these decisions: in discharges. During evenings and weekends, resources
are reduced, with limited access to decision-makers and
personnel with knowledge, existing relationships with
patients and insight into the care planning processes,
which were performed during weekdays. At best, the per-
sonnel might have access to written documentation, but
this could also be limited during weekends if secretar-
ies had not yet had the time to transcribe the physicians’
notes into the electronic patient records. It all depends on when in time you have the dis-
charge or care planning. Sometimes it happens
quickly and then things happen, and nothing
matches. Then you might even get a coordinated
care plan. One of our coordinators is doing a care
planning for this one gentleman today, where there
has already been a care planning meeting and he
wasn’t supposed to get any home care. Now he’s
gotten worse and will definitely need home care. […] So, it depends a bit on when in time it’s done. (Participant 25, registered nurse, municipal home care) If someone is discharged on a Friday afternoon and
then there’s just temporary staff all weekend and
then maybe, you’ll get a message about that on Mon-
day or something and then maybe you’ll talk it over
with the regular municipal personnel. But of course,
things can happen over the weekend and probably
do sometimes. And sometimes it’s not … sometimes
it takes a long time before the patient discharge sum-
mary is transcribed and sometimes things there are
missed. I think that’s a potential problem. (Participant 22, physician in primary care) However, if care planning is performed too late, the
personnel at the receiving end will have limited time
to arrange for the arrival at home. The timing of the
functions also relates to the concept of synchronisa-
tion, i.e., agreement between the sending unit and the
receiving unit. Precisionh The precision of the performed functions relates to the
quantity and quality of patient care. During a hospital
stay, many units and professions are involved in the pro-
cess of planning for sustainable continued care at home. The findings of the study suggest that gaps occur in this
process, obscuring possible problems which can surface
when the patient comes home. One such gap is the fact
that the social care personnel with the closest relation-
ships to the patient are neither invited nor informed first-
hand regarding care needs during the discharge process. Another illustrative example relates to information trans-
fer: too little information about a patient leads to knowl-
edge gaps, creating extra work to gather the missing
information. This may lead to unnecessary examinations
or tests or deficient care. However, too much or irrele-
vant information can also create problems, as important
information may be obscured. Yeah, things move quickly once they’re … when you
think that now such-and-such is done with treat-
ment and should go home and then it’s just wham-
bam…
(Participant 4, occupational therapist, municipal
rehabilitation services) Yeah, things move quickly once they’re … when you
think that now such-and-such is done with treat-
ment and should go home and then it’s just wham-
bam… (Participant 4, occupational therapist, municipal
rehabilitation services) Additionally, the time of day when functions are per-
formed can have major consequences for any worka-
rounds that must be made to gain control over a situation
and resolve it in the patient’s best interest. In particular,
the weekend is a vulnerable time where the personnel
must use ‘firefighting’ methods to meet care needs, until
more permanent solutions can be put in place: We had one person come home on a Friday, who
couldn’t get out of their wheelchair and did not have
a wheelchair lift at home. And then we got a hold of
… an emergency occupational therapist and physi-
cal therapist and had them bring a lift that they
could borrow and use over the weekend. And then
our occupational therapist and physical therapist
had to come over on the Monday. (Participant 37, assistant nurse, municipal social care) That the information is given, that it is relevant, a
bit brief. If there’s too much text, you realise that you
kind of lose track of what’s important. Straightfor-
ward, clear communication, with short pathways,
that they aren’t too long. Timing The analysis revealed the importance of
having time and opportunity for preparation, so that
everyone is ready when the patient is discharged. This
includes both social care personnel, the patient and
their family: Precisionh (Participant 13, registered nurse, municipal home care) And then I’ll come back a day or two later and talk
a bit more once they’ve settled in at home and when
there’s hopefully a bit more peace and quiet […] get
an idea of how things have actually gone during the
first few days at home. That follow-up is valuable, I
think, because then I can see quite a lot. (Participant 1, registered nurse, municipal home
care) There is an apparent gap in information transfer in the
system, which can be illustrated by the case of ambulance
personnel arriving at an older person’s home and try-
ing to get the bigger picture of what the patient’s usual
status was and what had been decided at hospital dis-
charge. Access to written information was limited or
non-existent, and there was difficulty getting in contact
with municipal healthcare personnel with knowledge of
the patient. The result was that the patient was returned
to hospital and readmitted, but the solution could have
been different: Precisionh (Participant 14, registered nurse, municipal home care) As emphasised in the quotation above, effective, clear
and concise communication is of utmost importance for
successful care transitions. The precision of information
and communication regarding relevant issues is equally Friday night was a time that was frequently mentioned
in the narratives of the participants as a known risk factor Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 22 of 28 important for social care personnel and when informing
the patient: important for social care personnel and when informing
the patient: The precision of the care planning is also a central
issue. Multiple stakeholders in various professions pre-
pare a plan of action, together with the patient and fam-
ily, regarding the care efforts needed when the patient
gets home. However, such planning is hypothetical, since
there are limited possibilities to know what everyday life
will be like for the patient after discharge: The patient is just buried in information and paper-
work when they are discharged from hospital, so they
can’t remember what’s important. They get confused
… it’s better if the important stuff is written down
and then they can read it afterward. (Participant 118, coordinator, municipal home care) The environment is different when you get home. The
conditions are completely different. (Participant 4, occupational therapist, municipal
rehabilitation services) The environment is different when you get home. The
conditions are completely different. (Participant 4, occupational therapist, municipal
rehabilitation services) The core issue of precision in care transitions may thus
emerge in the quality of information transfer and the
availability, procedure and use of systems where informa-
tion is stored. Availability of information in multiple sys-
tems can be considered a strength, but can also generate
extra work: To counteract this, municipal care personnel would
wait a few days to let the patient get acquainted with the
care efforts and aid tools and have time to recover from
the hospital visit: Yeah, the systems are … I kind of think that you
should have that information more available. There
are so many different systems that you have to access
to see it. Management of possible occurrences of uncontrolled
performance variability In the final step of the FRAM analysis, we propose
ways to manage possible occurrences of uncontrolled
performance variability as presented above. The preci-
sion of functions related to the planning of resources
was shown to vary greatly. This indicates that for
healthcare providers to have the ability to act even
in uncertain conditions, their possibilities for adap-
tation need to be increased. Functions like ‘Execute
coordinated care at home’ have several prerequisites
and controls originating from the output of the func-
tion ‘Execute care transition’. However, the analy-
sis indicates that variability in precision can also be
dealt with by increasing the flexibility in a patient’s
home after discharge. Thus, one way to dampen the
effects of functional resonance could be to redistribute
resources and increase them in a patient’s home the
first few days after homecoming and then perform reg-
ular and frequent reassessments, to adjust care flexibly
and accurately. This would enable adaption to the care
needs that emerge during the first few days as a way to
manage uncontrolled performance variability.hf It’s so hard to get information about the patient …
what has been decided … I’ll look through binders
and call, but I feel like I never get any information,
you just stand there with your papers and wonder …
So that’s what’s missing, from the side of the munici-
pality. I mean, if you’re supposed to call one of the
municipal nurses, then the person in charge will have
gone home or is off or it’s in the evening or there’s no
binder and there aren’t any papers and I don’t have
the medical records and I don’t have anything … a
care plan or whatever it is that you’re supposed to …
that’s been decided, and when I’m standing there and
I don’t have any idea, then maybe we’ll load up that
patient and head to the emergency room. And then
they’ll do something … small or, yeah, they’ll admit
the patient for a night or something and then it all
starts over. So, you get unnecessary care and unneces-
sary trips for these kinds of patients. (Participant 40, ambulance nurse) y
The timing of the care transition also affects the pos-
sibility of responding to unexpected events, as the Hedqvist et al. Management of possible occurrences of uncontrolled
performance variability BMC Health Services Research (2023) 23:851 Page 23 of 28 Page 23 of 28 planning, suggesting that detailed planning for care at
home is almost impossible. If viewed using the frame-
work of WAx [17], WAI in this study would rely on work-
as-prescribed and work-as-normative as presented in the
guidelines and policy documents by blunt-end operators. While the policy documents presented hospital discharge
as a linear process in eight consecutive steps, the FRAM
model revealed a total of 52 dynamic functions to realise
a transfer from hospital to home. In practice, the func-
tions are often performed in parallel, are intertwined, or
are performed in a back-and-forth, dynamic cooperation
between a multitude of professions and stakeholders. These people work in separate documentation systems,
with different technical solutions and access, regulated
by different laws and regulations, which further increases
complexity. Although the descriptions in WAI from ‘the
blunt-end operators’ (i.e., managers and policymakers)
[17] seek to give a direction and goal for the discharge
process, following a standardised and normative protocol
would be difficult – if at all desirable, given the diverse
needs of and requirements for the patients being dis-
charged from hospital to home. The findings reveal WAD
to be inherently different from WAI, despite the best
efforts of HSCPs. The differences in professional back-
grounds, values, and cultures, along with the patients’
varying recovery processes, home environments, and
supportive resources at home, mean that the reality is
almost always different from the plan [53–55]. This study
suggests that resources could be more efficiently utilised
to manage possible occurrences of uncontrolled perfor-
mance variability. Primarily, resources could be redis-
tributed to allow flexible adjustments during the first few
days after the return home, as some patients need less
resources than planned while others need more. system appears to be more vulnerable during evenings
and weekends. Variability in timing can mean that the
patient is discharged too early or too late, causing a ripple
effect and functional resonance with consequences for
downstream functions. One way of managing this could
be adding prerequisites and controls for the function
‘Execute care transition’ so that the time of discharge will
not be during the vulnerable periods. Discussionh The study aimed to map coordination of care across
healthcare and social care organisations, describe inter-
dependencies and system variability in the discharge
process for older people with complex care needs and
evaluate the alignment of the discharge planning with the
needs that arise at home. The modelling of the discharge
process reveals, in line with former studies [27, 29, 30],
that discharge planning is complex. There are multiple
task interactions and numerous functional dependen-
cies across organisations, with countless occasions where
things can go wrong. As shown in a study of complex
sociotechnical systems, a reductionist approach cannot
provide a sufficiently detailed and holistic picture of the
interacting components of a system to guide improve-
ment [52]. The results of the current study demonstrated
that the FRAM could help create an accurate and com-
prehensive view of the cross-organisational collaboration,
making it possible to identify areas of improvement and
changes that might lead to a more efficient and effective
discharge process. The FRAM provided insight into the
complexities of interdependencies and variability in the
components involved, such as how individuals, teams,
and organisational structures – as well as the technical
aspects of the discharge process – interact and influence
each other throughout the discharge process. This could
be used to identify potential barriers and to set strategies
to achieve better outcomes for patients.h Discrepancies between daily practice (WAD) and how
the process is described in protocols (WAI) have also
been found in other studies [41, 53, 54, 56–59]. A large
discrepancy between WAI and WAD may be an indica-
tor of brittleness in the system [60]. Thus, understand-
ing why deviations occur is fundamental to improving
patient safety [60, 61].i However, there is a fine line between adapting to vary-
ing conditions (i.e., being resilient) and going beyond the
realm of safety due to the pressure of a high workload,
limited resources, or the system’s expectations of high
efficiency [62]. The FRAM model highlights the limita-
tions in the system design that lead to ‘workarounds’ in
order to manage certain tasks in time and align WAD
with WAI [59]. This study reveals several gaps aris-
ing during the discharge process, though these are not
always visible until the patient faces reality at home, fur-
ther emphasising the importance of flexibility and adapt-
ability. Management of possible occurrences of uncontrolled
performance variability The downstream
function ‘Execute coordinated care at home’ requires
some components supplied from the hospital – such as
medical information regarding the care given, medica-
tions needed during the first few days, and an updated
medication list – and some components from the munic-
ipal organisation, such as availability of adapted care aids
for basic mobility and ergonomic work conditions for
personnel. The most critical functions in the system for
monitoring and directing efforts to manage variability are
‘Co-create plan of coordinated care’, ‘Prepare care transi-
tion to home’ and ‘Execute care transition’. These three
functions together constitute the grounds for the care
performed at home. Discussionh The process mapped in WAD works because This study adds to the knowledge from other studies
using the FRAM, by increasing our understanding of the
uncontrolled variability in the outcome of the discharge Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 24 of 28 Page 24 of 28 Other studies indicate that the time between the deci-
sion on discharge and the care transition is a poten-
tial barrier to information sharing, as time constraints
lead to greater time pressure, increased performance
demands and decreased flexibility [74]. Complex systems
are resilient when they have diversity, as different profes-
sionals deploy different behaviours to respond to actions
and situations based on evidence and their own experi-
ences [75]. However, according to complexity theory, a
system can never be fully described or fully controlled
[76]. This creates challenges in care transitions since
the findings suggest many interdependencies between
the actors involved, meaning that they depend on each
other and on collaboration. Rasmussen [77] emphasises
how circumstances may not be under the control of any
one person, and that there may be systematic but unseen
traps set by upstream decisions. individuals go beyond their responsibilities and do more
than expected to manage the negative effects of variabil-
ity. The findings thus indicate that the discharge process
as described in WAI is not adapted to the current health-
care system design, given existing staffing levels, struc-
tures, and information systems. Furthermore, gaps occur in the information exchange
between HSCPs in the planning and performance of care
transitions. In the findings, the most prominent gap is
that home care personnel, who often have the most up-
to-date information about a patient, are not included
in the information transfer to the same extent as other
parties. Instead, they are supplied with certain informa-
tion fragments after the patient has returned home [63]. Experiences of gaps in care are common, often arising
from failures in communication and coordination of care
[64] and interoperability of systems [65]. Cross-organi-
sational bridges may mitigate such gaps in care [66]. The
findings suggest that there are possibilities of creating
shared documentation of a patient’s current care needs
and plans. Strengths and limitations Care transitions for older people with complex care
needs involve multiple stakeholders and a diversity of
professionals and organisations. Furthermore, the sys-
tem’s adaptive complexity makes it difficult to over-
view, study, and evaluate. The FRAM showed potential
to uncover the complexity in collaborative work in the
dynamic discharge process, which can unfold in a vari-
ety of ways. This might have been difficult to do with
other methods. The FRAM allowed for a visualisation of
the everyday activities in the interprofessional team col-
laboration across organisational borders and the roles of
the different professionals during the discharge process. In this study, the FRAM offered a thick description of a
process that normally remains hidden and thus enabled
potential development of new guidelines. Further, it also
provided a detailed model of WAD and performance
variability of the complex coordination of transitional
care across care providers, which can be used as a guide
for improvements. However, using the FRAM requires
expert knowledge and experience along with access to
the research field. Therefore, the method cannot be used
for all types of processes [39] and may be too resource-
intensive and demanding for use in healthcare quality
improvement generally. Collaboration involving multiple individuals and teams
within and across organisational boundaries is expected
during the transition process, even though not all indi-
viduals and teams communicate. If the healthcare team
is seen as a CAS [72], healthcare professionals need to
expand the system boundaries to understand how any
action taken (or not taken) in their part of the system
has a ripple effect throughout the entire system. Health-
care professionals in hospital care must anticipate what
problems HSCPs at the receiving end will have when the
patient is discharged. In their turn, professionals in home
healthcare need to manage adaptations and moderation
of goals when care plans ‘as imagined’ do not fit with the
needs in the patient’s home. Systems thinking and antici-
pation of the consequences of processes across depart-
ments remain major challenges [73]. As regards the issues of timing in the discharge pro-
cess, the study shows that the time of day when the
patient is discharged strongly affects the ability of per-
sonnel to deal with unexpected situations. Friday after-
noons are mentioned as a particularly vulnerable time
for discharge of people with complex care needs. Discussionh Previous research indicates that sharing of
patient data across systems, if performed effectively, can
create the basis for safe care [67, 68], while also reduc-
ing the workload for healthcare providers [69], limiting
treatment delays [70] and decreasing overall costs for the
healthcare system [71]. Strengths and limitations Pre-
vious studies have shown that the time of day when a
patient is considered medically fit sets temporal condi-
tions and time pressures for subsequent actions [29]. Decisions later in the day were associated with increased
time pressure and variability, thus affecting precision. A key strength of this study was inclusion of multi-
ple perspectives across a variety of healthcare settings
to explore how different clinical teams successfully sup-
port care transitions. However, the heterogeneous sam-
ple may have limited the ability to achieve theoretical
saturation. On the other hand, we were not looking for
a representative sample, but one that encompassed par-
ticipants with as broad knowledge, skills, and expertise
as needed to answer the research question [78, 79]. The
number of participants is related to concerns about the Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 25 of 28 Page 25 of 28 always surface, making the planning of care at home a
hypothetical reality, left for HSCPs in the patient’s home
to deal with after discharge. To achieve a safe and secure
discharge and subsequent care at home, proactive work
during the discharge planning process needs to be facili-
tated. This may be done by increasing precision in the
assessment and planning of a patient’s care. Since this is
a time-sensitive process, considerations need to be made
as to at what time and on what day of the week the dis-
charge will be performed, so the variable needs can be
dealt with accordingly. It could be useful to have a clear
definition of what a secure discharge for patients with
complex care needs should be, and what parameters need
to be fulfilled. If security is intentionally created, the dis-
charge process can proceed and perform successfully. Furthermore, to ensure the ability to respond to unex-
pected events and variations by adapting care efforts at
home, a redistribution of resources during the first few
days after the return home would increase flexibility and
patient safety. richness and thickness of the data [79]. Thus, the large
amount of material represents a strength in this study. Multiple data collection methods were used as triangu-
lation enables qualitative researchers to study relatively
complex entities or phenomena in a holistic way [80]. The study was performed over the course of more than
a year. Strengths and limitations To reduce a possible ‘Hawthorne effect’ in obser-
vations [35], the researchers emphasised that it was not
the performance of individuals that was being observed
and that the participants were not in any way being
assessed in their work. Furthermore, the observer was
dressed in hospital scrubs, like other healthcare person-
nel, to blend in.f Constructing the FRAM model was a trade-off between
showing all the related functions and achieving a usable
and understandable model. Although in-depth informa-
tion was obtained on each function, some perspectives
to create a complete picture of the discharge process
may be lacking. To mitigate this, the model was checked
for sense by professionals with experience of working
with older people with complex care needs and the care
transitions involved. This expert audit was performed to
increase ecological validity.h By promoting insight and knowledge of other HSCPs’
areas and creating the basis for a resilient organisation
with HSCPs that anticipate and adapt by going outside
their boundaries, we could increase the possibility of
safely bridging the gaps in the system. This study was performed in a single region of south-
ern Sweden and the sampling in hospital was limited to
medical and geriatric wards, to gather data that were gen-
eral across the different geographical locations. Future
studies should be performed in other settings to elucidate
whether the results may be transferred. The study aimed
at mapping and describing intra- and inter-organisational
processes with a focus on coordination and collaboration
between different professionals and stakeholders. This
study contributed to increased understanding of the sys-
tem of hospital discharge and care transitions from the
perspective of healthcare professionals. It did not include
patients, family, or other informal caregivers. We want to
emphasise the importance of including patients and fam-
ilies in future studies [15, 81]. Conclusionsh The transition process from hospital to home for older
people is complex, encompassing couplings and inter-
dependencies in the exchange of information between
various individuals, teams, and organisations. The FRAM-
based approach revealed barriers and cracks in team col-
laborations. Discharge planning was carried out within
a system that lacked control because of limited access to
patient information and complexity in the communica-
tion channels between different organizations. Due to the
varying health conditions of patients, and as their com-
plex needs and requirements are difficult to predict and
plan for, a mismatch between discharge planning and the
needs at home may occur. This makes the arrival at home
the most vulnerable point of the transition process. To
optimise the outcome of the discharge process for patients
with complex needs, the system’s variability and adapta-
tions should not be stifled. Instead, the study suggests, the
system might be kept stable and resilient through flexibil-
ity, adjustments, and allocation of adequate resources at
the time of the patient’s return home.hi Implications for practiceh This study provides a comprehensive visualisation of
the most prominent gaps and vulnerabilities in the care
transition process indicating where special attention to
communication and coordination is needed. The findings
show the importance of consensus in the care planning
process and that all relevant information is collected, dis-
tributed and made available to the parties involved in the
decision process and particularly in patient care at home. Reinforcing the ability to bridge gaps through collabora-
tion may improve safety [82]. However, the possibility of
bridging the gaps depends on both access to informa-
tion and inclusion in the process. During the hospital
stay, possible problems or issues waiting at home do not The findings of this study can inform managers and
other decision-makers on how coordinated care is
designed and managed in care transitions to align with
the varying needs in patients’ homes after discharge. It can serve as a roadmap to future studies regarding
the development of guidelines and training, as well as Page 26 of 28 Page 26 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 multidisciplinary simulations to improve intra- and inter-
organisational collaboration and ultimately improve care
for people with complex care needs. multidisciplinary simulations to improve intra- and inter-
organisational collaboration and ultimately improve care
for people with complex care needs. Received: 28 October 2022 Accepted: 18 July 2023 Acknowledgements g
We thank Emelie Ingvarsson, PhD student, for her assistance in data collec‑
tion. We would also like to thank the participating healthcare and social care
professionals, who made this study possible. 7. Tinetti ME, Fried TR, Boyd CM. Designing health care for the most com‑
mon chronic condition—multimorbidity. JAMA. 2012;307(23):2493–4. 8. Kannampallil TG, Schauer GF, Cohen T, Patel VL. Considering complexity
healthcare systems. J Biomed Inform. 2011;44(6):943–7. Availability of data and materials The datasets generated and analysed in the current study are not publicly
available, to protect the privacy of the participants. Qualitative interview tran‑
scripts could be used to identify participants and cannot be shared publicly as
per the Research Ethics Board’s guidelines. 15. O’Hara J, Baxter R, Hardicre N. “Handing over to the patient”: a FRAM
analysis of transitional care combining multiple stakeholder perspectives. Appl Ergon. 2020;85:103060. 15. O’Hara J, Baxter R, Hardicre N. “Handing over to the patient”: a FRAM
analysis of transitional care combining multiple stakeholder perspectives
Appl Ergon. 2020;85:103060. 16. Woods D, Cook R. Incidents - markers of resilience or britleness? In: Holl‑
nagel E, Woods D, Leveson N, editors. Resilience Engineering: Concepts
and Precepts. Aldershot, UK: Ashgate; 2006. p. 69–76. 16. Woods D, Cook R. Incidents - markers of resilience or britleness? In: Holl‑
nagel E, Woods D, Leveson N, editors. Resilience Engineering: Concepts
and Precepts. Aldershot, UK: Ashgate; 2006. p. 69–76. Declarations 17. Patriarca R, Falegnami A, Costantino F, Di Gravio G, De Nicola A, Villani ML. WAx: an integrated conceptual framework for the analysis of cyber-socio-
technical systems. Saf Sci. 2021;136:105142. Funding
O 12. Augustinsson S, Petersson P. On discharge planning: dynamic com‑
plex processes – uncertainty, surprise and standardisation. J Res Nurs. 2015;20(1):39–53. 12. Augustinsson S, Petersson P. On discharge planning: dynamic com‑
plex processes – uncertainty, surprise and standardisation. J Res Nurs. 2015;20(1):39–53. Open access funding provided by Linnaeus University. This project was
funded through the Kamprad Family Foundation for Entrepreneurship,
Research, and Charity (No: 20190249) in Sweden. The funder had no role in the
design of the study, data collection, analysis, or interpretation of data, nor in
writing of the manuscript. 13. Jack BW. A reengineered hospital discharge program to decrease rehos‑
pitalization. Ann Intern Med. 2009;150(3):178. 13. Jack BW. A reengineered hospital discharge program to decrease rehos‑
pitalization. Ann Intern Med. 2009;150(3):178. 14. O’Hara JK, Aase K, Waring J. Scaffolding our systems? Patients and
families ‘reaching in’ as a source of healthcare resilience. BMJ Qual Saf J. 2019;28:3–6. 14. O’Hara JK, Aase K, Waring J. Scaffolding our systems? Patients and
families ‘reaching in’ as a source of healthcare resilience. BMJ Qual Saf J. 2019;28:3–6. Consent for publication
Not applicable. 21. Braithwaite J, Wears RL, Hollnagel E. Resilient health care: turning patient
safety on its head. Int J Qual Health Care. 2015;27(5):418–20. Authors’ contributions 9. Kuziemsky C. Decision-making in healthcare as a complex adaptive
system. Healthc Manage Forum. 2016;29(1):4–7. Funding acquisition and project administration: ME. Study design: ATH and
ME. Data collection and visualisation: ATH. All authors contributed to the anal‑
ysis and interpretation of the data. ATH drafted the original manuscript and all
authors contributed significantly to drafting and revising the manuscript and
agreed on the final version. 10. Plsek PE, Greenhalgh T. Complexity science: the challenge of complexity
in health care. BMJ. 2001;323(7313):625–8. 10. Plsek PE, Greenhalgh T. Complexity science: the challe
in health care. BMJ. 2001;323(7313):625–8. 11. Braithwaite J, Clay-Williams R, Nugus P, Plumb J. Health care as a complex
adaptive system. In: Hollnagel E, Braithwaite J, Wears R, editors. Resilient
Health Care. 1st ed. Surrey: Ashgate Publishing Limited; 2013. Supplementary Information Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1186/s12913-023-09832-7. The online version contains supplementary material available at https://doi.
org/10.1186/s12913-023-09832-7. 4. Starfield B, Lemke KW, Herbert R, Pavlovich WD, Anderson G. Comorbidity
and the use of primary care and specialist care in the elderly. Ann Fam
Med. 2005;3(3):215–22. 5. Szebehely M, Trydegård GB. Home care for older people in Sweden: a uni‑
versal model in transition. Health Soc Care Community. 2012;20(3):300–9. 5. Szebehely M, Trydegård GB. Home care for older people in Sweden: a uni‑
versal model in transition. Health Soc Care Community. 2012;20(3):300–9. 6. Roughead E, Vitry A, Caughey G, Gilbert A. Multimorbidity, care complex‑
ity and prescribing for the elderly. Aging Health. 2011;7:695–705. 6. Roughead E, Vitry A, Caughey G, Gilbert A. Multimorbidity, care complex‑
ity and prescribing for the elderly. Aging Health. 2011;7:695–705. Ethics approval and consent to participate The study was approved by the Swedish Ethical Review Authority (Registra‑
tion number 2020–01219). Information contained in the study was processed
in accordance with the EU General Data Protection Regulation [83]. The
study has adhered to the ethical guidelines of the Helsinki Declaration [84]
concerning requirements on information, consent, confidentiality, and use. All
participants gave fully informed written consent. 18. Adriaensen A, Patriarca R, Smoker A, Bergström J. A socio-technical analy‑
sis of functional properties in a joint cognitive system: a case study in an
aircraft cockpit. Ergonomics. 2019;62(12):1598–616. 19. Vincent C, Amalberti R. Safer healthcare: strategies for the real world. London: Springer Open; 2016. 19. Vincent C, Amalberti R. Safer he
London: Springer Open; 2016. g
20. Hollnagel E, Wears RL, Braithwaite J. From Safety-I to Safety-II: a white
paper. Middelfart, Denmark: Resilient Health Care Net; 2015. Abbreviations 1. Oeppen J, Vaupel JW. Demography Broken limits to life expectancy. Sci‑
ence. 2002;296(5570):1029–31. 1. Oeppen J, Vaupel JW. Demography Broken limits to life expectancy. Sci‑
ence. 2002;296(5570):1029–31. 1. Oeppen J, Vaupel JW. Demography Broken limits to life expectancy. Sci‑
ence. 2002;296(5570):1029–31. Abbreviations
CAS
Complex adaptive system
FRAM
Functional Resonance Analysis Method
HSCP
Healthcare and social care professional
WAD
Work-as-done
WAI
Work-as-imagined 2. Abdi S, Spann A, Borilovic J, De Witte L, Hawley M. Understanding the
care and support needs of older people: a scoping review and categori‑
sation using the WHO international classification of functioning, disability
and health framework (ICF). BMC Geriatr. 2019;19(1):195. 3. Barnett K, Mercer SW, Norbury M, Watt G, Wyke S, Guthrie B. Epi‑
demiology of multimorbidity and implications for health care,
research, and medical education: a cross-sectional study. Lancet. 2012;380(9836):37–43. Competing interests 22. Aase K, Waring J. Crossing boundaries: establishing a framework
for researching quality and safety in care transitions. Appl Ergon. 2020;89:103228. Competing interests
No competing interests have been declared by the authors. References Abbreviations
CAS
Complex adaptive system
FRAM
Functional Resonance Analysis Method
HSCP
Healthcare and social care professional
WAD
Work-as-done
WAI
Work-as-imagined
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12913-023-09832-7. Additional file 1. Author details Stockholm, Sweden; 2020. Available from: https://www.
socialstyrelsen.se/en/about-us/healthcare-for-visitors-to-sweden/about-
the-swedish-healthcare-system/. 60. Anderson JE, Ross AJ, Jaye P. Modelling resilience and researching the
gap between work as imagined and work as done. In: Braithwaite J,
Wears RL, Hollnagel E, editors. Resilient Health Care, Volume 3: Reconcil‑
ing Work-as-Imagined and Work-as-Done. 1st ed. Boca Raton: CRC Press;
2016. p. 133–41. y
38. Anell A, Glenngård AH, Merkur S. Sweden health system review. Health
Syst Transit. 2012;14(5):1–159. 39. Patriarca R, Di Gravio G, Woltjer R, Costantino F, Praetorius G, Ferreira P,
Hollnagel E. Framing the FRAM: A literature review on the functional reso‑
nance analysis method. Saf Sci. 2020;129:104827. p
61. Braithwaite J, Wears R, Hollnagel E. Resilient health care. Volume 3: recon‑
ciling work-as-imagined and work- as-done. Boca Raton: CRC Press; 2017. 40. McGill A, Smith D, McCloskey R, Morris P, Goudreau A, Veitch B. The func‑
tional resonance analysis method as a health care research methodology:
a scoping review protocol. JBI Evid Synth. 2020;18(0):1–7. 62. Cook R, Rasmussen J. “Going solid”: a model of system dynamics and
consequences for patient safety. Qual Saf Health Care. 2005;14(2):130–4. 63. Ekstedt M, Schildmeijer K, Backåberg S, Ljungholm L, Fagerström C. “We
just have to make it work”: a qualitative study on assistant nurses’ experi‑
ences of patient safety performance in home care services using forum
play scenarios. BMJ Open. 2022;12(5):e057261. 41. Hollnagel E. FRAM: The Functional Resonance Analysis Method: Model‑
ling Complex Socio-Technical Systems. Farnham: Ashgate Publishing, Ltd;
2012. 42. Hollnagel E, Slater D. The Functional Resonance Analysis Method and
Manual (Version 2). 2018. 64. Tarrant C, Windridge K, Baker R, Freeman G, Boulton M. ‘Falling through
gaps’: primary care patients’ accounts of breakdowns in experienced
continuity of care. Fam Pract. 2015;32(1):82–7. 43. Hollnagel E, Leonhardt J, Macchi L, Kirwan B. White paper on resilience
engineering (Eurocontrol). 2009. 65. Samal L, Dykes PC, Greenberg JO, Hasan O, Venkatesh AK, Volk LA, Bates
DW. Care coordination gaps due to lack of interoperability in the United
States: a qualitative study and literature review. BMC Health Serv Res. 2016;16:143–143. 44. Sandelowski M. Sample size in qualitative research. Res Nurs Health. 1995;18(2):179–83. 45. Ministry of Health and Social Affairs. Lag om samverkan vid utskrivning
från sluten hälso- och sjukvård (2017:612) [Internet]. Stockholm, Sweden;
2017. Available from: https://www.riksdagen.se/sv/dokument-och-lagar/
dokument/svensk-forfattningssamling/lag-2017612-om-samverkan-vid-
utskrivning-fran_sfs-2017-612/. 66. Fylan B, Marques I, Ismail H, Breen L, Gardner P, Armitage G, Blenkinsopp
A. Author details 23. Sheikh F, Gathecha E, Bellantoni M, Christmas C, Lafreniere JP, Arbaje AI. A
call to bridge across silos during care transitions. Jt Comm J Qual Patient
Saf. 2018;44(5):270–8. 1 Department of Health and Caring Sciences, Linnaeus University, Kalmar,
Sweden. 2 Ambulance Service, Region Kalmar County, Västervik, Sweden. 3 Swedish National Road and Transport Research Institute, Linköping, Sweden. 4 Department of Maritime Operations, University of South-Eastern Norway,
Borre, Norway. 5 Department of Learning, Informatics, Management and Ethics,
LIME, Karolinska Institutet, Stockholm, Sweden. 24. Shah MN, Hollander MM, Jones CM, Caprio TV, Conwell Y, Cush‑
man JT, Dugoff EH, Kind AJH, Lohmeier M, Mi R, et al. Improving
the ED-to-home transition: the community paramedic-delivered
care transitions intervention-preliminary findings. J Am Geriatr Soc. 2018;66(11):2213–20. Page 27 of 28 Page 27 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 25. Markle-Reid M, Valaitis R, Bartholomew A, Fisher K, Fleck R, Ploeg
J, Salerno J, Thabane L. Feasibility and preliminary effects of an
integrated hospital-to-home transitional care intervention for older
adults with stroke and multimorbidity: a study protocol. J Comorb. 2019;9:2235042X19828241. 48. Ministry of Health and Social Affairs. The Swedish Social Services Act
(2001:453) [Internet]. Stockholm, Sweden; 2001. Available from: https://
www.riksdagen.se/sv/dokument-och-lagar/dokument/svensk-forfattnin
gssamling/socialtjanstlag-2001453_sfs-2001-453/. 49. Hsieh H-F, Shannon SE. Three Approaches to Qualitative Content Analysis. Qual Health Res. 2005;15(9):1277–88. 26. Naylor MD, Aiken LH, Kurtzman ET, Olds DM, Hirschman KB. The care
span: the importance of transitional care in achieving health reform. Health Aff (Millwood). 2011;30(4):746–54. 50. Bowen GA. Document analysis as a qualitative research method. Qual Res
J. 2009;9(2):27–40. 27. Buikstra E, Strivens E, Clay-Williams R. Understanding variabil‑
ity in discharge planning processes for the older person. Saf Sci. 2020;121:137–46. 51. Patriarca R, Bergström J. Modelling complexity in everyday operations:
functional resonance in maritime mooring at quay. Cogn Technol Work. 2017;19(4):711–29. 28. Salehi V, Hanson N, Smith D, Mccloskey R, Jarrett P, Veitch B. Modeling
and analyzing hospital to home transition processes of frail older adults
using the functional resonance analysis method (FRAM). Appl Ergon. 2021;93:103392. 52. Woods D, Hollnagel E. Prologue: resilience engineering concepts. In: Holl‑
nagel E, Woods D, Leveson NG, editors. Resilience Engineering: Concepts
and Precepts. Aldershot: Ashgate Publishing; 2006. p. 1–6. 53. Iflaifel M, Lim RH, Crowley C, Greco F, Ryan K, Iedema R. Modelling
the use of variable rate intravenous insulin infusions in hospitals by
comparing work as done with work as imagined. Author details Res Social Adm Pharm. 2022;18(5):2786–95. 29. Laugaland K, Aase K, Waring J. Hospital discharge of the elderly-an obser‑
vational case study of functions, variability and performance-shaping
factors. BMC Health Serv Res. 2014;14(1):365. 30. Das P, Benneyan J, Powers L, Carmody M, Kerwin J, Singer S. Engineering
safer care coordination from hospital to home: lessons from the USA. Future Healthc J. 2018;5(3):164–70. 54. Carvalho PVRD, Righi AW, Huber GJ. Lemos CdF, Jatoba A, Gomes JO:
Reflections on work as done (WAD) and work as imagined (WAI) in an
emergency response organization: a study on firefighters training exer‑
cises. Applied Ergonomics. 2018;68:28–41. 31. Baxter R, Shannon R, Murray J, O’Hara JK, Sheard L, Cracknell A, Lawton R. Delivering exceptionally safe transitions of care to older people: a qualita‑
tive study of multidisciplinary staff perspectives. BMC Health Serv Res. 2020;20(1):780. 55. Van Dijk LM, Meulman MD, Van Eikenhorst L, Merten H, Schutijser BCFM,
Wagner C. Can using the functional resonance analysis method, as an
intervention, improve patient safety in hospitals?: a stepped wedge
design protocol. BMC Health Serv Res. 2021;21(1):1228. 32. Health Quality Ontario. Continuity of care to optimize chronic disease
management in the community setting: an evidence-based analysis. Ont
Health Technol Assess Ser. 2013;13(6):1–41. 56. Smith AF, Plunkett E. People, systems and safety: resilience and excellence
in healthcare practice. Anaesthesia. 2019;74(4):508–17. 33. Forster AJ, Murff HJ, Peterson JF, Gandhi TK, Bates DW. The incidence and
severity of adverse events affecting patients after discharge from the
hospital. Ann Intern Med. 2003;138(3):161–7. 57. Ashour A, Ashcroft DM, Phipps DL. Mind the gap: examining work-as-
imagined and work-as-done when dispensing medication in the com‑
munity pharmacy setting. Appl Ergon. 2021;93:103372. 34. Creswell JW, Plano Clark VL. Designing and conducting mixed methods
research. Los Angeles: SAGE; 2017. 58. Snooks HA. Gaps between policy, protocols and practice: a qualitative
study of the views and practice of emergency ambulance staff concern‑
ing the care of patients with non-urgent needs. Qual Saf Health Care. 2005;14(4):251–7. 35. Spradley JP. Participant observation. Long Grove, IL: Waveland Press; 2016. 36. Hollnagel E, Hounsgaard J, Coligan L. FRAM The Functional Resonance
Analysis Method a handbook for the practical use of the method. 2014. 59. Clay-Williams R, Hounsgaard J, Hollnagel E. Where the rubber meets the
road: using FRAM to align work-as-imagined with work-as-done when
implementing clinical guidelines. Implement Sci. 2015;10:125. 37. Ministry of Health and Social Affairs. About the Swedish healthcare
system [Internet]. Author details Sample size in qualitative
interview studies: guided by information power. Qual Health Res. 2016;26(13):1753–60. 79. Abrams LS. Sampling ‘hard to reach’ populations in qualitative research:
the case of incarcerated youth. Qual Soc Work. 2010;9(4):536–50. 79. Abrams LS. Sampling ‘hard to reach’ populations in qualitative research:
the case of incarcerated youth. Qual Soc Work. 2010;9(4):536–50. 80. Roller MR, Lavrakas PJ. Applied qualitative research design: a total quality
framework approach. New York: Guilford Press; 2015. 80. Roller MR, Lavrakas PJ. Applied qualitative research design: a total quality
framework approach. New York: Guilford Press; 2015. 81. O’Hara J, Canfield C, Aase K. Patient and family perspectives in
resilient healthcare studies: A question of morality or logic? Saf Sci. 2019;120:99–106. 81. O’Hara J, Canfield C, Aase K. Patient and family perspectives in
resilient healthcare studies: A question of morality or logic? Saf Sci. 2019;120:99–106. 82. Cook RI, Render M, Woods DD. Gaps in the continuity of care and pro‑
gress on patient safety. BMJ (Clinical research ed). 2000;320(7237):791–4. 83. General Data Protection Regulation (GDPR) 2016/679. http://eur-lex.
europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32016R0679. Accessed
20 Oct 2022. 84. World Medical Association. World Medical Association Declaration of
Helsinki. Ethical principles for medical research involving human sub‑
jects. Bull World Health Org. 2001;79(4):373-4. Author details Gaps, traps, bridges and props: a mixed-methods study of resilience
in the medicines management system for patients with heart failure at
hospital discharge. BMJ Open. 2019;9(2):e023440. g
46. Ministry of Health and Social Affairs. The Swedish Health and Social Ser‑
vices Act (2017:30) [Internet]. Stockholm, Sweden; 2017. Available from:
https://www.riksdagen.se/sv/dokument-och-lagar/dokument/svensk-
forfattningssamling/halso-och-sjukvardslag-201730_sfs-2017-30/. 67. Li E, Clarke J, Neves AL, Ashrafian H, Darzi A. Electronic health records,
interoperability and patient safety in health systems of high-income
countries: a systematic review protocol. BMJ Open. 2021;11(7):e044941. 68. Ghosh K, Dohan MS, Curl E, Goodwin M, Morrell P, Guidroz P. Informa‑
tion tools for care coordination in patient handover: is an electronic
medical record enough to support nurses? Health Care Manage Rev. 2022;47(2):100–8. 47. Ministry of Health and Social Affairs. The Swedish Patient Act. (2014:821)
[Internet]. Stockholm, Sweden; 2014. Available from: https://www.riksd
agen.se/sv/dokument-och-lagar/dokument/svensk-forfattningssamling/
patientsakerhetslag-2010659_sfs-2010-659/. 69. Fennelly O, Cunningham C, Grogan L, Cronin H, O’Shea C, Roche
M, Lawlor F, O’Hare N. Successfully implementing a national Page 28 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Hedqvist et al. BMC Health Services Research (2023) 23:851 electronic health record: a rapid umbrella review. Int J Med Inform. 2020;144:104281. electronic health record: a rapid umbrella review. Int J Med Inform. 2020;144:104281. 70. Rothman B, Leonard JC, Vigoda MM. Future of electronic health records:
implications for decision support. Mt Sinai J Med. 2012;79(6):757–68. 71. Highfill T. Do hospitals with electronic health records have lower
costs? A systematic review and meta-analysis. Int J Healthc Manag. 2020;13(1):65–71. gi
p
costs? A systematic review and meta-analysis. Int J Healthc Manag. 2020;13(1):65–71. 72. Pype P, Mertens F, Helewaut F, Krystallidou D. Healthcare teams as
complex adaptive systems: understanding team behaviour through team
members’ perception of interpersonal interaction. BMC Health Services
Res. 2018;18(1):570. 73. Amalberti R, Auroy Y, Berwick D, Barach P. Five system barriers to achiev‑
ing ultrasafe health care. Ann Intern Med. 2005;142(9):756. 74. Glenny C, Stolee P, Sheiban L, Jaglal S. Communicating during care
transitions for older hip fracture patients: family caregiver and health care
provider’s perspectives. Int J Integr Care. 2013;13(4):e044. 75. Dekker S. Drift into failure: from hunting broken components to under‑
standing complex systems. Farnham: Ashgate Pub; 2011. y
76. Cilliers P. Complexity, deconstruction and relativism. Theory Cult Soc. 2005;22:255–67. 77. Rasmussen J. The role of error in organizing behaviour. Qual Saf Health
Care. 2003;12(5):377–83. 78. Malterud K, Siersma VD, Guassora AD. Publisher’s Note
S Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations. •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from: •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from: •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from: •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from:
|
https://openalex.org/W3206000828
|
https://www.nature.com/articles/s41467-021-26354-0.pdf
|
English
| null |
COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models
|
Nature communications
| 2,021
|
cc-by
| 13,241
|
ARTICLE ARTICLE COVA1-18 neutralizing antibody protects against
SARS-CoV-2 in three preclinical models COVA1-18 neutralizing antibody protects against
SARS-CoV-2 in three preclinical models NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications A full list of author affiliations appears at the end of the paper. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 A A
cross the world, the Coronavirus Disease 19 (COVID-19)
pandemic caused by severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) continues to escalate1. Despite the progressive rollout of vaccines, there remains an
urgent need for both curative and preventive measures, especially
in individuals with high risk. Monoclonal neutralizing antibodies
(NAbs), isolated from convalescent COVID-19 patients, are one
of the most promising approaches and two NAb-based products
have already received emergency use authorizations by regulatory
agencies in both the US2,3 and Europe4,5. Although their clinical
efficacy in hospitalized patients remains to be fully assessed, their
capability to reduce viral loads and hospitalization in high risk
individuals shows that NAbs constitute an effective treatment
when administered early enough after symptom onset6–8. weaker binding to RBD compared to IgG (84 nM), with the
difference mainly caused by a faster Fab off-rate (Fig. 1a, Table 1),
as also observed in a different assay setting (Supplementary
Fig. 1d). With an IC50 of 199 ng ml−1, the COVA1-18 Fab was
237-fold less potent at neutralizing SARS-CoV-2 pseudovirus,
showing that the IgG avidity effect is important for COVA1-18
neutralization potency (Supplementary Fig. 1a, Table 1). COVA1-18 inhibits viral replication in rodents. We sought to
evaluate whether COVA1-18 could control SARS-CoV-2 viral
infection in a previously described Ad5-hACE2 mouse model22,23
using a 10 mg kg−1 dose. COVA1-18 administered intraper-
itoneally 24 h either prior to or after a SARS-CoV-2 challenge
with 104 plaque forming units (PFU) (n = 5 for treated groups,
n = 3 for control group) was fully protective with no detectable
viral replication in the lungs (Fig. 1b, c). We then tested the
efficacy of COVA1-18 in the golden Syrian hamster model (n = 5
per group), which is naturally susceptible to SARS-CoV-2 and
develops severe pneumonia upon infection24. We evaluated the
effect on lung viral loads of 10 mg kg−1 of COVA1-18 given 24 h
after a 105 PFU intranasal challenge (Fig. 1b, d). At 3 days post-
infection (d.p.i.), 3/5 animals had high serum neutralization,
while for 2/5 animals, low neutralization activity was observed
(Supplementary Fig. 1e). On day 3, the COVA1-18 treated group
had significantly lower median lung viral titers compared to the
control group (3.5 vs 6.7 log10 PFU g−1, respectively, p < 0.01)
with lowest viral titers corresponding to the higher neutralizing
serum activity (Fig. 1d). Results COVA1-18 in vitro potency is dependent on avidity. To
advance our earlier in vitro results9 on COVA1-18 and allow for
better comparability with other studies, we used two pseudovirus
assays, one using lentiviral pseudotypes with an ACE2-expressing
293 T cell line13, and one using VSV-pseudotypes with Vero E6
cells14, to confirm the potency of COVA1-18. With these assays,
we found that COVA1-18 IgG inhibited lentiviral SARS-CoV-2
pseudovirus with an IC50 of 1.7 ng ml−1 (11.3 pM) and VSV-
based pseudovirus with an IC50 of 9 ng ml−1 (60 pM), confirming
the remarkable potency previously observed against authentic
virus9 (Supplementary Fig. 1a, Table 1). These results were cor-
roborated in multiple independent labs and COVA1-18 was also
equipotent against the D614G variant (Table 1) that now dom-
inates worldwide15–19 as well as the recently emerged B.1.1.7
variant that includes the N501Y mutation in the RBD20,21
(Table 2). COVA1-18 PrEP prevents infection in NHP. We evaluated the
potential of COVA1-18 to prevent SARS-CoV-2 infection in
cynomolgus macaques in a pre-exposure prophylaxis (PrEP)
study. The animals were treated intravenously 24 h prior to viral
challenge with a dose of 10 mg kg−1 of COVA1-18 (Fig. 2a). Treated and control animals (n = 5 per group) were challenged
on day 0 with 106 PFU of SARS-CoV-2 via combined intranasal
and
intratracheal
routes
using
an
experimental
protocol
developed previously25,26. On the day of challenge, the mean
COVA1-18 serum concentration was 109 ± 2.7 μg ml−1 (Fig. 2b,
Supplementary Fig. 2a). COVA-18 was also detected in all
respiratory tract samples and rectal samples (Fig. 2c–e, Supple-
mentary Fig. 2b–d), and represented on average 1.5% and 1.2% of
the total IgG in heat-inactivated content in the nasopharyngeal
and tracheal mucosae, respectively. These levels remained con-
stant throughout the study period and similar levels were detected
at 3 d.p.i. in bronchoalveolar lavages (BAL) and saliva (Fig. 2e, f). As SARS-CoV-2 can cause damage to non-respiratory organs, we
performed a pharmacokinetic study on two additional macaques
to characterize the COVA1-18 distribution within the first 24 h
using non heat-inactivated samples (Fig. 2g and Supplementary COVA1-18 bound strongly to SARS-CoV-2 S protein and
showed no cross-reactivity with S proteins of SARS-CoV, MERS-
CoV and common cold coronaviruses HKU1-CoV, 229E-CoV
and NL63-CoV (Supplementary Fig. 1b)9. ARTICLE The time of treatment (24 h post-infec-
tion) and 3-day study period did not allow for prevention of lung
damage and recovery monitoring in this model (Supplementary
Fig. 1f, g and Supplementary Table 1). We and others have previously isolated and characterized
several highly potent monoclonal NAbs with half-maximum
inhibitory concentration (IC50) values in the picomolar range9–12,
with the majority of these targeting the receptor binding domain
(RBD) on the S1 subunit of the S protein. We previously iden-
tified COVA1-18, an RBD-specific monoclonal Ab, as one of the
most potent NAb in vitro9. In this work, we use three different experimental models as well
as mathematical modeling to demonstrate that COVA1-18 rapid
and extensive biodistribution is associated with a very potent
antiviral effect, and make it a promising candidate for clinical
evaluation, both as a prophylactic or therapeutic treatment of
COVID-19. COVA1-18 neutralizing antibody protects against
SARS-CoV-2 in three preclinical models Pauline Maisonnasse
1,21, Yoann Aldon
2,21, Aurélien Marc3, Romain Marlin
1,
Nathalie Dereuddre-Bosquet
1, Natalia A. Kuzmina
4,5, Alec W. Freyn
6, Jonne L. Snitselaar2,
Antonio Gonçalves
3, Tom G. Caniels
2, Judith A. Burger2, Meliawati Poniman2, Ilja Bontjer
2,
Virginie Chesnais7, Ségolène Diry7, Anton Iershov7, Adam J. Ronk
4,5, Sonia Jangra6, Raveen Rathnasinghe6,8,
Philip J. M. Brouwer2, Tom P. L. Bijl2, Jelle van Schooten
2, Mitch Brinkkemper2, Hejun Liu9, Meng Yuan
9,
Chad E. Mire
5,10, Mariëlle J. van Breemen2, Vanessa Contreras1, Thibaut Naninck
1, Julien Lemaître1,
Nidhal Kahlaoui1, Francis Relouzat1, Catherine Chapon1, Raphaël Ho Tsong Fang1, Charlene McDanal11,
Mary Osei-Twum12, Natalie St-Amant12, Luc Gagnon12, David C. Montefiori11, Ian A. Wilson
9, Eric Ginoux7,
Godelieve J. de Bree13, Adolfo García-Sastre
6,14,15,16, Michael Schotsaert
6,16, Lynda Coughlan
6,17,
Alexander Bukreyev
4,5,10, Sylvie van der Werf
18,19, Jérémie Guedj
3, Rogier W. Sanders2,20✉,
Marit J. van Gils2✉& Roger Le Grand
1✉ Effective treatments against Severe Acute Respiratory Syndrome coronavirus 2 (SARS-
CoV-2) are urgently needed. Monoclonal antibodies have shown promising results in
patients. Here, we evaluate the in vivo prophylactic and therapeutic effect of COVA1-18, a
neutralizing antibody highly potent against the B.1.1.7 isolate. In both prophylactic and
therapeutic settings, SARS-CoV-2 remains undetectable in the lungs of treated hACE2 mice. Therapeutic treatment also causes a reduction in viral loads in the lungs of Syrian hamsters. When administered at 10 mg kg-1 one day prior to a high dose SARS-CoV-2 challenge in
cynomolgus macaques, COVA1-18 shows very strong antiviral activity in the upper respira-
tory compartments. Using a mathematical model, we estimate that COVA1-18 reduces viral
infectivity by more than 95% in these compartments, preventing lymphopenia and extensive
lung lesions. Our findings demonstrate that COVA1-18 has a strong antiviral activity in three
preclinical models and could be a valuable candidate for further clinical evaluation. A full list of author affiliations appears at the end of the paper. 1 NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunicatio Results b Study design with n = 5 per group, except mouse control group (n = 3). Hamsters
were infected with 105 PFU on day 0 and treated on day 1. Mice received COVA1-18 24 h prior to or after exposure to 104 PFU. Lung viral titers at 3 days
post-infection are shown for mice (c) and hamsters (d). Bars indicate medians. Mann-Whitney unpaired two-tailed t-test, p values: *:0.0179, **:0.0079. Ctl. control group (black), KD dissociation constant, PFU Plaque forming unit, PrEP pre-exposure prophylaxis (dark blue), Ther. therapeutic (light blue). 0.01
0.1
1
10
BAL
3
Saliva
SARS-CoV-2
106 PFU
D-1
D0
Mucosal &
serum sampling
D28
a
b
c
d
e
COVA1-18
10 mg kg-1
Baseline
sampling
n = 5
0.01
0.1
1
10
-30 -15
0
5
15
20
25
0.01
0.1
1
10
10
-30-15
0
5
10
15
20
25
0.01
0.1
1
10
3
COVA1-18 (µg ml-1)
Serum
f
Control
COVA1-18
Days post-infection
COVA1-18
(% of total IgG)
Nasopharyngeal fluid
Tracheal fluid
-30 -15
0
5
10
15
20
25
0.1
1
10
100
Days post-infection
COVA1-18
(% of total IgG)
g
Accessory
Caudal L
Caudal R
Cranial L
Cranial R
Middle L
Middle R
Heart
Liver
Spleen
Kidney
Brain
Trachea
0.1
1
10
100
COVA1-18
(ng per mg of tissue)
Lungs
Biodistribution - PK
Fig. 2 COVA1-18 serum and mucosal pharmacokinetic in infected cynomolgus macaques. a Study design. Two groups of n = 5 were exposed to 106 PFU
of SARS-CoV-2 (intranasal and intratracheal routes). Treated animals received 10 mg kg−1of COVA1-18 1 day before challenge. b COVA1-18 serum
concentration (mean with range). COVA1-18 concentration reported as percent of total cynomolgus IgG in heat-inactivated (c) nasopharyngeal fluid, d
tracheal fluid (means with range), e bronchoalveolar lavage (BAL) and f saliva (means ± SEMs) with n = 5, except for the control group in (f) where n = 1. g
The two macaques from the pharmacokinetic study were euthanized at 24 h post-treatment and their organs analyzed to assess the biodistribution of
COVA1-18. The concentration of COVA1-18 was normalized to the weight of each sample for every organ. Bars represent means. The red dashed line
indicates challenge day. L left, PFU Plaque forming unit, PK pharmacokinetic, R right. Results Active viral replication, as
assessed by sgRNA levels, peaked at 1-2 d.p.i. in nasopharyngeal 50 nM
100 nM
200 nM
400 nM
ctl. 100 nM
200 nM
400 nM
800 nM
IgG
Fab
a
Response (nm)
0
1
2
3
4
5
KD = 7 nM
KD = 84 nM
0
100
200
0
100
200
Time (s)
Time (s) d
b
D0
D1
D3
SARS-
CoV-2
Lung viral titer
ctl. PrEP
Ther. 0
1
2
3
4
5
D-1
PrEP
Therapeutic
Hamsters
hACE2 mice
c
ctl. Ther. 2
3
4
5
6
7
8
Viral titer (log10 PFU g-1)
**
*
*
Viral titer (log10 PFU ml-1) 50 nM
100 nM
200 nM
400 nM
ctl. 100 nM
200 nM
400 nM
800 nM
IgG
Fab
a
d
Response (nm)
b
D0
D1
D3
SARS-
CoV-2
Lung viral titer
0
1
2
3
4
5
ctl. PrEP
Ther. 0
1
2
3
4
5
D-1
PrEP
Therapeutic
Hamsters
hACE2 mice
c
KD = 7 nM
KD = 84 nM
ctl. Ther. 2
3
4
5
6
7
8
Viral titer (log10 PFU g-1)
**
*
*
Viral titer (log10 PFU ml-1)
0
100
200
0
100
200
Time (s)
Time (s)
Fig. 1 COVA1-18 avidity and SARS-CoV-2 protection in rodents. a Biolayer interferometry sensorgrams comparing COVA1-18 IgG and Fab binding to
RBD. KDs are indicated. Representative of 3 independent experiments. b Study design with n = 5 per group, except mouse control group (n = 3). Hamsters
were infected with 105 PFU on day 0 and treated on day 1. Mice received COVA1-18 24 h prior to or after exposure to 104 PFU. Lung viral titers at 3 days
post-infection are shown for mice (c) and hamsters (d). Bars indicate medians. Mann-Whitney unpaired two-tailed t-test, p values: *:0.0179, **:0.0079. Ctl. control group (black), KD dissociation constant, PFU Plaque forming unit, PrEP pre-exposure prophylaxis (dark blue), Ther. therapeutic (light blue). d
ctl. PrEP
Ther. 0
1
2
3
4
5
Hamsters
hACE2 mice
c
ctl. Ther. 2
3
4
5
6
7
8
Viral titer (log10 PFU g-1)
**
*
*
Viral titer (log10 PFU ml-1) b Fig. 1 COVA1-18 avidity and SARS-CoV-2 protection in rodents. a Biolayer interferometry sensorgrams comparing COVA1-18 IgG and Fab binding to
RBD. KDs are indicated. Representative of 3 independent experiments. Results 2
3
4
5
6
7
8
Viral titer (log10 PFU g-1)
**
*
*
Viral titer (log10 PFU ml-1)
0
100
200
0
100
200
Time (s)
Time (s)
Fig. 1 COVA1-18 avidity and SARS-CoV-2 protection in rodents. a Biolayer interferometry sensorgrams comparing COVA1-18 IgG and Fab binding to
RBD. KDs are indicated. Representative of 3 independent experiments. b Study design with n = 5 per group, except mouse control group (n = 3). Hamsters
were infected with 105 PFU on day 0 and treated on day 1. Mice received COVA1-18 24 h prior to or after exposure to 104 PFU. Lung viral titers at 3 days
post-infection are shown for mice (c) and hamsters (d). Bars indicate medians. Mann-Whitney unpaired two-tailed t-test, p values: *:0.0179, **:0.0079. Ctl. control group (black), KD dissociation constant, PFU Plaque forming unit, PrEP pre-exposure prophylaxis (dark blue), Ther. therapeutic (light blue). Table 2 COVA1-18 and COVA1-16 neutralization potency
against variants in HEK293T hACE2 cells. IC50 (ng ml−1)
COVA1-18
COVA1-16
COVA1-18 + COVA1-16 (1:1)
Wild type
1.7
80.7
N/A
D614G
0.7
109.2
1.0
B.1.1.7
1.4
90.8
2.2
E484K
>12500
94.3
N/A
B.1.351
>50000
50.6
97.6
Mean IC50 values with n ≥3, except for the cocktail with n = 2. N/A, not assessed. Fig. 2e, f). COVA1-18 was found in all organs studied, including
the lungs, at concentrations of 4 to 22 ng mg−1 of tissue, except
for the brain where concentrations were substantially lower
(250 pg mg−1 of tissue) (Fig. 2g). Altogether, these data showed
that COVA1-18 administered intravenously was rapidly and
efficiently distributed to the natural sites of infection as well as to
organs affected by COVID-19 pathology. Table 2 COVA1-18 and COVA1-16 neutralization potency
against variants in HEK293T hACE2 cells. IC50 (ng ml−1)
COVA1-18
COVA1-16
COVA1-18 + COVA1-16 (1:1)
Wild type
1.7
80.7
N/A
D614G
0.7
109.2
1.0
B.1.1.7
1.4
90.8
2.2
E484K
>12500
94.3
N/A
B.1.351
>50000
50.6
97.6
Mean IC50 values with n ≥3, except for the cocktail with n = 2. N/A, not assessed. Table 2 COVA1-18 and COVA1-16 neutralization potency
against variants in HEK293T hACE2 cells. g
y
gy
Following viral challenge, control animals showed similar
genomic (g)RNA and subgenomic (sg)RNA levels and kinetics as
previously described25,26 with median peak viral loads (VL) of 6.4
and 6.2 log10 copies per ml at 1-2 d.p.i. in the nasopharyngeal and
tracheal swabs, respectively (Fig. 3a). Results Biolayer interferometry
experiments showed that COVA1-18 IgG bound to soluble SARS-
CoV-2 S protein with an apparent dissociation constant (KD) of
5 nM, and its affinity for RBD was similar at 7 nM (Fig. 1a,
Supplementary Fig. 1c, d, Table 1). Its Fab displayed a 12-fold Table 1 BLI and neutralization potency of COVA1-18 IgG vs Fab in HEK293T hACE2 cells. IC50
BLI
AMC (n ≥4)
Duke
(n = 1)
Duke
D614G
(n = 1)
Nexelis
(n = 1)
RBD loaded (n = 3)
Soluble S loaded (n = 3)
ng ml−1
pM
ng ml−1
KD (nM)
Ka (M−1s−1)
Kd (s−1)
KD (nM)
Ka (M−1s−1)
Kd (s−1)
1–18
IgG
1.7
11.3
9.0
7.0
9.0
7.0
1.7E+05
1.3E-03
5.0
3.7E+05
1.9E-03
Fab
199.0
3968.0
N/A
N/A
N/A
84.1
5.0E+04
4.1E-03
N/A
N/A
N/A
AMC and Duke neutralization assays use lentiviral pseudotyped particles and HEK293T hACE2 cells. Nexelis neutralization assay uses VSVΔG pseudotyped particles and Vero E6 cells. BLI biolayer
interferometry, RBD receptor binding domain. Table 1 BLI and neutralization potency of COVA1-18 IgG vs Fab in HEK293T hACE2 cells. 2 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 Fig. 2e, f). COVA1-18 was found in all organs studied, including
the lungs, at concentrations of 4 to 22 ng mg−1 of tissue, except
for the brain where concentrations were substantially lower
(250 pg mg−1 of tissue) (Fig. 2g). Altogether, these data showed
that COVA1-18 administered intravenously was rapidly and
efficiently distributed to the natural sites of infection as well as to
organs affected by COVID-19 pathology. Following viral challenge, control animals showed similar
genomic (g)RNA and subgenomic (sg)RNA levels and kinetics as
previously described25,26 with median peak viral loads (VL) of 6.4
and 6.2 log10 copies per ml at 1-2 d.p.i. in the nasopharyngeal and
tracheal swabs, respectively (Fig. 3a). Active viral replication, as
assessed by sgRNA levels, peaked at 1-2 d.p.i. in nasopharyngeal
50 nM
100 nM
200 nM
400 nM
ctl. 100 nM
200 nM
400 nM
800 nM
IgG
Fab
a
d
Response (nm)
b
D0
D1
D3
SARS-
CoV-2
Lung viral titer
0
1
2
3
4
5
ctl. PrEP
Ther. 0
1
2
3
4
5
D-1
PrEP
Therapeutic
Hamsters
hACE2 mice
c
KD = 7 nM
KD = 84 nM
ctl. Ther. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 Nasopharyngeal
Tracheal
a
b
-2
0
2
4
6
8
3
4
5
6
7
8
9
Viral load (log10 copies per ml)
**
**
sgRNA (log10 copies per ml)
**
**
*
0
1
2
3
2
3
4
5
6
7
8
Days post-infection
Days post-infection
-2
0
2
4
6
8
0
1
2
3
**
**
**
** **
** ** ** **
**
3
COVA1-18
LoD
MF1
MF2
MF3
MF4
MF5
MF6
MF7
MF8
MF9
MF10
BAL
Control
LoQ
3 Nasopharyngeal
Tracheal
a
-2
0
2
4
6
8
3
4
5
6
7
8
9
Viral load (log10 copies per ml)
Days post-infection
-2
0
2
4
6
8
**
**
**
** **
** ** ** **
**
BA
3 b
**
**
sgRNA (log10 copies per ml)
**
**
*
0
1
2
3
2
3
4
5
6
7
8
Days post-infection
Days post-infection
0
1
2
3
3
COVA1-18
LoD
MF1
MF2
MF3
MF4
MF5
MF6
MF7
MF8
MF9
MF10
Control d
0
2
4
6
8
10
Lymphocytes
(x103 cells per µl)
-4
2
0
4
6
7
**
Days post-infection
Control
COVA1-18 c
Lung lesions
D2
D5
ctl. 1-18
Historical ctl. D3
0
5
10
15
20
CT score d c Days post-infection D3 Historical ctl. Fig. 3 COVA1-18 pre-exposure prophylaxis protects cynomolgus monkeys against SARS-CoV-2 challenge and clinical symptoms. a Genomic (g)RNA
and b subgenomic (sg)RNA loads determined by PCR in nasopharyngeal fluids (left), tracheal fluids (middle) and bronchoalveolar lavages (BAL) (right). Individual values are plotted for nasopharyngeal and tracheal samples and bars represent medians for BAL. For b, boxes and whiskers representation with
min-max., median, 25th–75th percentile for n = 5 per group. c Chest CT scores were determined at 3 d.p.i. and at 2 or 5 d.p.i for historical controls (n = 8). d Absolute lymphocyte count in the blood (mean with range). Mann-Whitney unpaired two-tailed t-test, p values: * < 0.05, ** < 0.01. 1–18, COVA1-18; CT
Computed Tomography, Ctl. control group, LoD limit of detection, LoQ limit of quantification. the progressive elimination of the challenge inoculum, and does
not result from active replication. The gRNA and sgRNA loads in
BAL were also lower in COVA1-18 recipients compared to
controls but the difference did not reach statistical significance
(Fig. 3a, b). Cynomolgus anti-S IgM was detected as early as 6
d.p.i. Results SARS-CoV-2
106 PFU
D-1
D0
Mucosal &
serum sampling
D28
a
b
c
COVA1-18
10 mg kg-1
Baseline
sampling
n = 5
-30 -15
0
5
15
20
25
0.01
0.1
1
10
10
COVA1-18 (µg ml-1)
Serum
Control
COVA1-18
Nasopharyngeal fluid
-30 -15
0
5
10
15
20
25
0.1
1
10
100
Days post-infection
COVA1-18
(% of total IgG) 0.01
0.1
1
10
BAL
d
e
-30-15
0
5
10
15
20
25
0.01
0.1
1
10
3
Days post-infection
COVA1-18
(% of total IgG)
Tracheal fluid
Days post infec f f 3
Saliva
0.01
0.1
1
10
f
g
Accessory
Caudal L
Caudal R
Cranial L
Cranial R
Middle L
Middle R
Heart
Liver
Spleen
Kidney
Brain
Trachea
0.1
1
10
100
COVA1-18
(ng per mg of tissue)
Lungs
Biodistribution - PK d g Biodistribution - PK Lungs Fig. 2 COVA1-18 serum and mucosal pharmacokinetic in infected cynomolgus macaques. a Study design. Two groups of n = 5 were exposed to 106 PFU
of SARS-CoV-2 (intranasal and intratracheal routes). Treated animals received 10 mg kg−1of COVA1-18 1 day before challenge. b COVA1-18 serum
concentration (mean with range). COVA1-18 concentration reported as percent of total cynomolgus IgG in heat-inactivated (c) nasopharyngeal fluid, d
tracheal fluid (means with range), e bronchoalveolar lavage (BAL) and f saliva (means ± SEMs) with n = 5, except for the control group in (f) where n = 1. g
The two macaques from the pharmacokinetic study were euthanized at 24 h post-treatment and their organs analyzed to assess the biodistribution of
COVA1-18. The concentration of COVA1-18 was normalized to the weight of each sample for every organ. Bars represent means. The red dashed line
indicates challenge day. L left, PFU Plaque forming unit, PK pharmacokinetic, R right. NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 Nasopharyngeal
Tracheal
a
b
-2
0
2
4
6
8
3
4
5
6
7
8
9
Viral load (log10 copies per ml)
**
**
sgRNA (log10 copies per ml)
**
**
*
0
1
2
3
2
3
4
5
6
7
8
Days post-infection
Days post-infection
d
c
0
2
4
6
8
10
Lymphocytes
(x103 cells per µl)
Lung lesions
-4
2
0
4
6
7
**
-2
0
2
4
6
8
0
1
2
3
D2
D5
ctl. 1-18
Historical ctl. D3
0
5
10
15
20
Days post-infection
CT score
**
**
**
** **
** ** ** **
**
Control
COVA1-18
3
COVA1-18
LoD
MF1
MF2
MF3
MF4
MF5
MF6
MF7
MF8
MF9
MF10
BAL
Control
LoQ
3
Fig. 3 COVA1-18 pre-exposure prophylaxis protects cynomolgus monkeys against SARS-CoV-2 challenge and clinical symptoms. a Genomic (g)RNA
and b subgenomic (sg)RNA loads determined by PCR in nasopharyngeal fluids (left), tracheal fluids (middle) and bronchoalveolar lavages (BAL) (right). Individual values are plotted for nasopharyngeal and tracheal samples and bars represent medians for BAL. For b, boxes and whiskers representation with
min-max., median, 25th–75th percentile for n = 5 per group. c Chest CT scores were determined at 3 d.p.i. and at 2 or 5 d.p.i for historical controls (n = 8). d Absolute lymphocyte count in the blood (mean with range). Mann-Whitney unpaired two-tailed t-test, p values: * < 0.05, ** < 0.01. 1–18, COVA1-18; CT
Computed Tomography, Ctl. control group, LoD limit of detection, LoQ limit of quantification. NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications Discussion Despite the recent approval of several SARS-CoV-2 vaccines by
health authorities, the slow roll-out of vaccination campaigns will
not result in resolution of the pandemic in the immediate future. Furthermore, the emergence of viral escape mutants may lead to
reduced vaccine efficacy, and some individuals, such as immu-
nocompromised patients or the elderly, may not mount adequate
protective immune responses to vaccination. Thus, there is an
urgent need to develop effective therapeutics, in particular for
individuals with high risk of severe disease. Prediction models refine COVA1-18 dosage. Next, we used a
viral dynamic model previously developed in the same SARS-
CoV-2 NHP experimental model29 to evaluate the level of pro-
tection conferred by COVA1-18, and guide potential subsequent
studies on SARS-CoV-2 MAbs. The model considers a target cell
limited infection in both nasopharyngeal and tracheal compart-
ments. In addition to the previously developed model, we
assumed that sgRNA was a proxy for the total number of non-
productively and productively infected cells (see supplementary
methods) and we further assumed that COVA1-18 plasma drug
concentrations over time, noted C(t), was the driver of drug
efficacy. We modeled the changes in C(t) using a standard first
order absorption and elimination model, and we estimated the
half-life of COVA1-18 in plasma to be 12.6 days (Supplementary
Fig. 4a). We assumed that COVA1-18 reduces infectivity rate in
both tracheal and nasopharyngeal compartments with an efficacy,
noted η(t), determined by the following model ηðtÞ ¼
CðtÞ
CðtÞþEC50,
where EC50 is the plasma COVA1-18 concentrations corre-
sponding to a 50% reduction of viral infectivity. The model fitted
the viral kinetics well in all animals (Fig. 4a, Supplementary
Fig. 4b, Supplementary Table 2). The EC50 was estimated to be 2.2
and 0.053 µg ml−1 in the nasopharynx and trachea, respectively,
which is roughly 50 and 2000 times lower than the plasma drug
concentrations of 109 µg ml−1 observed on the day of infection
(see above). Thus, these results confirm that the efficacy of
COVA1-18 was very high, with efficacies above 95% and 99.9% in
nasopharyngeal and tracheal compartments on the day of infec-
tion, respectively (Fig. 4a, Supplementary Fig. 4b). Discussion Given the long
half-life of the drug, this efficacy was maintained over time, and
we estimated that the mean individual efficacy of the COVA1-18
in the first 10 days following infection ranged between 96.67%
and 97.50% in the nasopharynx and between 99.91% and 99.94%
in the trachea (Supplementary Fig. 4c). g
In hACE2-expressing mice and golden Syrian hamsters,
COVA1-18 showed remarkable control of SARS-CoV-2 infection. These promising results were confirmed in NHPs, with COVA1-
18 given one day prior to infection achieving nearly complete
protection in the upper respiratory tract in cynomolgus maca-
ques. Using a viral dynamic model, we estimated that COVA1-18
reduced viral infectivity by >95% and 99.9% in nasopharyngeal
and tracheal compartments, respectively. The robustness of these
results are reinforced by the high challenge dose that we used,
which was 10 to 100-fold higher than in other NHP studies
evaluating NAbs for PrEP against SARS-CoV-232–38. In fact, the
model allowed us to predict, without using additional animals,
that a high level of protection could be achieved with lower doses
of 5 mg kg−1 and 1 mg kg−1 with lower inoculum doses of 105 or
104 PFU, both in prophylactic and therapeutic settings (Supple-
mentary Fig. 4, Supplementary Fig. 5). How do these levels of efficacy greater than 95% translate into
clinical efficacy? In previous work, we estimated that achieving
90% efficacy would be sufficient to confer a high level of pro-
tection against infection acquisition if treatment can be admi-
nistered prophylactically or just after a high-risk contact38. In
hospitalized patients, where viral load kinetics after admission are
associated with the risk of death, we estimated that administra-
tion of treatment with an efficacy higher than 90% could reduce
the time to viral clearance by more than 3 days in patients over 65
years of age, which could translate into significantly lower rates of
mortality in this population39. y
p p
Several NAbs are being developed and some have achieved
clinical endpoints, such as the reduction of the risk of hospitaliza-
tion in patients that initiate treatment within 5 days of symptom
onset6–8, leading to their approval for emergency use22,32–37,40. However, the narrow efficacy range of FDA-approved NAbs41–43,
together with rapidly spreading new variants complicate treatment
strategies30,31,44,45, highlighting the need for additional treatment
options, including potent NAbs, such as COVA1-18, that could be
used in combination with other NAbs. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 One mutation (E725G) was detected
in the S gene in the MF7 BAL sample when applying standard
quality filters, but this mutation has not been previously
implicated in immune escape and located outside the epitope of
COVA1-18 (Supplementary Fig. 3 and Supplementary Informa-
tion). The high efficacy of COVA1-18 treatment prevented
recovery of viral genetic information past 3 d.p.i. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 in control animals, while no IgM was detected in treated
animals at early timepoints (Supplementary Fig. 2g). Some IgM
was detected at 28 d.p.i. in 3 treated animals (MF6, MF7, MF9),
although levels remained lower than controls at 6 d.p.i. No anti-S
specific cynomolgus IgG was detected up to the day of euthanasia
in control animals (7 d.p.i.) or in treated animals up to 28 d.p.i. (Supplementary Fig. 2h). Overall, these results demonstrate that a
10 mg kg−1 dose of COVA1-18 PrEP dramatically reduced the
acquisition and/or early spread of SARS-CoV-2 in the different
respiratory compartments. and tracheal swabs with median values of 4.6 and 4.0 log10 copies
per ml, respectively (Fig. 3b). At 3 d.p.i., viral loads were detected
in the BAL with a median value of 4.9 log10 copies per ml of
gRNA and 3.2 log10 copies per ml of sgRNA, including 3 animals
with no detectable sgRNA. In comparison, treated animals had a reduction of 2.2 and 3.4
log10 median gRNA VL in tracheal swabs on days 1 and 2 (both
p < 0.01 to controls), and had undetectable VL after day 4
(Fig. 3a). The difference was also evident in nasopharyngeal
swabs, with treated animals having a reduction of 1.5 and 2.2
log10 gRNA VL on days 1 and 2 (both p < 0.01 to controls). By
day 4, 4/5 treated animals had undetectable gRNA in the
nasopharyngeal swabs while one animal (MF7) remained positive
with a low residual gRNA signal up to 7 d.p.i. COVA1-18
treatment dramatically hindered viral replication in the upper
respiratory tract as evidenced by the absence of detectable sgRNA
in the nasopharyngeal and tracheal swabs for all treated animals
with the exception of animal (MF9) that showed a low signal at 1
d.p.i. only in the tracheal swabs (Fig. 3b). Therefore, in the treated
group, most upper respiratory tract gRNA VL likely represents p
y
p
Analysis of lung lesions by chest computed tomography (CT)
showed that all treated animals had few and small lung lesions as
recorded by low CT scores at 3 d.p.i. while 2/5 controls showed
mild pulmonary lesions characterized by non-extended ground-
glass opacities (GGOs) with scores superior to 5, consistent with 4 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 emerging variants-of-concern (VOC), in particular E484K30,31. We evaluated the ability of COVA1-18 to neutralize VOCs B.1.1.7
and B.1.351 as well as a E484K single mutant virus. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 While
COVA1-18 retains its high potency against the B.1.1.7 strain, it
lost its capacity to neutralize the B.1.351 strain due primarily to
the RBD E484K mutation in the spike (Supplementary Fig. 6). Therefore, we also evaluated the in vitro potency of COVA1-18 in
a cocktail with COVA1-16, an antibody that neutralizes B.1.351
as well as SARS-CoV-1, but is less potent than COVA1-189
(Supplementary Fig. 6 and Table 2). This mAb cocktail retained
the high potency of COVA1-18 against wild-type, D614G and
B.1.1.7 and also efficiently neutralized B.1.351, providing an
avenue for broad mAb prophylaxis and treatment against VOCs. what was observed in historic controls25 and mirroring the
heterogeneity of COVID-19 infection in humans27 (Fig. 3c). In
addition, we observed that all control animals were lymphopenic
at 2 d.p.i., consistent with previous studies25,26, while all treated
animals had normal lymphocyte counts throughout the study
(p < 0.01 for the comparison) (Fig. 3d and Supplementary Fig. 2i). One concern about SARS-CoV-2 vaccines and NAb treatments
is the possible generation of suboptimal concentrations of NAb in
individuals, which could foster viral escape28. Sequencing analysis
of nasopharyngeal, tracheal and BAL samples at 3 d.p.i. showed
that COVA1-18 treatment resulted in enrichment of subclonal
variations in N and ORF1ab. One mutation (E725G) was detected
in the S gene in the MF7 BAL sample when applying standard
quality filters, but this mutation has not been previously
implicated in immune escape and located outside the epitope of
COVA1-18 (Supplementary Fig. 3 and Supplementary Informa-
tion). The high efficacy of COVA1-18 treatment prevented
recovery of viral genetic information past 3 d.p.i. what was observed in historic controls25 and mirroring the
heterogeneity of COVID-19 infection in humans27 (Fig. 3c). In
addition, we observed that all control animals were lymphopenic
at 2 d.p.i., consistent with previous studies25,26, while all treated
animals had normal lymphocyte counts throughout the study
(p < 0.01 for the comparison) (Fig. 3d and Supplementary Fig. 2i). One concern about SARS-CoV-2 vaccines and NAb treatments
is the possible generation of suboptimal concentrations of NAb in
individuals, which could foster viral escape28. Sequencing analysis
of nasopharyngeal, tracheal and BAL samples at 3 d.p.i. showed
that COVA1-18 treatment resulted in enrichment of subclonal
variations in N and ORF1ab. NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 LoQ
LoQ
y
p
Days post-infection
b
c
0
-2
4
6
2
8
0
-2
4
6
2
8
Therapeutic
Inoculum: 106 PFU
Nasopharynx
Trachea
3
4
5
6
7
8
9
2
3
4
5
6
7
8
9
2
3
4
5
6
7
8
9
2
3
4
5
6
7
8
9
2
0
-2
4
6
2
8
0
-2
4
6
2
8
PrEP
Inoculum: 106 PFU
Nasopharynx
Trachea gRNA
sgRNA
Viral load (log10 copies per ml)
Control
10 mg kg-1
5 mg kg-1
1 mg kg-1
0.1 mg kg-1
COVA1-18 dose
Injection i.v. Days post-infection
b
3
4
5
6
7
8
9
2
3
4
5
6
7
8
9
2
0
-2
4
6
2
8
0
-2
4
6
2
8
PrEP
Inoculum: 106 PFU
Nasopharynx
Trachea LoQ
LoQ
c
0
-2
4
6
2
8
0
-2
4
6
2
8
Therapeutic
Inoculum: 106 PFU
Nasopharynx
Trachea
3
4
5
6
7
8
9
2
3
4
5
6
7
8
9
2 b c Days post-infection
0
2
4
6
2 Fig. 4 Modeling of viral dynamics and treatment efficacy. a Individual prediction of the nasopharyngeal genomic (g)RNA and subgenomic (sg)RNA in
control (top) and treated animals (bottom) with individual efficacy prediction indicated (green line). The dashed red line indicates the time of infection. gRNA (squares) and sgRNA (circles) data are indicated as plain (above LoQ) or open (below LoQ). b Model predictions of gRNA and sgRNA dynamics
with 4 doses of COVA1-18 given 24 h prior to challenge (arrow). c Simulation as in (b) with COVA1-18 given 24 h post-infection. Black dotted lines indicate
LoQ (limit of quantification), i.v. intravenous, PFU plaque forming units, PrEP Pre-exposure prophylaxis. lower inoculum doses of 104 or 105 PFU, as used in other
studies32,33,37 (Supplementary Table 3), a dose of 10 mg kg−1
COVA1-18 could reduce the viral load even more dramatically
(Supplementary Fig. 4d and Supplementary Fig. 5a, b). Although it
is difficult to compare results obtained with different experimental
and virological models, this model shows that the in vivo efficacy of
COVA1-18 is comparable with what has been obtained for other
advanced NAbs in clinical development. found in the B.1.1.28 lineage, similar to what has been found with
first wave convalescent plasma and many NAbs30,31. Discussion The plasma half-life was
12.6 days, albeit lower to what is found typically for human NAbs in
humans37, ranging from 15 to 25 days, and consistent with values
reported for other human NAbs in the macaque model (Supple-
mentary Table 3). The efficacy in this model was high, despite the
high challenge dose (106 PFU) used here. We estimated that with Next, we used our model to investigate changes in experimental
conditions, such as COVA1-18 dose being administered at a lower
dose and/or after the viral challenge (see methods). In all scenarios
considered, a dose of 5 mg kg−1 was determined to provide nearly
similar results than 10 mg kg−1 (Fig. 4b, c, Supplementary Fig. 5a, b). A dose of 1 mg kg−1 could be sufficient to prevent active viral
replication as long as treatment is given prior to infection, but might
be insufficient in a therapeutic setting. However, this dose could be
relevant if lower doses of virus were used for infection, such as 104 or
105 PFU (Supplementary Fig. 4d–g). COVA1-18/1-16 cocktail neutralizes B.1.351. Many highly
potent RBD-targeting mAbs are affected by mutations in 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 gRNA
sgRNA
Viral load (log10 copies per ml)
Control
10 mg kg-1
5 mg kg-1
1 mg kg-1
0.1 mg kg-1
COVA1-18 dose
Injection i.v. LoQ
LoQ
MF1
MF2
MF3
MF4
MF5
gRNA
sgRNA
MF6
MF7
MF8
MF9
3
4
5
6
7
8
9
2
3
4
5
6
7
8
9
2
99.99
99.9
99
90
99.99
99.9
99
90
Viral load (log10 copies per ml)
% Efficacy
Control
COVA1-18
0
2
4
6
8
Days post-infection
0
2
4
6
8
0
2
4
6
8
0
2
4
6
8
0
2
4
6
8
a
MF10
gRNA
sgRNA
Efficacy
Days post-infection
b
c
0
-2
4
6
2
8
0
-2
4
6
2
8
Therapeutic
Inoculum: 106 PFU
Nasopharynx
Trachea
3
4
5
6
7
8
9
2
3
4
5
6
7
8
9
2
3
4
5
6
7
8
9
2
3
4
5
6
7
8
9
2
0
-2
4
6
2
8
0
-2
4
6
2
8
PrEP
Inoculum: 106 PFU
Nasopharynx
Trachea
Fig. 4 Modeling of viral dynamics and treatment efficacy. a Individual prediction of the nasopharyngeal genomic (g)RNA and subgenomic (sg)RNA in
control (top) and treated animals (bottom) with individual efficacy prediction indicated (green line). The dashed red line indicates the time of infection. gRNA (squares) and sgRNA (circles) data are indicated as plain (above LoQ) or open (below LoQ). b Model predictions of gRNA and sgRNA dynamics
with 4 doses of COVA1-18 given 24 h prior to challenge (arrow). c Simulation as in (b) with COVA1-18 given 24 h post-infection. Black dotted lines indicate
LoQ (limit of quantification), i.v. intravenous, PFU plaque forming units, PrEP Pre-exposure prophylaxis. MF1
MF2
MF3
MF4
MF5
gRNA
sgRNA
MF6
MF7
MF8
MF9
3
4
5
6
7
8
9
2
3
4
5
6
7
8
9
2
99.99
99.9
99
90
99.99
99.9
99
90
Viral load (log10 copies per ml)
% Efficacy
Control
COVA1-18
0
2
4
6
8
Days post-infection
0
2
4
6
8
0
2
4
6
8
0
2
4
6
8
0
2
4
6
8
a
MF10
gRNA
sgRNA
Efficacy % Efficacy gRNA
sgRNA
Viral load (log10 copies per ml)
Control
10 mg kg-1
5 mg kg-1
1 mg kg-1
0.1 mg kg-1
COVA1-18 dose
Injection i.v. Methods
I G F b All mice were housed in a temperature con-
trolled environment (68–72 degrees Fahreheit, 50–60% humidity) with twelve
hours of light per day at the Center for Comparative Medicine and Surgery
(CCMS) at Icahn School of Medicine at Mount Sinai (New York, NY, USA). All
experiments involving viral infections were carried out in a CDC/ USDA-approved
BSL-3 facility at CCMS and animals were transferred into the facility four days
prior to onset of experiments. Mice were housed in Allentown individually ven-
tilated cages with ad libitum access to food and water. The mouse experimental
study was approved by the Icahn School of Medicine at Mount Sinai Institutional
Animal Care and Use Committee (IACUC-2017-0170 and IACUC-2017-0330). Hamsters were housed in the ABSL-4 facility of the Galveston National
Laboratory. The animal protocol # 2004049 was approved by the Institutional
Animal Care and Use Committee (IACUC) of the University of Texas Medical
Branch at Galveston (UTMB). Ni-NTA-capture ELISA. SARS-CoV-2, SARS-CoV, MERS, HKU1, 229E and NL63
S His-tagged proteins were loaded at 2 µg ml−1 in TBS/2% skimmed milk (100 µl/
well) on 96-well Ni-NTA plates (Qiagen) for 2 h at room temperature (RT). Three-
fold serially diluted COVA NAb were then added onto the plates for 2 h at RT
followed by the addition goat anti-human IgG-HRP (Jackson Immunoresearch)
secondary Ab (1:3000) for 1 h at RT. The plates were developed for 3 min using
TMB solution and then stopped. Optical densities were measured at 450 nm on a
spectrophotometer and data graphed using GraphPad Prism software (v8.3.0). Hamsters were housed in the ABSL-4 facility of the Galveston National
Laboratory. The animal protocol # 2004049 was approved by the Institutional
Animal Care and Use Committee (IACUC) of the University of Texas Medical
Branch at Galveston (UTMB). Cynomolgus macaques (Macaca fascicularis) originating from Mauritian
AAALAC certified breeding centers were used in this study. All animals were
housed in IDMIT infrastructure facilities (CEA, Fontenay-aux-roses), under BSL-2
and BSL-3 containment when necessary (Animal facility authorization #D92-032-
02, Préfecture des Hauts de Seine, France) and in compliance with European
Directive 2010/63/EU, the French regulations and the Standards for Human Care
and Use of Laboratory Animals, of the Office for Laboratory Animal Welfare
(OLAW, assurance number #A5826-01, US). The protocols were approved by the
institutional ethical committee “Comité d’Ethique en Expérimentation Animale du
Commissariat à l’Energie Atomique et aux Energies Alternatives” (CEtEA #44)
under statement number A20-011. Methods
I G F b IgG, Fab, and soluble viral protein expression. COVA1-18 was isolated from a
participant in the “COVID-19 Specific Antibodies” (COSCA) study as described9. The COSCA study was conducted at the Amsterdam University Medical Centre,
location AMC, the Netherlands, and approved by the local ethical committee of the
AMC (NL 73281.018.20). COVA1-18 IgG was produced in HEK293F suspension
cells as previously described9. COVA1-18 His-tagged Fab was produced in Expi-
CHO cells as previously described50. Spike and RBD proteins were produced and
purified as previously described9. Briefly, cells were transfected at a density of
0.8–1.2 million cells per mL by addition of a mix of PEImax (1 μg μl−1) with
expression plasmids (312.5 μg l−1) in a 3:1 ratio in OptiMEM. Supernatants of
glycoproteins were harvested six days post transfection, centrifuged for 30 min at
4000 rpm and filtered). Constructs with a his-tag were purified by affinity pur-
ification using Ni-NTA agarose beads. Protein eluates were concentrated and buffer
exchanged to PBS using Vivaspin filters with a 100 kDa molecular weight cutoff
(GE Healthcare) for Spike proteins or 10 kDa for RBD. Protein concentrations were
determined by the Nanodrop method using the proteins peptidic molecular weight
and extinction coefficient as determined by the online ExPASy software
(ProtParam). Pseudovirus neutralization assay. Neutralization assays were performed using
SARS-CoV-2 S-pseudotyped HIV-1 virus and HEK293T hACE2 cells as described
previously13. In brief, pseudotyped virus was produced by co-transfecting expression
plasmids of SARS-CoV-2Δ19 S proteins (GenBank MT449663.1) with an HIV
backbone expressing NanoLuc luciferase (pHIV-1NL4-3 ΔEnv-NanoLuc) in
HEK293T cells (ATCC, CRL-11268). After 2 days, the cell culture supernatants
containing SARS-CoV-2 S-pseudotyped HIV-1 viruses were harvested and stored at
−80 °C. HEK293T hACE2 cells were seeded 20,000 cells/well in a flat-bottom 96-well
plates one day prior to the start of the neutralization assay. COVA1-18 IgG and His6-
tagged Fab as well as heat-inactivated serum samples were serially diluted in 3-fold
steps using cell culture medium and then mixed with pseudotyped virus in a 1:1 ratio
and incubated for 1 h at 37 °C. The mixtures were then added to the HEK293T
hACE2 cells in a 1:1 medium to mixture ratio. The final starting concentration for
IgGs was 20 µg ml−1 and 13.33 µg ml−1 for Fab. The cells were then incubated at
37 °C for 48 h followed by one PBS wash and lysis buffer addition. Methods
I G F b The luciferase
activity in the cell lysates was measured using the Nano-Glo Luciferase Assay System
(Promega) and GloMax Discover microplate reader. Relative luminescence units
(RLU) were normalized to those from positive control wells where cells were infected
with SARS-CoV-2 pseudovirus without IgG, Fab or serum. The inhibitory con-
centration (IC50) and neutralization titers (ID50) were determined as the IgG/Fab
concentration or serum dilution at which infectivity was inhibited by 50%. d
d
l
(
)
l
d
l Bio-layer interferometry. The affinity of COVA1-18 IgG and His-tagged Fab
versions were determined using Ni-NTA biosensors (ForteBio) onto which
20 µg ml−1 of SARS-CoV-2 RBD was in running buffer (PBS, 0.02% Tween-20,
0.1% BSA) was loaded for 300 s as previously described50. The association rate and
dissociation step were assessed over a 120 s step each. Serially diluted IgG (50, 100,
200, and 400 nM) and Fab (100, 200, 400, and 800 nM) were tested and an anti-
HIV-1 His-tagged Fab at 800 nM in running buffer was included as negative
control. KDs were determined using ForteBio Octet CFR software using a 1:2 fitting
model for IgGs and a 1:1 fitting model for Fabs. The apparent affinity of COVA1-
18 IgG to the SARS-CoV-2 S trimer was determined as described above except that
20 µg ml−1 SARS-CoV-2 S 2 P Fld His protein was loaded instead of RBD. The
COVA1-18 IgG avidity effect was further evaluated by titrating the loaded SARS-
CoV-2 RBD (5, 1, 0.2, and 0.04 μg ml−1). An additional loading step using His-
tagged HIV-1 gp41 was performed to minimize background binding of His-tagged
Fabs to the biosensor and both the COVA1-18 IgG and Fab concentrations were
set at 250 nM. All other steps were performed as described above. Data were
acquired with Octet Data Acquisition 10.0.03.12 and analyzed with Octet Analysis
HT 10.0.3.7 (ForteBio). Pseudotyped Vesicular Stomatitis Virus (VSVΔG) particles displaying SARS-
CoV-2Δ19 S and containing a luciferase reporter were used as previously
described14. Two-fold dilution series of COVA1-18 were prepared in complete
medium, pseudotyped virus added and the mixture incubated for 1 h at 37 °C. The
virus-antibody mixtures were then loaded onto plates seeded with Vero E6 cells
24 h prior this step. Following a 20 h incubation at 37 °C, the luciferase substrate
was added to lysed cells and RLU determined and analyzed as described above. Ethics and biosafety statement. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 ARTICLE In conclusion, our COVA1-18 in vitro data translated into a
powerful protective drug in three preclinical models to prevent
SARS-CoV-2 replication. Together with our prediction model,
these data showed that COVA1-18 could be used in patients at
low doses either to prevent infection or to reduce viral loads in a
therapeutic setting, with a potential greater impact in high-risk
patients. The high in vivo efficacy of COVA1-18 and its
demonstrated potency against the B.1.1.7. isolate also suggests
that it is a promising candidate for a NAb cocktail. Cynomolgus monkey IgG ELISA. Half area high binding 96-well plates were
coated overnight (4 °C) with goat anti-Human IgG λ and goat anti-Human IgG κ
(Southern Biotech), 1:2000 (each) in PBS, 50 µl/well. The plates were washed (1X
TBS – 0,05% Tween20) and blocked for 2 h at RT with 50 µl/well casein buffer. Serially diluted mucosal and serum samples were loaded onto the plates. Serially
diluted polyclonal cynomolgus IgG (Molecular Innovations) was used as standard. Following a 1 h incubation at RT, mouse anti-Monkey IgG Fc-BIOT (Southern
Biotech) was loaded onto the plate (1:50000). After 1 h at RT, poly-HRP40 was
added (1:10000) and the plates incubated for 1 h. Finally, the plates were washed 5
times, developed for 5 min, and analyzed as described above. Cynomolgus anti-S IgG and IgM ELISA were performed as described above
except that 2 µg ml−1 SARS-CoV-2 S Fld His-tagged protein were coated in the
sample wells instead of goat anti-Human IgG λ and goat anti-Human IgG κ. For
the IgM ELISA, the standard was obtained from Molecular Innovations and the
detection goat anti-Monkey IgM (μ-chain specific)-Biotin antibody from Sigma
Aldrich and used at 1:20000 dilution. Strep-HRP (R&D systems) at 1:500 was used
for IgG and poly-HRP40 for IgM. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 This finding
highlights the necessity of using NAbs cocktails targeting distinct
epitopes and we propose the use of the SARS-CoV-1 cross-
neutralizing antibody COVA1-16, which can effectively neutralize
B.1.351, in a 1:1 cocktail with COVA1-18. In addition, the half-
life of COVA1-18 can be extended by incorporating the LS or
YTE47 mutations, which can further reduce the protective dose
required and reduce the cost of treatment. An optimal cocktail should not only be based on intrinsic
efficacy against wild-type virus of each NAbs, but rather whether
synergy could be achieved in terms of binding domain and/or
spectrum of efficacy. Indeed, the increasing prevalence of mutant
strains has reduced the sensitivity to pre-existing NAbs, including
those given in combination30. Escape mutations can arise fol-
lowing single NAb treatment as recently demonstrated37,46 and
the one S mutation found in a unique sample from one animal
treated with COVA1-18 is not in the epitope of COVA-18. Importantly, we and others have determined that COVA1-18
retains high potency against the B.1.1.7 variant, which includes
the N501Y mutation20,21. However, COVA1-18 lost its potency
against B.1.351 which harbors the E484K mutation that is also q
While approved SARS-CoV-2 mAbs are given intravenously,
other therapeutic mAbs are given intramuscularly or by sub-
cutaneous injection48. SARS-CoV-2 mAbs could potentially also
be administered intranasally or delivered via gene therapy to the
airways49, to provide protection where it is most needed, i.e. the
respiratory tract. The biodistribution of COVA1-18 by different
routes of administration would also have to be investigated. In
addition, we note that COVA1-18 and numerous potent neu-
tralizing Abs isolated to date against SARS-CoV-2 have very low
levels of somatic hypermutation. Thus, these antibodies are very
close to the germline precursor and unlikely to trigger anti-
idiotypic response in patients. NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications 6 ARTICLE Lesions and scoring were assessed in each lung lobe
blindly and independently by two persons and the final results were established by
consensus. Overall CT scores include the lesion type (scored from 0 to 3) and lesion
volume (scored from 0 to 4) summed for each lobe as previously described25,26. Animals and study design. Seven week old female Balb/cJ mice (Jackson
Laboratories Bar Harbor, ME) were anesthetized before being administered with
2.5 × 108 PFU of human adenovirus type 5 encoding the human angiotensin
converting enzyme-2 receptor (Ad5-hACE2) 5-days prior to challenge with SARS-
CoV-2, as previously described29,30. Animals were transferred to the BSL-3 facility
where two groups of n = 5 mice per group received 10 mg kg−1 of COVA1-18
intraperitoneally 24 h prior to, or post-infection with 104 PFU SARS-CoV-2 in
50 μl PBS. A control group of n = 3 mice received 50 μl PBS. Mice were euthanized
3 d.p.i. and lungs harvested to quantify viral lung titers. Lungs were homogenized
in PBS using a Beadblaster Microtube homogenizer (Benchmark Scientific). SARS-
CoV-2 plaque assay was performed on 10-fold serial dilutions of lung homogenates
prepared in 0.2% bovine serum albumin (BSA) in PBS that were plated onto a Vero
E6 cells monolayer and incubated with shaking for 1 h. Inoculum was removed and
plates were overlaid with Minimal Essential Media (MEM) containing 2% FBS/
0.05% oxoid agar and incubated for 72 h at 37 °C. Plates were fixed with 4%
formaldehyde overnight, stained with a mAb cocktail composed of SARS-CoV-2
spike and SARS-CoV-2 nucleoprotein (Center for Therapeutic Antibody Dis-
covery; NP1C7C7) followed by anti-Mouse IgG-HRP (Abcam ab6823) and
developed using KPL TrueBlue peroxidase substrate (Seracare; 5510-0030). Viral sequencing. 30 RNA samples from nasopharyngeal and tracheal swabs as
well as BAL fluids at 3 d.p.i. were selected for sequencing along with the inoculum. cDNA and multiplex PCR reactions were prepared following the ARTIC SARS-
CoV-2 sequencing protocol v256. V3 primer scheme (https://github.com/artic-
network/primer-schemes/tree/master/nCoV-2019/V3) was used to perform the
multiplex PCR for SARS-CoV-2. All samples were run for 35 cycles in the two
multiplex PCRs. Pooled and cleaned PCR reactions were quantified using QubitTM
fluorometer (Invitrogen). The Ligation Sequencing kit (SQK-LSK109; Oxford
Nanopore Technologies) was used to prepare the library following the manu-
facturer’s protocol (“PCR tiling of COVID-19 virus”, release F; Oxford Nanopore
Technologies). ARTICLE Twenty-four samples were multiplexed using Native Barcoding
Expansion 1–12 and Native Barcoding Expansion 13–24 kits (EXP-NBD104 and
EXP-NBD114; Oxford Nanopore Technologies). Two libraries of 24 samples were
prepared independently and quantified by QubitTM fluorometer (Invitrogen). After the quality control, two R9.4 flowcells (FLO-MIN106; Oxford Nanopore
Technologies) were primed as described in the manufacturer’s protocol and loaded
with 45 and 32 ng of library. Sequencing was performed on a GridION (Oxford
Nanopore Technologies) for 72 h with high-accuracy Guppy basecalling (v3.2.10). After sequencing, demultiplexing was performed using Guppy v4.0.14 with the
option -require_barcodes_both_ends to ensure high quality demultiplexing. Reads
were then filtered by Nanoplot v1.28.1 based on length and quality to select high
quality reads. Then, reads were aligned on the SARS-CoV-2 reference genome
NC_045512.2 using minimap2 v2.17. Primary alignments were filtered based on
reads length alignment and reads identity. Reads were basecalled and demulti-
plexed with Guppy 4.0.14. The potential clonal and subclonal variants were
detected with a custom pipeline based on ARTIC network workflow. Longshot
v0.4.1 was used for variant detection. The potential subclonal variants were
manually curated by comparing the generated VCF files and visual inspection of
the alignments in IGV browser. p
g
p
Female golden Syrian hamsters, aged 6–7 weeks, were randomly assigned to two
groups of n = 5 and microchipped 24 h before SARS-CoV-2 challenge. On the day
of challenge, hamsters were anesthetized with ketamine/xylazine and challenged by
the intranasal route with 105 PFU of SARS-CoV-2 diluted in sterile PBS in the total
volume 100 µl. Body weight and body temperature were measured each day, starting
at day 0. Twenty four hours post-challenge, hamsters were treated with 10 mg kg−1
of COVA1-18 diluted in 0.5 ml of sterile PBS via the intraperitoneal route. The
control group of animals received an equal volume of sterile PBS via the
intraperitoneal route. All animals were euthanized 72 h post-infection with an
overdose of anesthetic (isoflurane or ketamine/xylazine) followed by bilateral
thoracotomy, and terminal blood and lungs were collected at necropsy. Right lungs
were frozen in 5 ml L-15 Leibowitz medium (Gibco) with 10% FBS. Tissue sections
were homogenized in bead beater tubes, weighed, and supernatants were titrated per
standard protocol. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 Respiratory Viruses (Institut Pasteur, Paris, France) as previously described51 and
produced by two passages on Vero E6 cells in DMEM (Dulbecco’s Modified Eagles
Medium) without FBS, supplemented with 1% P/S (penicillin at 10,000 U ml−1 and
streptomycin at 10,000 μg ml−1) and 1 μg ml−1 TPCK-trypsin at 37 °C in a Respiratory Viruses (Institut Pasteur, Paris, France) as previously described51 and
produced by two passages on Vero E6 cells in DMEM (Dulbecco’s Modified Eagles
Medium) without FBS, supplemented with 1% P/S (penicillin at 10,000 U ml−1 and
streptomycin at 10,000 μg ml−1) and 1 μg ml−1 TPCK-trypsin at 37 °C in a
humidified CO2 incubator and titrated on Vero E6 cells. Whole genome sequen-
cing was performed as described51 with no modifications observed compared with
the initial specimen and sequences were deposited after assembly on the GISAID
EpiCoV platform under accession number ID EPI_ISL_406596. Sequencing ana-
lysis revealed two clonal mutations, one in the S gene (22661 G > T: V367F, non-
synonymous) and one in the ORF3a gene (26144 G > T: G251V, non-synonymous),
which were already present in the challenge inoculum. epiglottis. All specimens were stored between 2 °C and 8 °C until analysis by RT-
qPCR with a plasmid standard concentration range containing an RdRp gene
fragment including the RdRp-IP4 RT-PCR target sequence. SARS-CoV-2 E gene
subgenomic mRNA (sgRNA) levels were assessed by RT-qPCR using primers and
probes previously described52,53 (Supplementary Table 4). The protocol describing
the procedure for the detection of SARS-CoV-2 is available on the WHO website54. p
y
μg
μg
yp
humidified CO2 incubator and titrated on Vero E6 cells. Whole genome sequen-
cing was performed as described51 with no modifications observed compared with
the initial specimen and sequences were deposited after assembly on the GISAID
EpiCoV platform under accession number ID EPI_ISL_406596. Sequencing ana-
lysis revealed two clonal mutations, one in the S gene (22661 G > T: V367F, non-
synonymous) and one in the ORF3a gene (26144 G > T: G251V, non-synonymous),
which were already present in the challenge inoculum. Chest CT and image analysis. Lung images were acquired using a computed
tomography (CT) system (Vereos-Ingenuity, Philips) as previously described25,26,
andanalysed using INTELLISPACE PORTAL 8 software (Philips Healthcare). All
images had the same window level of enuity, window width of 1,600. Lesions were
defined as ground glass opacity, crazy-paving pattern, consolidation or pleural thick-
ening as previously described35,55. ARTICLE All animals were then exposed to a total dose of 106 PFU of SARS-
CoV-2 (BetaCoV/France/IDF/0372/2020; passaged twice in VeroE6 cells) via the
combination of intranasal and intratracheal routes (day 0), using atropine
(0 04
k
1) f
di
i
d k
i
(
k
1)
i h
d
idi Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article. Data availability The viral sequencing data used in Supplementary Fig. 3 have been deposited in the SRA
repository under the accession code PRJNA758764 (PRJNA758764 - SRA - NCBI
(nih.gov)). All the other raw data generated in this study are provided in the Source Data
file. Source data are provided with this paper. Code availability The code used for the modeling part has been deposited in the Zenodo repository57
under the https://doi.org/10.5281/zenodo.5140032. Received: 15 February 2021; Accepted: 24 September 2021; Received: 15 February 2021; Accepted: 24 September 2021; One male and one female cynomolgus macaques aged 3–6 years received the
treatment as described above for the pharmacokinetic and pharmacodynamics
(PK/PD) study. Blood was sampled before and 2, 4, 6, and 24 h post-treatment. Saliva, nasopharyngeal and tracheal fluids were sampled before and 24 h post-
treatment. Twenty-four hours post-treatment, animals were euthanized and their
lungs, heart, kidney, liver, spleen, trachea, and brain were sampled, rinsed with PBS
and around 100 mg of tissue was homogenized in 500 µl of PBS with a Precellys
and stored at −80 °C. Methods
I G F b The study was authorized by the “Research,
Innovation and Education Ministry” under registration number APAFIS#24434-
2020030216532863. All information on the ethics committee is available at https://
cache.media.enseignementsup-recherche.gouv.fr/file/utilisation_des_animaux_
fins_scientifiques/22/1/comiteethiqueea17_juin2013_257221.pdf. Detection of human IgG in NHP fluid. Detection of COVA1-18 in NHP samples
determined by ELISA using a protocol adapted from others33. Briefly, half area
high binding 96-well plates (Greiner Bio-One) were coated overnight with goat
anti-Human IgG H+L (monkey pre-adsorbed) at 1 µg ml−1 in PBS. The plates
were then blocked in casein buffer (Thermo Scientific) for 2 h at RT. Serum and
mucosal samples were serially diluted and loaded onto the plates as well as serially
diluted COVA1-18 as the standard. Following a 1 h RT incubation, goat anti-
Human IgG (monkey adsorbed)-HRP secondary antibody (Southern Biotech) was
added for serum samples (1:4000). For mucosal samples, goat anti-Human IgG
(monkey adsorbed)-BIOT (Southern Biotech) was added at 1:10000 dilution. After
1 h RT incubation, serum sample plates were ready for development. For mucosal
samples, an additional 1 h incubation with poly-HRP40 (Fitzgerald) (1:10000) was
necessary. The plates were then developed for 5 min, and the optical densities
measured at 450 nm on a spectrophotometer. The raw data were exported and
analyzed using Microsoft Excel and GraphPad Prism (v8.3.0) software. The
COVA1-18 concentration in a specific sample was determined by interpolating OD
values from dilutions that fell into the linear range of the standard curve of the
matching ELISA plate. Viruses and cells. For the macaques studies, SARS-CoV-2 virus (hCoV-19/
France/ lDF0372/2020 strain) was isolated by the National Reference Center for 7 7 TURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications ARTICLE Briefly, of 10-fold dilutions of supernatants at 100 µl per well
were placed atop of Vero-E6 monolayers in 96-well plates, the plates were incubated
for 1 h, supernatants were replaced by methyl cellulose overlay, incubated for 3 days
at 5% CO2 and 37 °C. The plates were fixed with formalin, removed from BSL-4
according the approved protocol, and plaques counted to determine the viral titers. Statistical analysis. Statistical analysis of Syrian hamsters and hACE2 mice lung
viral titers as well as for NHP gRNA and sgRNA were carried out using Mann-
Whitney unpaired t-test in GraphPad Prism software (v8.3.0). g
pp
p
p q
Ten female cynomolgus macaques aged 3–6 years were randomly assigned
between the control and treated groups to evaluate the efficacy of COVA1-18
prophylaxis. The treated group (n = 5) received one bolus dose of COVA-18
human IgG1 monoclonal antibody (10 mg kg−1) by the intravenous route in the
saphenous vein one day prior to challenge, while control animals (n = 5) received
no treatment. All animals were then exposed to a total dose of 106 PFU of SARS-
CoV-2 (BetaCoV/France/IDF/0372/2020; passaged twice in VeroE6 cells) via the
combination of intranasal and intratracheal routes (day 0), using atropine
(0.04 mg kg−1) for pre-medication and ketamine (5 mg kg−1) with medetomidine
(0.05 mg kg−1) for anesthesia. Animals were observed daily and clinical exams were
performed at baseline, daily for one week, and then twice weekly, on anaesthetized
animals using ketamine (5 mg kg−1) and metedomidine (0.05 mg kg−1). Body
weight and rectal temperature were recorded and blood, as well as nasopharyngeal,
tracheal and rectal swabs, were collected. Broncho-alveolar lavages (BAL) were
performed using 50 ml sterile saline on 3 d.p.i. Chest CT was performed at 3 d.p.i. in anesthetized animals using tiletamine (4 mg kg−1) and zolazepam (4 mg kg−1). Blood cell counts, hemoglobin, and hematocrit, were determined from EDTA
blood using a DHX800 analyzer (Beckman Coulter). Ten female cynomolgus macaques aged 3–6 years were randomly assigned
between the control and treated groups to evaluate the efficacy of COVA1-18
prophylaxis. The treated group (n = 5) received one bolus dose of COVA-18
human IgG1 monoclonal antibody (10 mg kg−1) by the intravenous route in the
saphenous vein one day prior to challenge, while control animals (n = 5) received
no treatment. URE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications Acknowledgements p
26. Brouwer, P. J. M. et al. Two-component spike nanoparticle vaccine protects
macaques from SARS-CoV-2 infection. Cell 184, 1188–1200 e1119 (2021). g
We thank Benoit Delache, Sébastien Langlois, Joanna Demilly, Emma Burban, Quentin
Sconosciuti, Maxime Potier, Nina Dhooge, Pauline Le Calvez, Jean-Marie Robert, Tierry
Prot and Christina Dodan for their help with the macaque experiments; Laetitia Bos-
sevot, Marco Leonec, Laurine Moenne-Loccoz and Julie Morin for the RT-qPCR, and for
the preparation of reagents; Blanche Fert and Céline Mayet for her help with the CT
scans; Céline Aubenque, Karine Storck, Mylinda Barendji, Julien Dinh and Elodie Guyon
for the macaque sample processing; Sylvie Keyser for the transports organization; Nas-
tasia Dimant and Brice Targat for their help with the experimental studies in the context
of COVID-19-induced constraints; Frédéric Ducancel and Yann Gorin for their help
with the logistics and safety management; Isabelle Mangeot for her help with resources
management. We thank Sylvie Behillil and Vincent Enouf for contribution to viral stock
challenge production, Antoine Nougairede for sharing the plasmid used for the sgRNA
assays standardization and Paul Bieniasz for donating cells and reagents for pseudovirus
neutralization assays. We thank Matt Hyde and Julie Williams (Animal Resource Center,
University of Texas Medical Branch at Galveston) who performed technical procedures
with animals. We acknowledge support from CoVIC supported by the Bill and Melinda
Gates Foundation. We thank staff at the ISMMS CCMS vivarium for their assistance. We
also thank Randy Albrecht for support with the BSL-3 facility and procedures at the
ISMMS and Richard Cadagan for excellent technical assistance. The macaque image used
in Fig. 2 has been obtained from BioRender. This study was supported by the Nether-
lands Organization for Scientific Research (NWO) Vici grant (to R.W.S.), the Bill &
Melinda Gates Foundation through the Collaboration for AIDS Vaccine Discovery
(CAVD) grant INV-002022 (to R.W.S.), the Fondation Dormeur, Vaduz (to R.W.S. and
to M.J.v.G.) and Health Holland PPS-allowance LSHM20040 (to M.J.v.G.). M.J.v.G. is a
recipient of an AMC Fellowship, Amsterdam UMC and a COVID-19 grant of the
Amsterdam Institute of Infection and Immunity, the Netherlands. R.W.S and M.J.v.G. We thank Benoit Delache, Sébastien Langlois, Joanna Demilly, Emma Burban, Quentin
Sconosciuti, Maxime Potier, Nina Dhooge, Pauline Le Calvez, Jean-Marie Robert, Tierry
Prot and Christina Dodan for their help with the macaque experiments; Laetitia Bos-
sevot, Marco Leonec, Laurine Moenne-Loccoz and Julie Morin for the RT-qPCR, and for 27. Zarifian, A. ARTICLE Lancet Infect. Dis. 20, 697–706 (202 52. Corman, V. M. et al. Detection of 2019 novel coronavirus (2019-nCoV) by
real-time RT-PCR. Euro Surveill 25 (2020). y
p
g
y
19. Yurkovetskiy, L. et al. Structural and functional analysis of the D614G SARS-
CoV-2 spike protein variant. Cell 183, 739–751 e738 (2020). 53. Wolfel, R. et al. Virological assessment of hospitalized patients with COVID-
2019. Nature 581, 465–469 (2020). 20. Rees-Spear, C. et al. The effect of spike mutations on SARS-CoV-2
neutralization. Cell Rep. 34, 108890 (2021). 54. Pasteur I. Protocol: Real-time RT-PCR assays for the detection of SARS-CoV-
2 Institut Pasteur, Paris. (2020). p
21. Shen, X. et al. SARS-CoV-2 variant B.1.1.7 is susceptible to neutralizing antibodies
elicited by ancestral spike vaccines. Cell Host Microbe 29, 529–539 e523 (2021). 55. Pan, F. et al. Time course of lung changes at chest CT during recovery from
coronavirus disease 2019 (COVID-19). Radiology 295, 715–721 (2020). y
p
22. Hassan, A. O. et al. A SARS-CoV-2 infection model in mice demonstrates
protection by neutralizing antibodies. Cell 182, 744–753 e744 (2020). 56. Tyson, J. R. et al. Improvements to the ARTIC multiplex PCR method for
SARS-CoV-2 genome sequencing using nanopore. Preprint at https://
www.biorxiv.org/content/10.1101/2020.09.04.283077v1 (2020). 23. Rathnasinghe, R. et al. Comparison of transgenic and adenovirus hACE2
mouse models for SARS-CoV-2 infection. Emerg. Microbes Infect. 9,
2433–2445 (2020). 57. Guedj, J. Marc A. Code used to estimate viral kinetic parameters of NHPs
treated with COVA1-18. Zenodo https://doi.org/10.5281/zenodo.5140032
(2021). 24. Munoz-Fontela, C. et al. Animal models for COVID-19. Nature 586, 509–515
(2020). 25. Maisonnasse, P. et al. Hydroxychloroquine use against SARS-CoV-2 infection
in non-human primates. Nature 585, 584–587 (2020). ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 4. Ema. EMA issues advice on use of REGN-COV2 antibody combination
(casirivimab / imdevimab). (2021). 38. Czuppon, P. et al. Success of prophylactic antiviral therapy for SARS-CoV-2:
Predicted critical efficacies and impact of different drug-specific mechanisms
of action. PLoS Comput Biol. 17, e1008752 (2021). 5. Ema. EMA issues advice on use of antibody combination (bamlanivimab /
etesevimab). (2021). p
39. Néant, N. et al. Modeling SARS-CoV-2 viral kinetics and association with
mortality in hospitalized patients: results from the French Covid-19 cohort. Proc. Natl Acad. Sci. USA (2021). 6. Gsk. Vir Biotechnology and GSK announce VIR-7831 reduces hospitalisation
and risk of death in early treatment of adults with COVID-19. (2021). 6. Gsk. Vir Biotechnology and GSK announce VIR-7831 reduces hospitalisation
d i k
f d
th i
l t
t
t
f d lt
ith COVID 19 (2021) y
7. prnewswire. Lilly’s bamlanivimab and etesevimab together reduced
h
i li
i
d d
h i
Ph
i l f
l COVID
(
) 40. Fagre, A. C. et al. A potent SARS-CoV-2 neutralizing human monoclonal
antibody that reduces viral burden and disease severity in syrian hamsters. Front Immunol. 11, 614256 (2020). y
7. prnewswire. Lilly’s bamlanivimab and etesevimab together reduced
hospitalizations and death in Phase 3 trial for early COVID-19. (2021). 7. prnewswire. Lilly s bamlanivimab and etesevimab together reduced
hospitalizations and death in Phase 3 trial for early COVID-19. (2021). hospitalizations and death in Phase 3 trial for early COVID- 8. Roche. New phase III data shows investigational antibody cocktail casirivimab
and imdevimab reduced hospitalisation or death by 70% in non-hospitalised
patients with COVID-19.) (2021). (
)
41. Weinreich, D. M. et al. REGN-COV2, a neutralizing antibody cocktail, in
outpatients with Covid-19. N. Engl. J. Med. 384, 238–251 (2021). p
9. Brouwer, P. J. M. et al. Potent neutralizing antibodies from COVID-19
patients define multiple targets of vulnerability. Science 369, 643–650 (2020). p
g
42. Chen, P. et al. SARS-CoV-2 neutralizing antibody LY-CoV555 in outpatients
with Covid-19. N. Engl. J. Med. 384, 229–237 (2021). g
y
with Covid-19. N. Engl. J. Med. 384, 229–237 (2021). 10. Zost, S. J. et al. Rapid isolation and profiling of a diverse panel of human
monoclonal antibodies targeting the SARS-CoV-2 spike protein. Nat. Med 26,
1422–1427 (2020). 43. Group, A.-T. L.-C. S. et al. A neutralizing monoclonal antibody for
hospitalized patients with Covid-19. N. Engl. J. Med. References 1. COVID-19 Dashboard by the Center for Systems Science and Engineering
(CSSE) at Johns Hopkins University (JHU). (2021). 2. Fda. Coronavirus (COVID-19) Update: FDA Authorizes Monoclonal
Antibodies for Treatment of COVID-19. (2021). 3. Fda. Coronavirus (COVID-19) Update: FDA Authorizes Monoclonal
Antibody for Treatment of COVID-19. (2020). 1. COVID-19 Dashboard by the Center for Systems Science and Engineering
(CSSE) at Johns Hopkins University (JHU). (2021). y
2. Fda. Coronavirus (COVID-19) Update: FDA Authorizes Monoclonal
Antibodies for Treatment of COVID-19. (2021). Virus quantification in NHP samples. Upper respiratory (nasopharyngeal and
tracheal) and rectal specimens were collected with swabs (Viral Transport Medium,
CDC, DSR-052-01). Tracheal swabs were performed by insertion of the swab above
the tip of the epiglottis into the upper trachea at approximately 1.5 cm of the 3. Fda. Coronavirus (COVID-19) Update: FDA Authorizes Monoclonal
Antibody for Treatment of COVID-19. (2020). NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunicatio 8 ARTICLE 384, 905–914 (2021). 44. Wu, K. et al. mRNA-1273 vaccine induces neutralizing antibodies against
spike mutants from global SARS-CoV-2 variants. Preprint at http://
biorxiv.org/content/early/2021/01/25/2021.01.25.427948.abstract (2021). 11. Cao, Y. et al. Potent Neutralizing Antibodies against SARS-CoV-2 Identified
by High-Throughput Single-Cell Sequencing of Convalescent Patients’ B Cells. Cell 182, 73–84 e16 (2020). 45. Wibmer, C. K. et al. SARS-CoV-2 501Y.V2 escapes neutralization by South
African COVID-19 donor plasma. Nat. Med. 27, 622–625 (2021). 12. Hansen, J. et al. Studies in humanized mice and convalescent humans yield a
SARS-CoV-2 antibody cocktail. Science 369, 1010–1014 (2020). 46. Baum, A. et al. Antibody cocktail to SARS-CoV-2 spike protein prevents rapid
mutational escape seen with individual antibodies. Science 369, 1014–1018
(2020). y
13. Schmidt, F. et al. Measuring SARS-CoV-2 neutralizing antibody activity using
pseudotyped and chimeric viruses. J. Exp. Med. 217 (2020). 47. Saunders, K. O. Conceptual approaches to modulating antibody effector
functions and circulation half-life. Front Immunol. 10, 1296 (2019). 14. Almahboub, S. A., Algaissi, A., Alfaleh, M. A., ElAssouli, M. Z. & Hashem, A. M. Evaluation of neutralizing antibodies against highly pathogenic
coronaviruses: a detailed protocol for a rapid evaluation of neutralizing
antibodies using vesicular stomatitis virus pseudovirus-based assay. Front
Microbiol 11, 2020 (2020). 48. Wang, W., Wang, E. Q. & Balthasar, J. P. Monoclonal antibody
pharmacokinetics and pharmacodynamics. Clin. Pharm. Ther. 84, 548–558
(2008). 15. Korber, B. et al. Tracking changes in SARS-CoV-2 spike: evidence that D614G
increases infectivity of the COVID-19 virus. Cell 182, 812–827 e819 (2020). 49. Tse, L. V., Meganck, R. M., Graham, R. L. & Baric, R. S. The current and
future state of vaccines, antivirals and gene therapies against emerging
coronaviruses. Front Microbiol. 11, 658 (2020). y
16. Mansbach, R. A. et al. The SARS-CoV-2 Spike variant D614G favors an open
conformational state. Sci. Adv. 7, eabf3671–eabf3671 (2021). 50. Liu, H. et al. Cross-neutralization of a SARS-CoV-2 antibody to a functionally
conserved site is mediated by avidity. Immunity 53, 1272–1280 e1275 (2020). 17. Plante, J. A. et al. Spike mutation D614G alters SARS-CoV-2 fitness. Nature
592, 116–121 (2021). y
y
y
(
)
51. Lescure, F. X. et al. Clinical and virological data of the first cases of COVID-19
in Europe: a case series. Lancet Infect. Dis. 20, 697–706 (2020). 18. Volz, E. et al. Evaluating the effects of SARS-CoV-2 spike mutation D614G on
transmissibility and pathogenicity. Cell 184, 64–75 e11 (2021). in Europe: a case series. 1Université Paris-Saclay, Inserm, CEA, Center for Immunology of Viral, Auto-immune, Hematological and Bacterial diseases (IMVA-HB/IDMIT),
Fontenay-aux-Roses & Le Kremlin-Bicêtre, Paris, France. 2Departments of Medical Microbiology of the Amsterdam UMC, University of
Amsterdam, Amsterdam Institute for Infection and Immunity, Amsterdam, The Netherlands. 3Université de Paris, INSERM, IAME, Paris, France.
4Department of Pathology, University of Texas Medical Branch at Galveston, Galveston, TX, USA. 5Galveston National Laboratory, Galveston, TX,
USA. 6Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 7Life and Soft, Le Plessis-Robinson, France.
8Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 9Department of Integrative Structural
and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. 10Department of Microbiology, University of Texas Medical Branch
at Galveston, Galveston, TX, USA. 11Duke Human Vaccine Institute & Department of Surgery, Durham, NC, USA. 12Nexelis, Laval, Québec, Canada.
13Internal Medicine of the Amsterdam UMC, University of Amsterdam, Amsterdam Institute for Infection and Immunity, Amsterdam, The Netherlands.
14Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 15The Tisch Cancer Institute,
Icahn School of Medicine at Mount Sinai, New York, NY, USA. 16Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount
Sinai, New York, NY, USA. 17University of Maryland School of Medicine, Department of Microbiology and Immunology and Center for Vaccine
Development and Global Health (CVD), Baltimore, MD, USA. 18Molecular Genetics of RNA Viruses, Department of Virology, Institut Pasteur, CNRS UMR
3569, Université de Paris, Paris, France. 19National Reference Center for Respiratory Viruses, Institut Pasteur, Paris, France. 20Department of Microbiology
and Immunology, Weill Medical College of Cornell University, New York, NY, USA. 21These authors contributed equally: Pauline Maisonnasse,
Yoann Aldon. ✉email: r.w.sanders@amsterdamumc.nl; m.j.vangils@amsterdamumc.nl; roger.le-grand@cea.fr Author contributions Reprints and permission information is available at http://www.nature.com/reprints Additional information Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41467-021-26354-0. Correspondence and requests for materials should be addressed to Rogier W. Sanders,
Marit J.van Gils or Roger Le Grand. Peer review information Nature Communications thanks the anonymous reviewer(s) for
their contribution to the peer review of this work. Peer reviewer reports are available. Competing interests Amsterdam UMC filed a patent application on SARS-CoV-2 monoclonal antibody
COVA1-18. The García-Sastre Laboratory has received research support from Pfizer,
Senhwa Biosciences, 7Hills Pharma, Pharmamar, Blade Therapeutics, Avimex, Johnson
& Johnson, Dynavax, Kenall Manufacturing, ImmunityBio, Merck, and Nanocomposix. Adolfo García-Sastre has consulting agreements for the following companies involving
cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto,
Pagoda, Accurius, Farmak, Pfizer, and Esperovax. The remaining authors declare no
competing interests. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 are recipients of support from the University of Amsterdam Proof of Concept fund
(contract no 200421) as managed by Innovation Exchange Amsterdam (IXA). The
Infectious Disease Models and Innovative Therapies (IDMIT) research infrastructure is
supported by the “Programme Investissements d’Avenir”, managed by the ANR under
reference ANR-11-INBS-0008. The Fondation Bettencourt Schueller and the Region Ile-
de-France contributed to the implementation of IDMIT’s facilities and imaging tech-
nologies. The NHP study received financial support from REACTing, the Fondation pour
la Recherche Médicale (FRM, France; AM-CoV-Path) and the European Infrastructure
TRANSVAC2 (730964). The virus stock used in NHPs was obtained through the EVAg
platform (https://www.european-virus-archive.com/), funded by H2020 (653316). Work
performed at Duke University was supported by the CoVPN grant (NIH AI46705) (to
D.C.M.). The Ad5-hACE2 mouse work was supported in part by NIAID R21AI157606
(L.C.), and was partially supported by CRIP (Center for Research for Influenza Patho-
genesis), an NIAID supported Center of Excellence for Influenza Research and Sur-
veillance (CEIRS, contract # HHSN272201400008C) (A.G.S.), by supplements to NIAID
grant U19AI135972 and DoD grant W81XWH-20-1-0270, by the Defense Advanced
Research Projects Agency (HR0011-19-2-0020), and by the generous support of the JPB
Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384) and
anonymous donors to A.G.S. Part of this study was supported by the Bill and Melinda
Gates Foundation through grants OPP1170236 and INV-004923 (I.A.W.) and through
the Global Health Vaccine Accelerator Platforms (GH-VAP) and the Coronavirus
Immunotherapy Consortium (CoVIC) (Nexelis). The funders had no role in study
design, data collection, data analysis, data interpretation, or data reporting. R.L.G, and R.W.S. conceived, designed, supervised the project, acquired funding, pro-
vided resources, and wrote the manuscript. All authors contributed to the review and
editing of the final manuscript. R.L.G, and R.W.S. conceived, designed, supervised the project, acquired funding, pro-
vided resources, and wrote the manuscript. All authors contributed to the review and
editing of the final manuscript. Acknowledgements et al. Chest CT findings of coronavirus disease 2019 (COVID-19):
a comprehensive meta-analysis of 9907 confirmed patients. Clin. Imaging 70,
101–110 (2021). 28. Andreano, E. et al. SARS-CoV-2 escape from a highly neutralizing COVID-19
convalescent plasma. Proc. Natl Acad. Sci. USA. 118 (2021). 29. Goncalves, A. et al. SARS-CoV-2 viral dynamics in non-human primates. PLoS Comput Biol. 17, e1008785 (2021). p
30. Wang, P. et al. Antibody resistance of SARS-CoV-2 variants B.1.351 and
B.1.1.7. Nature 593, 130–135 (2021). 31. Starr, T. N. et al. Prospective mapping of viral mutations that escape
antibodies used to treat COVID-19. Science 371, 850–854 (2021). 32. Baum, A. et al. REGN-COV2 antibodies prevent and treat SARS-CoV-2
infection in rhesus macaques and hamsters. Science 370, 1110–1115 (2020). 33. Zost, S. J. et al. Potently neutralizing and protective human antibodies against
SARS-CoV-2. Nature 584, 443–449 (2020). 34. Wang, S. et al. Characterization of neutralizing antibody with prophylactic
and therapeutic efficacy against SARS-CoV-2 in rhesus monkeys. Nat. Commun. 11, 5752 (2020). 35. Shi, R. et al. A human neutralizing antibody targets the receptor-binding site
of SARS-CoV-2. Nature 584, 120–124 (2020). 36. Li, D. et al. In vitro and in vivo functions of SARS-CoV-2 infection-enhancing
and neutralizing antibodies. Cell 184, 4203–4219 e4232 (2021). 37. Jones, B. E. et al. The neutralizing antibody, LY-CoV555, protects against
SARS-CoV-2 infection in nonhuman primates. Sci. Transl. Med. 13,
eabf1906–eabf1906 (2021). 9 NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications © The Author(s) 2021 Reprints and permission information is available at http://www.nature.com/reprints P.M. and Y.A. conceived, designed, performed experiments, analyzed data, managed the
project, and wrote the manuscript (original draft). A.M. conceived and developed the
predictive model, and wrote the manuscript. R.M., N.D.B. performed, supervised, and
analyzed macaque experiments. N.A.K. designed, performed, and analyzed the hamster
experiment. M.S. designed, performed, and analyzed the mouse experiment. A.W.F. performed the mouse experiment. J.L.S. produced antibodies and performed ELISAs. A.G. contributed to the predictive model development. J.A.B., M.P., I.B., C.M., M.O.T.,
N.S.A., and L.G. performed neutralization assays. V.Ch., S.D., and A.I. performed
sequencing, analyzed, and interpreted the data. A.J.R. analyzed the hamster histology
data. S.J. and R.R. performed the mouse experiment. T.G.C., P.J.M.B., T.P.L.B., J.v.S.,
M.B., M.J.v.B., H.L., and M.Y. produced proteins. C.E.M. contributed to the hamster
experiment. V.Co. contributed to performing and supervising macaque studies. T.N. and
J.L. contributed to the macaque experiments and analysis. N.K. and F.R. contributed to
the macaque experiment. C.C. and R.H.T.F. provided resources and supervision for the
macaque studies contributed to the macaque experiments and analysis. D.C.M., I.A.W.,
G.J.d.B., and A.G.S. provided resources and funding. E.G. provided resources and
supervision for the sequencing. L.C. conceived, designed, and performed the mouse
study; acquired funding. A.B. designed and supervised the hamster study, provided
funding. S.v.d.W. provided the virus for the macaque study; J.G. conceived and developed
the predictive model, supervised, provided funding, and wrote the manuscript. M.J.v.G, Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/. © The Author(s) 2021 10 NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunicatio
|
https://openalex.org/W4224064109
|
https://www.mdpi.com/2076-393X/10/4/585/pdf?version=1649658973
|
English
| null |
Impact of Moderna mRNA-1273 Booster Vaccine on Fully Vaccinated High-Risk Chronic Dialysis Patients after Loss of Humoral Response
|
Vaccines
| 2,022
|
cc-by
| 7,714
|
Citation: Patyna, S.; Eckes, T.; Koch,
B.F.; Sudowe, S.; Oftring, A.; Kohmer,
N.; Rabenau, H.F.; Ciesek, S.;
Avaniadi, D.; Steiner, R.; et al. Impact
of Moderna mRNA-1273 Booster
Vaccine on Fully Vaccinated
High-Risk Chronic Dialysis Patients
after Loss of Humoral Response. Vaccines 2022, 10, 585. https://
doi.org/10.3390/vaccines10040585
Academic Editors: Lo Presti Elena
and Trizzino Antonino
Received: 17 February 2022
Accepted: 6 April 2022
Published: 11 April 2022
Publisher’s Note: MDPI stays neutral *
Correspondence: patyna@em.uni-frankfurt.de Citation: Patyna, S.; Eckes, T.; Koch,
B.F.; Sudowe, S.; Oftring, A.; Kohmer,
N.; Rabenau, H.F.; Ciesek, S.;
Avaniadi, D.; Steiner, R.; et al. Impact
of Moderna mRNA-1273 Booster
Vaccine on Fully Vaccinated
High-Risk Chronic Dialysis Patients
after Loss of Humoral Response. Vaccines 2022, 10, 585. https://
doi.org/10.3390/vaccines10040585 †
These authors contributed equally to this work. Abstract: The long-term effect of protection by two doses of SARS-CoV-2 vaccination in patients
receiving chronic intermittent hemodialysis (CIHD) is an urging question. We investigated the
humoral and cellular immune response of 42 CIHD patients who had received two doses of SARS-
CoV-2 vaccine, and again after a booster vaccine with mRNA-1273 six months later. We measured
antibody levels and SARS-CoV-2-specific surrogate neutralizing antibodies (SNA). Functional T
cell immune response to vaccination was assessed by quantifying interferon-γ (IFN-γ) and IL-2
secreting T cells specific for SARS-CoV-2 using an ELISpot assay. Our data reveal a moderate immune
response after the second dose of vaccination, with significantly decreasing SARS-CoV-2-specific
antibody levels and less than half of the study group showed neutralizing antibodies six months
afterwards. Booster vaccines increased the humoral response dramatically and led to a response rate
of 89.2% for antibody levels and a response rate of 94.6% for SNA. Measurement in a no response/low
response (NR/LR) subgroup of our cohort, which differed from the whole group in age and rate
of immunosuppressive drugs, indicated failure of a corresponding T cell response after the booster
vaccine. We strongly argue in favor of a regular testing of surrogate neutralizing antibodies and
consecutive booster vaccinations for CIHD patients to provide a stronger and persistent immunity. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Keywords: COVID-19; SARS-CoV-2; vaccination; hemodialysis; booster; mRNA-1273; seroconversion;
T cell response 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/). Impact of Moderna mRNA-1273 Booster Vaccine on Fully
Vaccinated High-Risk Chronic Dialysis Patients after Loss of
Humoral Response Sammy Patyna 1,2,*,†, Timon Eckes 2,†
, Benjamin F. Koch 1
, Stephan Sudowe 3, Anke Oftring 2, Niko Kohmer 4,
Holger F. Rabenau 4, Sandra Ciesek 4,5,6
, Despina Avaniadi 1, Rahel Steiner 1, Ingeborg A. Hauser 1,
Josef M. Pfeilschifter 2 and Christoph Betz 1 1
Division of Nephrology, Department of Internal Medicine III, University Hospital Frankfurt,
Goethe University Frankfurt, 60590 Frankfurt, Germany; benjamin.koch@kgu.de (B.F.K.);
despina.avaniadi@kgu.de (D.A.); rahel.steiner@kgu.de (R.S.); ingeborg.hauser@kgu.de (I.A.H.);
christoph.betz@kgu.de (C.B.) 2
Institute of General Pharmacology and Toxicology, University Hospital Frankfurt,
Goethe University Frankfurt, 60590 Frankfurt, Germany; eckes@em.uni-frankfurt.de (T.E.);
oftring@med.uni-frankfurt.de (A.O.); pfeilschifter@em.uni-frankfurt.de (J.M.P.) g
p
3
GANZIMMUN Diagnostics AG Mainz, 55128 Mainz, Germany; dr.sudowe@ganzimmun.de 4
Institute for Medical Virology, University Hospital Frankfurt, Goethe University Frankfurt,
60590 Frankfurt, Germany; niko.kohmer@kgu.de (N.K.); rabenau@em.uni-frankfurt.de (H.F.R.);
sandra.ciesek@kgu.de (S.C.) 5
German Centre for Infection Research, External Partner Site, 60323 Frankfurt, Germany 6
Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Branch Translational Medicine
and Pharmacology, 60596 Frankfurt, Germany 2.1. Study Design and Cohorts In a prospective, non-interventional study approach, 42 long-term dialysis patients
were observed from January 2021 to December 2021 in a tertiary care dialysis unit at
the Department of Nephrology and Dialysis at Frankfurt University Hospital (Frankfurt,
Germany). A healthy control group (n = 11; median (IQR) age 34 years (26–49), 27.3% male)
was included to compare cellular immune responses after immunization with two doses of
BNT162b2 (BioNTech/Pfizer) mRNA vaccine. Antibody levels and SNA were measured four weeks after the second dose (=t0, either
with BNT162b2 or ChAdOx1), six months after second dose (=t1) and 14 days after a third
dose (=t2, booster vaccine) of mRNA-1273 (Moderna) (Figure 1), exploring the time course
of SARS-CoV-2-specific antibody levels and the percentage of neutralizing antibodies after
vaccination against SARS-CoV-2 during the COVID-19 pandemic. The study was conducted in accordance with the principles of the Declaration of
Helsinki and approved by the local ethics committee (reference number: 20/658). All study
participants provided written informed consent before study entry. 1. Introduction The current coronavirus disease 2019 (COVID-19) pandemic confronts our society
with a global threat that particularly concerns patients with compromised immune systems,
either due to immunosuppressive therapy or diseases associated with impaired immune
response. Patients with end-stage renal disease (ESRD) undergoing chronic intermittent https://www.mdpi.com/journal/vaccines Vaccines 2022, 10, 585. https://doi.org/10.3390/vaccines10040585 Vaccines 2022, 10, 585 2 of 9 hemodialysis (CIHD) are affected by lower humoral and cellular immunity [1]. For this rea-
son, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and chronic
dialysis patients make a particular dangerous relationship. Patients undergoing CIHD
represent a vulnerable population which are at an increased risk of SARS-CoV-2 infection
and threatened by an almost four-fold higher mortality rate compared to the general pop-
ulation [2,3]. Thus, CIHD patients were prioritized for early SARS-CoV-2-vaccination in
several countries [4]. Sahin et al. showed that vaccination with BNT162b2 (Tozinameran,
BioNTech/Pfizer, Mainz, Germany/New York, NY, USA) induced both a strong antibody
and T cell response in healthy adults [5,6]. However, recent data reveal a significantly lower
SARS-CoV-2 spike protein antibody titer and reduced vaccination success rates of patients
receiving CIHD compared to the general population after vaccination with BNT162b2 [7]. An effective short-term seroconversion rate after SARS-CoV-2 vaccination in the CIHD
population was shown but recently published results indicate rapidly decreasing SARS-
CoV-2-specific antibody levels in the long term [8]. There is evidence for a beneficial effect
of a third dose of a COVID-19 mRNA vaccine on antibody titers in this specific high-risk
population [9] and, in addition to the humoral immune response, T cells may also con-
tribute to a protective immunity against SARS-CoV-2 and improve vaccine efficacy [10,11]. However, data regarding cellular immune response in this cohort are scarce. g
g
p
Here, we investigated the initial SARS-CoV-2-specific serological and functional T
cell immune response after two doses of SARS-CoV-2 vaccination (either BNT162b2 or
ChAdOx1 (AstraZeneca, Cambridge, UK)) and its longevity during a time course of up
to six months after the second dose in a cohort of high-risk CIHD patients in a tertiary
care dialysis unit. In addition, we analyzed antibody titers and surrogate neutralizing
antibodies (SNA) after a third dose, often referred to as booster vaccination, with Spikevax
(mRNA-1273, Moderna, Cambridge, MA, USA) and measured its effect on the SARS-CoV-
2-reactive T cell response in a subgroup of CIHD patients with no response/low response
(NR/LR) (n = 10) after their second vaccination. o positivity is set to .
AU/
. We e evated t e cut o
to 8.52
AU/
“threshold” range in between.
2.4. Assessment of Cytokine-Producing SARS-CoV-2-Reactive T Cell Responses 2.3. Assessment of SARS-CoV-2-Specific Surrogate Neutralizing Antibodies (SNA)
The binding of S-RBD to angiotensin-converting enzyme 2 allowing detection of
SARS-CoV-2-neutralizing antibodies was performed using the ELISA-based GenScript
SARS-CoV-2 Surrogate Virus Neutralization Test Kit (GenScript Biotech, Piscataway
Township, NJ, USA) [12]. Results are expressed as percentage inhibition (%-INH). The adaptive immune response was analyzed by quantifying interferon-γ (IFN-γ)
and IL-2 secreting T cells specific for SARS-CoV-2 using a multicolor fluorescence enzyme-
linked immunospot (ELISpot) assay (CoV-iSpot, AID GmbH, Strassberg, Germany) [13]. Highly specific sequences of immunodominant epitopes of SARS-CoV-2 structural proteins
(=SARS-CoV-2-peptide-mix) were used for stimulation of T cells. Results are expressed as
spot forming cells (SFC)/106 lymphocytes. 2.2. Assessment of SARS-CoV-2-Specific Antibodies
2.3. Assessment of SARS-CoV-2-Specific Surrogate Neutralizing Antibodies (SNA) 2.2. Assessment of SARS-CoV-2-Specific Antibodies
2.3. Assessment of SARS-CoV-2-Specific Surrogate Neutralizing Antibodies (SNA) Levels of antibodies targeted against SARS-CoV-2 spike receptor binding domain (S-
RBD) were measured with a chemiluminescent microparticle based immunoassay (SARS-
CoV-2 IgG II Quant, Abbott GmbH, Wiesbaden, Germany). Results are expressed as
standardized binding antibody units (BAU)/mL. According to the manufacturer, a cut-off
f
i i i
i
7 1 BAU/
L W
l
d h
ff
8 52 BAU/
L b
ddi
The binding of S-RBD to angiotensin-converting enzyme 2 allowing detection of SARS-
CoV-2-neutralizing antibodies was performed using the ELISA-based GenScript SARS-
CoV-2 Surrogate Virus Neutralization Test Kit (GenScript Biotech, Piscataway Township,
NJ, USA) [12]. Results are expressed as percentage inhibition (%-INH). 2.2. Assessment of SARS-CoV-2-Specific Antibodies 2.2. Assessment of SARS-CoV-2-Specific Antibodies Levels of antibodies targeted against SARS-CoV-2 spike receptor binding domain (S-
RBD) were measured with a chemiluminescent microparticle based immunoassay (SARS-
CoV-2 IgG II Quant, Abbott GmbH, Wiesbaden, Germany). Results are expressed as
standardized binding antibody units (BAU)/mL. According to the manufacturer, a cut-off
for positivity is set to 7.1 BAU/mL. We elevated the cut-off to 8.52 BAU/mL by adding a
“threshold” range in between. 3 of 9
3 of 9 Vaccines 2022, 10, 585
Vaccines 2022, 10, x FO Figure 1. Illustration of the study setup. Samples were collected at three different time points to
evaluate the dynamic of the patients’ immunization status by different immunoassays. Abbrevia-
tions: CIHD (chronic intermittent hemodialysis); S-RBD (spike receptor binding domain); ELISpot
(enzyme-linked immunospot). Figure 1. Illustration of the study setup. Samples were collected at three different time points to
evaluate the dynamic of the patients’ immunization status by different immunoassays. Abbreviations:
CIHD (chronic intermittent hemodialysis); S-RBD (spike receptor binding domain); ELISpot (enzyme-
linked immunospot). Figure 1. Illustration of the study setup. Samples were collected at three different time points to
evaluate the dynamic of the patients’ immunization status by different immunoassays. Abbrevia-
tions: CIHD (chronic intermittent hemodialysis); S-RBD (spike receptor binding domain); ELISpot
(enzyme-linked immunospot). Figure 1. Illustration of the study setup. Samples were collected at three different time points to
evaluate the dynamic of the patients’ immunization status by different immunoassays. Abbreviations:
CIHD (chronic intermittent hemodialysis); S-RBD (spike receptor binding domain); ELISpot (enzyme-
linked immunospot). 2.2. Assessment of SARS-CoV-2-Specific Antibodies
2.3. Assessment of SARS-CoV-2-Specific Surrogate Neutralizing Antibodies (SNA) 2.2. Assessment of SARS-CoV-2-Specific Antibodies
2.3. Assessment of SARS-CoV-2-Specific Surrogate Neutralizing Antibodies (SNA) 3.1. Basic Study Cohort Characteristics We prospectively enrolled 45 CIHD patients, of which 42 met inclusion criteria, with a
median (IQR) age of 62 (52–72.5) years. Three patients were excluded due to SARS-CoV-2
positivity (real-time reverse transcriptase PCR). The main clinical and demographic charac-
teristics are depicted in Table 1. Due to the incidence of comorbidities, this study cohort is
representing a highly vulnerable CIHD group. Five patients were not available for follow-
up because of death unrelated to COVID-19 (n = 2, sepsis and cardiac arrest) and change of
the dialysis center (n = 3). We observed a good vaccine tolerability, without serious adverse
events in this cohort. No patient developed critical side effects or was hospitalized. Table 1. Baseline patient characteristics. NR/LR (no response/low response) group with insufficient
humoral response after two doses of SARS-CoV-2 vaccine (t0). Abbreviations: m (male); GN (glomeru-
lonephritis); ADPKD (autosomal dominant polycystic kidney disease); Tx (transplant). Data expressed
as number (n) and percentage (%) or median (med) and interquartile range (IQR), respectively. Table 1. Baseline patient characteristics. NR/LR (no response/low response) group with insufficient
humoral response after two doses of SARS-CoV-2 vaccine (t0). Abbreviations: m (male); GN (glomeru-
lonephritis); ADPKD (autosomal dominant polycystic kidney disease); Tx (transplant). Data expressed
as number (n) and percentage (%) or median (med) and interquartile range (IQR), respectively. 2.4. Assessmen
2.5. Statistics f
y
g
p
The adaptive immune response was analyzed by quantifying interferon-γ (IFN-γ)
and IL-2 secreting T cells specific for SARS-CoV-2 using a multicolor fluorescence
enzyme-linked immunospot (ELISpot) assay (CoV-iSpot, AID GmbH, Strassberg,
Germany) [13]. Highly specific sequences of immunodominant epitopes of SARS-CoV-2
structural proteins (=SARS-CoV-2-peptide-mix) were used for stimulation of T cells. Results are expressed as spot forming cells (SFC)/106 lymphocytes. 2.5. Statistics
Continuous variables are shown as median and interquartile range (IQR), categorical
variables are reported as frequencies and percentages. Differences between patient
cohorts were determined using the Fisher’s exact test for categorical variables; for
quantitative variables, a t-test or a Mann–Whitney-U test was used for parametric and
nonparametric data, respectively. When comparing more than two groups, one-way
ANOVA f ll
d b B
f
i’
h
i
K
k l W lli
f ll
d b
Continuous variables are shown as median and interquartile range (IQR), categor-
ical variables are reported as frequencies and percentages. Differences between patient
cohorts were determined using the Fisher’s exact test for categorical variables; for quan-
titative variables, a t-test or a Mann–Whitney-U test was used for parametric and non-
parametric data, respectively. When comparing more than two groups, one-way ANOVA
followed by Bonferroni’s post hoc correction or Kruskal–Wallis test followed by Dunn’s
post hoc test was performed for parametric and nonparametric data, respectively. For
the comparison of related samples comprising more than two groups, a Friedman test
was performed. Spearman’s rank correlation analysis was used to determine bivariate
relationships by calculating Spearman’s Rho (correlation coefficient). Normality was as-
sessed by a Kolmogorov–Smirnov test. All p-values reported are two-sided, the level of
significance was set p < 0.05. Statistical analyses were performed using GraphPad Prism
version 9.3.1 (GraphPad Software, San Diego, CA, USA) and BiAS (v11.01; epsilon-Verlag,
Nordhasted, Germany). Vaccines 2022, 10, 585 4 of 9 3. Results 3.1. Basic Study Cohort Characteristics 3.1. Basic Study Cohort Characteristics 3.1. Basic Study Cohort Characteristics All Patients
NR/LR Group
p-Value
n = 42
n = 10
Age (years)
med (IQR)
62
(52–72.5)
74.5
(67.3–79.3)
0.0041
Gender (male)
n (%)
29
(69)
6
(60)
0.7109
Vintage on dialysis (years)
med (IQR)
2.4
(1–7.2)
6.2
(0.9–12)
0.3893
Days after 2nd vaccination (days)
med (IQR)
34.5
(30.5–47.8)
34
(29–47)
0.8189
Ethnicity
Caucasian
n (%)
31
(73.8)
8
(80)
1.0000
Black
n (%)
6
(14.3)
2
(20)
0.6415
Other
n (%)
5
(11.9)
/
Reason for HD
Diabetic nephropathy
n (%)
9
(21.4)
3
(30)
0.6792
Hypertensive nephropathy
n (%)
4
(9.5)
1
(10)
1.0000
ADPKD
n (%)
6
(14.3)
2
(20)
0.6415
GN
n (%)
3
(7.1)
1
(10)
1.0000
Amyloidosis
n (%)
5
(11.9)
/
Chronic interstitial nephritis
n (%)
6
(14.3)
/
Nephrosclerosis
n (%)
6
(14.3)
/
Vasculitis
n (%)
1
(2.4)
/
Others/unknown
n (%)
2
(4.8)
3
(30)
0.0432
Comorbidities
Diabetes mellitus
n (%)
13
(31.0)
6
(60)
0.1424
Lung disease
n (%)
5
(11.9)
2
(20)
0.6079
Smoker
n (%)
14
(33.3)
3
(30)
1.0000
Cancer
n (%)
5
(11.9)
/
Severe cardiovascular disease
n (%)
23
(54.8)
6
(60)
1.0000
Hypertension
n (%)
38
(90.5)
9
(90)
1.0000
Kidney or liver-Tx
n (%)
6
(14.3)
4
(40)
0.0847
Immunosupression
n (%)
9
(21.4)
6
(60)
0.0243
Data: (%) percentage; (med) median; (IQR) interquartile range; m (male); GN (glomerulonephritis), ADPKD
(autosomal dominant polycystic kidney disease). Data: (%) percentage; (med) median; (IQR) interquartile range; m (male); GN (glomerulonephritis), ADPKD
(autosomal dominant polycystic kidney disease). 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients Four weeks after the second dose of vaccination (=t0), 37 patients (88.1%) developed
antibodies against the SARS-CoV-2 spike protein with a median (IQR) of 128 BAU/mL
(43–1027.5), and 32 patients (76.2%) showed functional neutralizing capacity of SNA
with a median (IQR) of 69%-INH (15–89), above the predefined threshold for positivity
(>8.52 BAU/mL and >30%-INH, respectively; Figure 2). Vaccines 2022, 10, 585 5 of 9 Figure 2. Antibody response in plasma samples of patients taken at three different time points (t0 =
four weeks after second dose, t1 = six months after second dose and t2 = 14 days after third dose
(=booster vaccine) of mRNA-1273 (Moderna)). Abbreviations: S-RBD-ab (spike receptor binding
domain-antibodies); SNA (surrogate neutralizing antibodies). Data are expressed as median (IQR)
* p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001 (Friedman test). Figure 2. Antibody response in plasma samples of patients taken at three different time points
(t0 = four weeks after second dose, t1 = six months after second dose and t2 = 14 days after third dose
(=booster vaccine) of mRNA-1273 (Moderna)). Abbreviations: S-RBD-ab (spike receptor binding
domain-antibodies); SNA (surrogate neutralizing antibodies). Data are expressed as median (IQR). * p ≤0.05, *** p ≤0.001, **** p ≤0.0001 (Friedman test). Figure 2. Antibody response in plasma samples of patients taken at three different time points (t0 =
four weeks after second dose, t1 = six months after second dose and t2 = 14 days after third dos
(=booster vaccine) of mRNA-1273 (Moderna)). Abbreviations: S-RBD-ab (spike receptor binding
domain-antibodies); SNA (surrogate neutralizing antibodies). Data are expressed as median (IQR)
* p ≤0.05, *** p ≤0.001, **** p ≤0.0001 (Friedman test). Figure 2. Antibody response in plasma samples of patients taken at three different time points
(t0 = four weeks after second dose, t1 = six months after second dose and t2 = 14 days after third dose
(=booster vaccine) of mRNA-1273 (Moderna)). Abbreviations: S-RBD-ab (spike receptor binding
domain-antibodies); SNA (surrogate neutralizing antibodies). Data are expressed as median (IQR). * p ≤0.05, *** p ≤0.001, **** p ≤0.0001 (Friedman test). During the six-month period, the response rate of positive serology did not change
(87.8%) but antibody levels decreased significantly compared to t0 (median (IQR) of 32.3
BAU/mL (9.9–224.1), p < 0.014). 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients Less than half of this study group showed neutralizing
antibodies (48.8%) with a median (IQR) of 0%-INH (0–87.8) six months after the second
dose of the vaccine. Measuring antibody levels and SNA after the booster vaccine (=t2), we
observed a significant elevation compared to timepoint t0 (p < 0.001) and t1 (p < 0.001) with
a response rate of 89.2% for antibody levels (median (IQR): 4560 BAU/mL (646.7–7272.5)
and a response rate of 94.6% for SNA (median (IQR) of 97%-INH (0–97)). This correspond
to a 36 fold i
ea e i
edia
a tibody tite
afte the thi d do e o
a ed to t
During the six-month period, the response rate of positive serology did not change
(87.8%) but antibody levels decreased significantly compared to t0 (median (IQR) of
32.3 BAU/mL (9.9–224.1), p < 0.014). Less than half of this study group showed neutralizing
antibodies (48.8%) with a median (IQR) of 0%-INH (0–87.8) six months after the second
dose of the vaccine. Measuring antibody levels and SNA after the booster vaccine (=t2), we
observed a significant elevation compared to timepoint t0 (p < 0.001) and t1 (p < 0.001) with
a response rate of 89.2% for antibody levels (median (IQR): 4560 BAU/mL (646.7–7272.5))
and a response rate of 94.6% for SNA (median (IQR) of 97%-INH (0–97)). This corresponds
to a 36-fold increase in median antibody titers after the third dose compared to t0. to a 36-fold increase in median antibody titers after the third dose compared to t0. To get a more detailed insight into the distribution of the humoral immune response
we predefined different threshold values depending on the level of antibodies o
neutralization capacity. Applying these antibodies and SNA cut-off levels (<100 BAU/mL
or <50%-INH, no response/low response; 100–500 BAU/mL or 50–70%-IHN, moderat
response; >500 BAU/mL or >70%-IHN, strong response) the overall response rates at t0, t
and t2 are described in Figure 3, demonstrating a significant reduction in the humora
immunity six months after the second dose of the vaccine (=t1), followed by a distinc
y
p
To get a more detailed insight into the distribution of the humoral immune response we
predefined different threshold values depending on the level of antibodies or neutralization
capacity. 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤0.05, ** p ≤0.01, *** p ≤0.001 compared to t0; ### p ≤0.001 compared to t1. neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from
our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are
significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p <
0.001 and t2 rho: 0.58; p < 0.001). Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤0.05, ** p ≤0.01, *** p ≤0.001 compared to t0; ### p ≤0.001 compared to t1. neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from
our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are
significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p <
0.001 and t2 rho: 0.58; p < 0.001). The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important
than only the magnitude of antibody levels. For this reason, we analyzed the fraction of
neutralizing capacity for different antibody levels, as illustrated in Figure 4. 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important
than only the magnitude of antibody levels. For this reason, we analyzed the fraction of
neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from
our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are
significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p <
0.001 and t2 rho: 0.58; p < 0.001). Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤0.05, ** p ≤0.01, *** p ≤0.001 compared to t0; ### p ≤0.001 compared to t1. Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important
than only the magnitude of antibody levels. For this reason, we analyzed the fraction of
neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from
our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are
significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p <
0.001 and t2 rho: 0.58; p < 0.001). p
y
de of an vels. F d the Figure 3. 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important
than only the magnitude of antibody levels. For this reason, we analyzed the fraction of
neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from
our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are
significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p <
0.001 and t2 rho: 0.58; p < 0.001). Figure 4. Fraction of neutralizing capacity for different antibody levels. The inner circle is showing
levels of antibody response (BAU/mL) with the corresponding percentage of neutralizing antibodies
(%-INH) in the outer circle for t0, t1 and t2, respectively. Data are expressed as percentages. Applying
the following antibody and SNA cut-off levels: negative (<7.1 BAU/mL or <30%-INH), border-
line (>7.1–8.5 BAU/mL or >30–35%-IHN), low (>8.5–100 BAU/mL or >35–50%-INH), moderate
(>100–500 BAU/mL or >50–70%-INH), strong (>500 BAU/mL or >70%-INH). Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤0.05, ** p ≤0.01, *** p ≤0.001 compared to t0; ### p ≤0.001 compared to t1. Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients Applying these antibodies and SNA cut-off levels (<100 BAU/mL or <50%-
INH, no response/low response; 100–500 BAU/mL or 50–70%-IHN, moderate response;
>500 BAU/mL or >70%-IHN, strong response) the overall response rates at t0, t1 and t2
are described in Figure 3, demonstrating a significant reduction in the humoral immunity
six months after the second dose of the vaccine (=t1), followed by a distinct increase two
weeks after the booster vaccine (=t2) with a significant difference, respectively. increase two weeks after the booster vaccine (=t2) with a significant difference
respectively. g
p
y
The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important
than only the magnitude of antibody levels. For this reason, we analyzed the fraction of
neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from our
study group reveal that levels of SARS-CoV-2-spike-specific antibodies are significantly
correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p < 0.001 and
t2 rho: 0.58; p < 0.001). Vaccines 2022, 10, 585
Vaccines 2022, 10, x FOR 6 of 9
6 of 9 Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important
than only the magnitude of antibody levels. For this reason, we analyzed the fraction of
neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from
our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are
significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p <
0.001 and t2 rho: 0.58; p < 0.001). Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate
(light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH)
humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤0.05, ** p ≤0.01, *** p ≤0.001 compared to t0; ### p ≤0.001 compared to t1. Figure 3. 3.3. T Cell Response after Vaccination
3.3. T Cell Response after Vaccination
F
k
ft
th
d d Four weeks after the second dose, SARS-CoV-2-specific T cell reactivity of CIHD
patients showed significantly lower levels of IFN-γ release compared to the healthy control
group (HC, n = 11) (median (IQR) 7.5 SFC/106 cells (2.5–42.5) vs. 72.5 SFC/106 cells
(21.25–101.25) (p < 0.001)), but no difference in IL-2 release between both groups (median
(IQR) 40 SFC/106 cells (8.75–111.25) vs. 47.5 SFC/106 cells (28.75–68.75) (p < 0.001)); Figure 5. A positive IFN-γ release was only observed for nine patients (21.4%). A positive release of
IL-2 after the booster vaccine occurred in 17 patients (40.5%). Four weeks after the second dose, SARS-CoV-2-specific T cell reactivity of CIHD
patients showed significantly lower levels of IFN-γ release compared to the healthy
control group (HC, n = 11) (median (IQR) 7.5 SFC/106 cells (2.5–42.5) vs. 72.5 SFC/106 cells
(21.25–101.25) (p < 0.001)), but no difference in IL-2 release between both groups (median
(IQR) 40 SFC/106 cells (8.75–111.25) vs. 47.5 SFC/106 cells (28.75–68.75) (p < 0.001)); Figure
5. A positive IFN-γ release was only observed for nine patients (21.4%). A positive release
of IL-2 after the booster vaccine occurred in 17 patients (40.5%). Figure 5. Cytokine profile of T cell response specific to SARS-CoV-2 at different time points and in
comparison to a healthy control group. Abbreviations: ELISpot (enzyme-linked immunospot); ctrl
(control); IFN-γ (Interferon-γ); IL-2 (Interleukin-2). Data are expressed as median (IQR). Intragroup
paired analysis: * p ≤ 0.05, ** p ≤ 0.01 as indicated (Kruskal–Wallis test). Figure 5. Cytokine profile of T cell response specific to SARS-CoV-2 at different time points and in
comparison to a healthy control group. Abbreviations: ELISpot (enzyme-linked immunospot); ctrl
(control); IFN-γ (Interferon-γ); IL-2 (Interleukin-2). Data are expressed as median (IQR). Intragroup
paired analysis: * p ≤0.05, ** p ≤0.01 as indicated (Kruskal–Wallis test). Figure 5. Cytokine profile of T cell response specific to SARS-CoV-2 at different time points and in
comparison to a healthy control group. Abbreviations: ELISpot (enzyme-linked immunospot); ctrl
(control); IFN-γ (Interferon-γ); IL-2 (Interleukin-2). Data are expressed as median (IQR). Intragroup
paired analysis: * p ≤ 0.05, ** p ≤ 0.01 as indicated (Kruskal–Wallis test). Figure 5. Cytokine profile of T cell response specific to SARS-CoV-2 at different time points and in
comparison to a healthy control group. Abbreviations: ELISpot (enzyme-linked immunospot); ctrl
(control); IFN-γ (Interferon-γ); IL-2 (Interleukin-2). Data are expressed as median (IQR). Intragroup
paired analysis: * p ≤0.05, ** p ≤0.01 as indicated (Kruskal–Wallis test). 3.3. T Cell Response after Vaccination
3.3. T Cell Response after Vaccination
F
k
ft
th
d d We measured the T cell response again after the booster vaccination (t2) in a no
response/low response NR/LR subgroup (n = 10) of t0. One patient showed a positive IFN-
γ release after the second dose, but none of the NR/LR group showed a positive release of
IL-2. Although data of NR/LR patients present a robust serological response to the booster
vaccination (median (IQR): 990 BAU/mL (235.3–5033.3); 94%-INH (64.3–97)), only two
patients showed a positive release of IFN-γ and only one patient for IL-2. Compared to
the remaining study group (median (IQR): 5261 BAU/mL (970.8–10,238)), we measured
significant lower antibody levels in the NR/LR subgroup (p < 0.049). In summary, only
one patient in the NR/LP subgroup developed a T cell response after booster vaccination
(IFN-γ: 132.5 SFC/106 lymphocytes; IL-2: 165 SFC/106 lymphocytes). We measured the T cell response again after the booster vaccination (t2) in a no
response/low response NR/LR subgroup (n = 10) of t0. One patient showed a positive
IFN-γ release after the second dose, but none of the NR/LR group showed a positive release
of IL-2. Although data of NR/LR patients present a robust serological response to the
booster vaccination (median (IQR): 990 BAU/mL (235.3–5033.3); 94%-INH (64.3–97)), only
two patients showed a positive release of IFN-γ and only one patient for IL-2. Compared
to the remaining study group (median (IQR): 5261 BAU/mL (970.8–10,238)), we measured
significant lower antibody levels in the NR/LR subgroup (p < 0.049). In summary, only
one patient in the NR/LP subgroup developed a T cell response after booster vaccination
(IFN-γ: 132.5 SFC/106 lymphocytes; IL-2: 165 SFC/106 lymphocytes). 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients Data from
our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are
significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p <
0.001 and t2 rho: 0.58; p < 0.001). The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important
than only the magnitude of antibody levels. For this reason, we analyzed the fraction of
neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from
our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are
significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p <
0.001 and t2 rho: 0.58; p < 0.001). Figure 4. Fraction of neutralizing capacity for different antibody levels. The inner circle is showing
levels of antibody response (BAU/mL) with the corresponding percentage of neutralizing antibodies
(%-INH) in the outer circle for t0, t1 and t2, respectively. Data are expressed as percentages. Applying
the following antibody and SNA cut-off levels: negative (<7.1 BAU/mL or <30%-INH), border-
line (>7.1–8.5 BAU/mL or >30–35%-IHN), low (>8.5–100 BAU/mL or >35–50%-INH), moderate
(>100–500 BAU/mL or >50–70%-INH), strong (>500 BAU/mL or >70%-INH). izing capacity of SARS-CoV
agnitude of antibody levels th ntibodies is even more imp
son we analyzed the frac ntibodies is even more imp
son we analyzed the frac ntibodies is even more imp
son we analyzed the frac Figure 4. Fraction of neutralizing capacity for different antibody levels. The inner circle is showing
levels of antibody response (BAU/mL) with the corresponding percentage of neutralizing antibodies
(%-INH) in the outer circle for t0, t1 and t2, respectively. Data are expressed as percentages. Applying
the following antibody and SNA cut-off levels: negative (<7.1 BAU/mL or <30%-INH), border-
line (>7.1–8.5 BAU/mL or >30–35%-IHN), low (>8.5–100 BAU/mL or >35–50%-INH), moderate
(>100–500 BAU/mL or >50–70%-INH), strong (>500 BAU/mL or >70%-INH). Figure 4. Fraction of neutralizing capacity for different antibody levels. The inner circle is showing
levels of antibody response (BAU/mL) with the corresponding percentage of neutralizing antibodies
(%-INH) in the outer circle for t0, t1 and t2, respectively. Data are expressed as percentages. Applying
the following antibody and SNA cut-off levels: negative (<7.1 BAU/mL or <30%-INH), border-
line (>7.1–8.5 BAU/mL or >30–35%-IHN), low (>8.5–100 BAU/mL or >35–50%-INH), moderate
(>100–500 BAU/mL or >50–70%-INH), strong (>500 BAU/mL or >70%-INH). Vaccines 2022, 10, 585 7 of 9
derline
00–500 3.3. T Cell Response after Vaccination
3.3. 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients T Cell Response after Vaccination
F
k
ft
th
d d 4. Discussion
4. Discussion Patients undergoing CIHD represent a vulnerable population with an increased risk
of SARS-CoV-2 infection, higher disease severity and COVID-19-related mortality risk
Patients undergoing CIHD represent a vulnerable population with an increased risk
of SARS-CoV-2 infection, higher disease severity and COVID-19-related mortality risk [14]. For this group, there is still lacking data for long-term humoral and cellular immune
responses, especially relating to the presence of neutralizing antibodies. In this study,
we analyzed the magnitude and kinetics of antibody and T cell responses to the initial
immunization (t0), the status quo after 6 months (t1) and the state following a booster
with Moderna’s mRNA-1273 (t2). In our cohort of hemodialysis patients, 88.1% developed
antibodies against the SARS-CoV-2 spike protein 4 weeks after the primary vaccine series,
and 76.2% showed functional neutralizing capacity of SNA (surrogate neutralizing antibod- Vaccines 2022, 10, 585 8 of 9 ies). It was reported that neutralizing antibody titers decreased over time in patients after
SARS-CoV-2 infection [15]. However, after 6 months we can clearly see a significant decline,
not only in antibody levels, but even more decisively, a loss in neutralizing capacity. Impor-
tantly, more than half of our patients (51.2%) had no levels of SARS-CoV-2-neutralizing
antibodies at that time. Comparable results have been reported by Ducloux et al. and
Kohmer et al. for Pfizer-BioNTech’s BNT162b2 [9,16]. In line with our data, both studies
confirm that levels of SARS-CoV-2-specific antibodies were significantly increased by ad-
ministering a third dose of a COVID-19 mRNA vaccine. However, cell-mediated immunity
was not analyzed in these studies. y
We conclude that a decline in neutralizing antibody levels could be prevented by a
timed booster intervention for our cohort. However, measurement in a NR/LR subgroup
of our cohort also indicates failure of a corresponding T cell response after the booster
vaccine. These findings are in line with data of Espi et al. that describe a missing increase
in T cell response after third dose vaccination in dialysis patients [17]. Notably, the NR/LR
subgroup showed a higher rate of immunosuppressive medication (21.4% vs. 60%, p = 0.024)
and a higher age (median (IQR): 62 (52–72.5) vs. 74.5 (67.3–79.3)) compared to the whole
study group, which could be a confounder and an explanation for the diminished T cell
response after booster vaccination. 4. Discussion
4. Discussion To analyze the influence of these possible confounders,
a higher number of participants in the NR/LR subgroup would be required, which poses a
limitation of this study. y
Our data underline the importance of SNA when screening for the response of pa-
tient´s adaptive immunity after SARS-CoV-2 vaccination, especially in this highly vulner-
able group of CIHD patients. Hence, CIHD patients are not only particularly threatened
by the COVID-19 pandemic because of a more severe course of disease and a higher risk
of infection caused by higher frequency of medical consultations but, most notably, by a
lower response after the second dose of vaccination, combined with a relevant loss of both
serological and specific T cell immunity. T cell mediated immunity seems to play an important role in SARS-CoV-2 infection. However, to what extent is not clear. The presented results are highlighting the importance
of an early booster vaccination of CIHD patients. Further studies are required to generate convincing data for CIHD patients on long-
term T cell response after SARS-CoV-2 vaccination. This study only provides data on T cell
response after booster vaccination in a rather small subgroup of initial NR/LR patients. In addition, more investigations regarding protection against newly arising variants (e.g.,
Omicron) are needed. References 1. Losappio, V.; Franzin, R.; Infante, B.; Godeas, G.; Gesualdo, L.; Fersini, A.; Castellano, G.; Stallone, G. Molecular Mechanisms of
Premature Aging in Hemodialysis: The Complex Interplay Between Innate and Adaptive Immune Dysfunction. Int. J. Mol. Sci. 2020, 21, 3422. [CrossRef] [PubMed] 2. Taji, L.; Thomas, D.; Oliver, M.J.; Ip, J.; Tang, Y.; Yeung, A.; Cooper, R.; House, A.A.; McFarlane, P.; Blake, P.G. COVID-19 in
patients undergoing long-term dialysis in Ontario. Can. Med Assoc. J. 2021, 193, E278–E284. [CrossRef] [PubMed] 3. Hilbrands, L.B.; Duivenvoorden, R.; Vart, P.; Franssen, C.F.; Hemmelder, M.H.; Jager, K.J.; Kieneker, L.M.; Noordzij, M.; Pena, M.J.;
Gansevoort, R.T.; et al. COVID-19-related mortality in kidney transplant and dialysis patients: Results of the ERACODA
collaboration. Nephrol. Dial. Transplant. 2020, 35, 1973–1983. [CrossRef] [PubMed] p
p
4. Weinhandl, E.D.; Wetmore, J.B.; Peng, Y.; Liu, J.; Gilbertson, D.T.; Johansen, K.L. Initial Effects of COVID-19 on Patients with
ESKD. J. Am. Soc. Nephrol. 2021, 32, 1444–1453. [CrossRef] [PubMed]
Ö p
5. Sahin, U.; Muik, A.; Derhovanessian, E.; Vogler, I.; Kranz, L.M.; Vormehr, M.; Baum, A.; Pascal, K.; Quandt, J.; Türeci, Ö.; et al. COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses. Nature 2020, 586, 594–599. [CrossRef] [PubMed]
6. Sahin, U.; Muik, A.; Vogler, I.; Derhovanessian, E.; Kranz, L.M.; Vormehr, M.; Quandt, J.; Bidmon, N.; Ulges, A.; Türeci, Ö.; et al. BNT162b2 vaccine induces neutralizing antibodies and poly-specific T cells in humans. Nature 2021, 595, 572–577. [CrossRef]
[PubMed] 5. Sahin, U.; Muik, A.; Derhovanessian, E.; Vogler, I.; Kranz, L.M.; Vormehr, M.; Baum, A.; Pascal, K.; Quandt, J.; Türeci, Ö.; et al. COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses. Nature 2020, 586, 594–599. [CrossRef] [PubMed]
6. Sahin, U.; Muik, A.; Vogler, I.; Derhovanessian, E.; Kranz, L.M.; Vormehr, M.; Quandt, J.; Bidmon, N.; Ulges, A.; Türeci, Ö.; et al. 5. Sahin, U.; Muik, A.; Derhovanessian, E.; Vogler, I.; Kranz, L.M.; Vormehr, M.; Baum, A.; Pascal, K.; Quandt, J.; Türeci, Ö.; et al. COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses. Nature 2020, 586, 594–599. [CrossRef] [PubMed]
Ö COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses. Nature 2020, 586, 594–599. [CrossRef] [PubMed]
6. Sahin, U.; Muik, A.; Vogler, I.; Derhovanessian, E.; Kranz, L.M.; Vormehr, M.; Quandt, J.; Bidmon, N.; Ulges, A.; Türeci, Ö.; et al. BNT162b2 vaccine induces neutralizing antibodies and poly-specific T cells in humans. Nature 2021, 595, 572–577. [CrossRef]
[PubMed] 7. References Simon, B.; Rubey, H.; Treipl, A.; Gromann, M.; Hemedi, B.; Zehetmayer, S.; Kirsch, B. Haemodialysis patients show a highly
diminished antibody response after COVID-19 mRNA vaccination compared to healthy controls. Nephrol. Dial. Transplant. 2021,
36, 1709–1716. [CrossRef] [PubMed] 8. Speer, C.; Schaier, M.; Nusshag, C.; Töllner, M.; Buylaert, M.; Kälble, F.; Reichel, P.; Grenz, J.; Süsal, C.; Benning, L.; et al. Longitudinal Humoral Responses after COVID-19 Vaccination in Peritoneal and Hemodialysis Patients over Twelve Weeks. Vaccines 2021, 9, 1130. [CrossRef] [PubMed] 9. Ducloux, D.; Colladant, M.; Chabannes, M.; Yannaraki, M.; Courivaud, C. Humoral response after 3 doses of the BNT162b2
mRNA COVID-19 vaccine in patients on hemodialysis. Kidney Int. 2021, 100, 702–704. [CrossRef] [PubMed]
l
dd
h
d
l
h
l 10. Kalimuddin, S.; Tham, C.Y.; Qui, M.; de Alwis, R.; Sim, J.X.; Lim, J.M.; Tan, H.-C.; Syenina, A.; Zhang, S.L.; Low, J.G.; et al. Early T cell and binding antibody responses are associated with COVID-19 RNA vaccine efficacy onset. Med 2021, 2, 682–688.e4. [CrossRef] [PubMed] [
] [
]
11. Moss, P. The T cell immune response against SARS-CoV-2. Nat. Immunol. 2022, 23, 186–193. [CrossRef] [PubMed] ]
ell immune response against SARS-CoV-2. Nat. Immunol. 2022, 23, 186–193. [CrossRef] [PubMed] 11. Moss, P. The T cell immune response against SARS-CoV-2. Nat. Immunol. 2022, 23, 186–193. [CrossRef] 12. Kohmer, N.; Rühl, C.; Ciesek, S.; Rabenau, H.F. Utility of Different Surrogate Enzyme-Linked Immunosorbent Assays (sELISAs)
for Detection of SARS-CoV-2 Neutralizing Antibodies. J. Clin. Med. 2021, 10, 2128. [CrossRef] [PubMed] 13. Lehmann, P.V.; Zhang, W. Unique strengths of ELISPOT for T cell diagnostics. Methods Mol. Biol. 2012, 792, 3–23. [PubMed]
14. Jdiaa, S.S.; Mansour, R.; El Alayli, A.; Gautam, A.; Thomas, P.; Mustafa, R.A. COVID–19 and chronic kidney disease: An updated
overview of reviews. J. Nephrol. 2022, 35, 69–85. [CrossRef] [PubMed] p
15. Wu, L.-P.; Wang, N.-C.; Chang, Y.-H.; Tian, X.-Y.; Na, D.-Y.; Zhang, L.-Y.; Zheng, L.; Lan, T.; Wang, L.-F.; Liang, G.-D. Duration of
Antibody Responses after Severe Acute Respiratory Syndrome. Emerg. Infect. Dis. 2007, 13, 1562–1564. [CrossRef] [PubMed]
16
K h
N R b
H F Ci
k S K ä
B K Gött
U K ll
C R
D Bl
C Th
M L
t A
t l y
p
p
y y
g
f
16. Kohmer, N.; Rabenau, H.F.; Ciesek, S.; Krämer, B.K.; Göttmann, U.; Keller, C.; Rose, D.; Blume, C.; Thomas, M.; Lammert, A.; et al. 5. Conclusions In summary, a third dose of SARS-CoV-2 mRNA-1273 vaccine in high-risk CIHD
patients increased the magnitude and the neutralizing capacity of SARS-CoV-2-specific
antibodies, even in the NR/LR subgroup. The relevance of a repeated booster vaccination
after a certain period to enhance immunity against SARS-CoV-2 provides an incent for the
next inoculation campaign for these immunocompromised CIHD patients. Author Contributions: Conceptualization, C.B. and S.P.; methodology, S.P., B.F.K. and S.S.; validation,
T.E., H.F.R. and S.S.; formal analysis, S.P; investigation, S.P., S.S., H.F.R., D.A. and R.S.; resources, C.B.,
I.A.H., S.S. and J.M.P.; data curation, S.P.; writing—original draft preparation, S.P.; writing—review
and editing, T.E., B.F.K., I.A.H., J.M.P., A.O., N.K., H.F.R., S.S. and S.C.; visualization, S.P. and A.O.;
supervision, C.B.; project administration, C.B. and I.A.H. All authors have read and agreed to the
published version of the manuscript. Funding: The APC was funded by the University Library of Goethe University Frankfurt, Germany. Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and approved by the Ethics Committee of University Hospital Frankfurt, Germany
(reference number: 20/658; 14 January 2021). Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and approved by the Ethics Committee of University Hospital Frankfurt, Germany
(reference number: 20/658; 14 January 2021). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Vaccines 2022, 10, 585 9 of 9 Acknowledgments: We thank Rosemarie Preyer and Ulrike Loewe from AID Autoimmun Diagnos-
tika (Strassberg, Germany) for their contribution to this study. Conflicts of Interest: The authors declare no conflict of interest. References Heterologous immunization with BNT162b2 followed by mRNA-1273 in dialysis patients: Seroconversion and presence of
neutralizing antibodies. Nephrol. Dial. Transplant. 2022, gfac144. [CrossRef] [PubMed] 17. Espi, M.; Charmetant, X.; Barba, T.; Mathieu, C.; Pelletier, C.; Koppe, L.; Chalencon, E.; Kalbacher, E.; Mathias, V.; Ovize, A.; et al. A prospective observational study for justification, safety, and efficacy of a third dose of mRNA vaccine in patients receiving
maintenance hemodialysis. Kidney Int. 2022, 101, 390–402. [CrossRef] [PubMed]
|
https://openalex.org/W4291653993
|
https://zenodo.org/records/6990634/files/IJHSS%203(8)%201-6.pdf
|
English
| null |
Motivation in Writing Scientific Articles: A Case Study at Junior Higher School (SMP) PGRI 6 Surabaya
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,022
|
cc-by
| 5,189
|
INTRODUCTION Writing scientific articles is an academic
activity for teachers, mainly junior high school (SMP)
teachers. This is an activity that gives teachers skills in
writing their ideas in the form of popular scientific
articles. This can be the same skill as writing textbooks
as student textbooks if these scientific writing skills are
owned by every teacher (Moeliono, 2017). In terms of
writing skills, the activity of writing popular scientific
articles can also become a habit of reading or literacy. This is because writing activities will be carried out
smoothly if their reading activities are also higher. In
essence, writing requires sufficient reference by reading
so that writers can express their ideas in words that are
appropriate to their context or field (Handayani et al.,
2014). Therefore, the activity of writing scientific
papers is a teacher's skill that supports mainly when
writing textbooks for the rest and this must be strongly
supported by reading activities. Based on the condition of the location when
observed twice, namely on May 21, 2022 and July 18,
2022 at SMP PGRI 6 Surabaya, there are several
obstacles as follows. First, there is the principal's
interest in improving the quality of teachers. It is
expected that the improvement of the quality of teachers
is also related to the writing of scientific papers. Second, there are no activities held by the government
specifically related to the writing of popular scientific
papers so far. And, thirdly, there is high interest in
teachers when they were visited by researchers,
especially the second visit on July 18, 2022. Therefore,
this research is also related to the popular scientific
writing training program for teachers of SMP PGRI 6
Surabaya. First, they want to know the extent of their
knowledge and skills in writing popular scientific
papers. Second, this research will provide strategic
suggestions for improving the writing skills of popular
scientific papers so that they are accustomed to writing
and directly try to read as a source of writing material. So far, not all schools have got training in
scientific writing skills in the places where their schools
they are teaching. Therefore, not all of them have the
opportunity to even write in the media (Interview with
the Principal of SMP PGRI 6 Surabaya). In addition,
there are not always trainings that teach them to write
scientific papers. Research Article Research Article Citation Dijuwari., Saputri, T., Authar,
N., Hardiningrum, A., & Arifah,
L. Z. (2022). Motivation in
Writing Scientific Articles: A
Case Study at Junior Higher
School (SMP) PGRI 6 Surabaya. Indiana Journal of Humanities
and Social Sciences, 3(8), 1-6. Keywords: Scientific writing, internal motivation, external motivation, Policy. and Social Sciences, 3(8), 1-6. Copyright © 2022 The Author(s): This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY-NC
4 0) and Social Sciences, 3(8), 1-6. Copyright © 2022 The Author(s): This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY-NC
4 0) and Social Sciences, 3(8), 1-6. Copyright © 2022 The Author(s): This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 Interna
4 0) , ( ),
Copyright © 2022 The Author(s): This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY-NC
4.0). promoting school literacy is very intense (Priyasti &
Suyatno, 2021). It is based on their research at the
Sidereja 1 State Elementary School. It was stated that
this program was also developed through a literacy
strategy with professional development related to the
teaching of teachers and students in their schools. Environmental
conditions
also
support
literacy
activities. Therefore, reading and later activities to
improve writing skills will be in line with the training
activities for teachers of SMP PGRI 6 Surabaya. Article History Received: 04.08.2022
Accepted: 08.08.2022
Published: 12.08.2022
Citation
Dijuwari., Saputri, T., Authar,
N., Hardiningrum, A., & Arifah,
L. Z. (2022). Motivation in
Writing Scientific Articles: A
Case Study at Junior Higher
School (SMP) PGRI 6 Surabaya. Indiana Journal of Humanities
and Social Sciences, 3(8), 1-6. *Corresponding Author: Djuwari OPEN ACCESS JOURNALS
Indiana Journal of Humanities and Social Sciences
Abbriviate Tittle- Ind J Human Soc Sci
ISSN (Online)- 2582-8630
Journal Homepage Link- https://indianapublications.com/journal/IJHSS
DOI: 10.5281/zenodo.6990634
INDIANA
PRODUCTIVE AND QUALITY RESEARCH OPEN ACCESS JOURNALS
Indiana Journal of Humanities and Social Sciences
Abbriviate Tittle- Ind J Human Soc Sci
ISSN (Online)- 2582-8630
Journal Homepage Link- https://indianapublications.com/journal/IJHSS
DOI: 10.5281/zenodo.6990634
INDIANA
PRODUCTIVE AND QUALITY RESEARCH OPEN ACCESS JOURNALS
Indiana Journal of Humanities and Social Sciences
Abbriviate Tittle- Ind J Human Soc Sci
ISSN (Online)- 2582-8630
Journal Homepage Link- https://indianapublications.com/journal/IJHSS
DOI: 10.5281/zenodo.6990634
INDIANA
PRODUCTIVE AND QUALITY RESEARCH INDIANA Volume-03|Issue-08|2022 Volume-03|Issue-08|2022 Volume-03|Issue-08|2022 uwari*1, Tiyas Saputri1, Nailul Authar1, Andini Hardiningrum1, Lia Zannuba Arifah1 Djuwari*1, Tiyas Saputri1, Nailul Authar1, Andini Hardiningrum1, Lia Zannuba Arifah1 1Universitas Nahdlatul Ulama Surabaya (UNUSA), Indonesia 1Universitas Nahdlatul Ulama Surabaya (UNUSA), Indonesia Abstract: Writing scientific articles is a good way for improving the teachers’ quality in their profession. Therefore, teachers need this skill in order they can increase their reasoning for the profession. This study tried to
explore the teachers’ interest in writing scientific articles at Junior High School (SMP) PGRI 6 Surabaya,
Indonesia. It also tried to find out what factors made them interested in writing scientific articles offered to them. This study took 22 teachers as the respondents. They were interviewed in focus-group discussion (FGD). The data
were also taken from the interview with the School Principal. These two sources of the data triangulated, and
interpreted. The results showed that both the teachers and the school principal have the same interest and
willingness to have training on scientific articles. It was also found out that there was no policy taken by the
school and the government for that school related to increasing their skill of writing scientific articles. It is
recommended that the training program on writing scientific articles be done soon. The school and government
policy is needed. This can increase the teachers’ motivation in writing scientific article. For further research,
exploring the effectiveness of the training program is required. Article History
Received: 04.08.2022
Accepted: 08.08.2022
Published: 12.08.2022 INTRODUCTION This is actually in accordance with the
condition in Indonesia, in general, regarding student
literacy in primary and secondary schools. Due to this
condition, currently the government's program for 1 1 Djuwari et al.; Ind J Human Soc Sci; Vol-3, Iss-8 (Aug, 2022): 1-6 School Policy The policy of the school where the teacher
teaches is also an external motivation. The awareness of
teachers and schools in terms of the importance of
writing is also an attraction that can encourage someone
to write. In essence, school work programs can
encourage students' interest in writing. This can be
implemented in the form of school policies so that
students have an interest in writing. Policies are very
important related to motivation to write (Akyol &
Aktaş, 2018). In relation to this research, there is also
research conducted by Wandasari et al. (2019). They
stated that one of the external factors that could increase
students' interest in writing was also school policy. If
the school has policies related to scientific writing, for
example literacy programs, the students' interest in
writing will increase. So, teachers and schools and even
the government are also factors that can increase
students' writing creativity. This is implemented in the
form of school policies. In the framework, this study
can be drawn in Figure 1. Writing Interest Writing strategies will be more effective if the
topic of writing is related to one's daily life. In relation
to writing interest, Boscolo & Hidi (2007), in his
research, it was revealed that there was a relationship
between interest in writing and interest in the chosen
topic. The chosen topic will help the authors if the topic
is interesting. Interesting topics are topics that are liked
by the authors. Therefore, so that there is an attraction
for teachers' writing motivation, it would be better if
they were given interesting topics for them. Meanwhile,
according to Akyol & Aktaş (2018), interest in writing
is also determined by themes related to stories related to
students' lives. In their research, it was found that
stories of students' lives can provide them with high
motivation in writing. From this empirical evidence, the
writing strategy will be more effective if the topic is
interesting and related to the daily life of writers, such
as teachers or students. Interest in writing is very important for all
scientists, ranging from high school students, students,
teachers, lecturers, and scientists. This is also according
to Afianto (2018); it is also very helpful in the
development of emotional intelligence as well as to
increase interest in writing. So, writing skills or
intelligence, for example, writing arguments also
supports emotional intelligence and interest in writing. If this is supported by the results of research such as
that conducted by Afianto, then scientific writing
training is an activity program that greatly supports not
only writing skills but also develops a person's emotions
and interest in writing. Meanwhile, there are several factors that
influence the increasing interest in writing in human
life, thus Jatmika (2019), in his research, recommends
that to increase literacy and interest in reading and
especially writing, preconditions are needed, such as
infrastructure in schools, communities, and parents. From these findings and recommendations, it seems that
there are many factors, including the condition of the
school in terms of infrastructure, the condition of the
community, and even the parents. In relation to these
factors, the training of writing scientific papers is one of
the supports for the increasing interest in reading and
writing for teachers as well (Moeliono et al., 2017). Theoretical Framework Writing strategies will be more effective if the
topic of writing is related to one's daily life. In relation
to writing interest, Boscolo & Hidi (2007), in his
research, it was revealed that there was a relationship
between interest in writing and interest in the chosen
topic. The chosen topic will help the authors if the topic
is interesting. Interesting topics are topics that are liked
by the authors (Djuwari, 2021). Therefore, there is an
attraction for teachers' writing motivation, it would be
better if they were given interesting topics for them. Meanwhile, according to Akyol & Aktaş (2018),
interest in writing is also determined by themes related
to stories related to students' lives. In their research, it
was found that stories of students' lives can provide
them with high motivation in writing. From this
empirical evidence, the writing strategy will be more
effective if the topic is interesting and related to the
daily life of writers, such as teachers or students
(Djuwari, 2013). Scientific writing can be divided into several
types. In relation to the type of scientific writing,
Djuwari (2021) explains that there are pure scientific
works (academic writing) such as dissertations, theses,
theses, and scientific papers in seminars. These papers
are often done by researchers, lecturers, students, and
other scientists. In addition, there are also informal
scientific writing. This type, for example, is a scientific
work in the form of work reports. Finally, there are also
popular scientific works. Examples of this popular
scientific work are the articles published in the mass
media, namely newspapers, magazines, or tabloids
which are written according to systematic procedures as
well (Djuwari, 2010). Therefore, there are many kinds
or types of scientific works. The writing of popular
scientific works is included in informal scientific works
as a systematic expression of ideas as well. *Corresponding Author: Djuwari First Visit Data It was done on the first visit (25 May 2022). Some information was obtained from taking the policy,
in this case the school represented by the Principal
related to the policies and real conditions of the school
regarding the scientific work writing program as
follows. Table 1: Summary of the Data from the School Represented by Principal
Question
Theme
Data or information
1
Policy
(External Motivation)
Considering the importance of writing scientific for increasing the
teachers quality but it is important for accreditation status
2
Policy
(External Motivation)
Awareness of the benefits of writing scientific articles for the
teachers quality and school accreditation status
3
School Condition
There is no formal program that is stipulated in the school plan and
also the problem about the time schedule Table 1: Summary of the Data from the School Represented by Principal Question 1: Sir, what if this school held a scientific
writing training program for its teachers? motivation in the form of policy programs (Wandasari
et al., 2019). It was answered that the program offered
would have an impact on the quality of teachers and the
quality of schools. It's scientifically like that. Principal: We consider that scientific writing for
teachers in our school is very important. Writing is the
same as creativity. That's why, yes, we will improve it. If there is a program, please hold it at our school. We'll
schedule it later. In addition, there is an awareness of the
principal that writing skills are important because it is
the same as increasing the reasoning power of teachers. If they have high reasoning power, then it is the same as
increasing their creative power. The point is, from the
principal's point of view, there are indications that so far
there has been no policy for writing scientific papers in
schools. However, he realized that this program was
very important for the creativity of teachers in
increasing the power of reasoning through writing
popular scientific papers. So, this is the initial capital to
improve the quality of teachers through external
encouragement in the form of policies (Jatnika, 2019). From question 1 and the principal's answer,
there is data information related to school policies and
conditions related to the interests and programs of
writing scientific papers for teachers at the school. First,
there is no formal policy from schools and the
government. RESEARCH METHODOLOGY There are several findings in the field from the
results of interviews when the researchers conducted
visits to schools. From the results of this visit,
researchers obtained data both in the form of data from
the Principal and Teachers. The first visit, the interview
was addressed to the Principal. The second visit, the
interview was addressed to a group of teachers (Focus
Group Discussion/FGD) This research is a qualitative research that aims
to explore the interest in writing scientific papers of the
teachers of SMP PGRI 6 Surabaya. Research seeks to
explore related to writing interest and strategies to
increase writing creativity (Williams & Moser, 2019). They are the teachers who teach at the school. Data
were taken from field surveys and interviews with
principals as representatives of school policy makers
and interviews with teachers. Data were collected by
means of field visits and discussions or interviews with
the principal and teachers at the school. The information
from both sides of sources were summarized in t= terms
of the themes (Policy, Motivation, and Problem), Then
these themes were described. There were 22 teachers in
this school and then they were interviewed in the form
of a discussion with them. The first visit was conducted
on May 25, 2022 and the second visit was conducted on Writing Strategy There are two ways to increase someone's
interest in writing. The strategy can be started by
finding internal motivation and external motivation. Internal motivation can be generated from within
through for example someone's interest or interest in the
topic or story of their life. In relation to this motivation,
Esra & Sevilen (2021), in their research, state that
internal motivation is related to the likes and daily lives
of students while external motivation is related to even
organization and policies where students learn. 2 Djuwari et al.; Ind J Human Soc Sci; Vol-3, Iss-8 (Aug, 2022): 1-6 June 16, 2022. Apart from the visit, additional data was
also obtained by asking through WhatsApp (WA)
related to school problems and policies. The, they were
triangulated. This is one way in qualitative research
(Orel & Mayerhoffer, 2021). Figure 1. Research Framework The data were then analyzed and inferred
based on the problems and objectives of this study,
namely the condition of their writing interest and how
to increase their writing interest. In the end, they could
be given training on how to write scientific papers,
especially popular scientific works in the media. Figure 1. Research Framework *Corresponding Author: Djuwari First Visit Data For example, the principal approves the
program plan offered by the researcher to the school. This will encourage the success of the scientific paper
writing program as external motivation or external 3 Djuwari et al.; Ind J Human Soc Sci; Vol-3, Iss-8 (Aug, 2022): 1-6 Question 2: Do you think it has any use or benefits
other than the teachers, sir? Question 2: Do you think it has any use or benefits
other than the teachers, sir? From the third question, information related to
quality improvement problems was obtained through
the teacher's scientific writing training program. It was
stated that so far there has been no training program for
writing scientific papers for teachers in their schools. In
addition,
there
is
information
concerning
their
awareness about the importance of writing skills for
teachers because they are considered it the same as
increasing the reasoning skill of teachers. More
importantly, there is an indication that the principal is
very interested in holding scientific writing training for
his school teachers. He also wants to set a time or
schedule for the program. In essence, there are factors
that support the program in terms of the policies that
will be adopted by the principal at the school. This is
the same as the internal motivation of the principal. This motivation is also important for increasing
creativity in the form of writing scientific papers
(Jatnika, 2019). Principal: What is clear, in addition to improving the
quality of teachers in scientific writing, this training
program can also increase school accreditation. It is
because our schools are also currently accredited by
the government. From the second question, the researchers
obtained a confirmation of the principal's intention for
his commitment to the program to improve scientific
writing skills. He has historically clearly stated that his
school's scientific writing training program will be
useful for improving the quality of teachers in writing
scientific papers. In addition, he is also aware that scientific
writing training will support the improvement of school
accreditation. This means, scientifically, it is found that
the motivational factor for writing also comes not only
from school policies, but also government policies. The
policy is disclosed in the form of school evaluation
related to accreditation status. It is also realized by the
principal that with the existence of a scientific writing
training program, it is not directly the same as
increasing the school's accreditation status. First Visit Data So, the
government and its policies can also not great support
increasing motivation to write scientific papers of
teachers in schools. This is what is suggested by the
results of the studies by Wandasai et al. (2019). Thus, the survey data for the first visit,
addressed to the principal, can be said that so far there
has been no real policy related to special training in
scientific writing. Second, it is also realized that writing
scientific articles is very important for improving the
quality of teachers. Indirectly, by improving the quality
of teachers, it can also obtain an increase in the quality
of schools through the status of school accreditation. This means that there is a relationship between school
policy and improving the quality of teachers through
increasing scientific writing skills. This is in accordance
with the recommendation of the research results of
Wandasri et al. (2019) Question 3: Are there any obstacles experienced in
improving the quality, especially writing scientific
papers for teachers in this school. *Corresponding Author: Djuwari Second Visit Data Second visit, it was an interview for data from
teachers (18 June, 2022). This particular visit, at that
time, was attended by 9 teachers. Researchers were
accepted into their study and sat at a table. At this time,
initially, researchers only talked about strategies for
writing popular scientific papers. Researchers were
warmly received and even took pictures with them at
one table. Principal: The problem is, so far, there is no such
program. However, we really like the training program
to improve the quality of teachers through writing
scientific papers. That's important. Please hold. We will
arrange the schedule later and we will adjust it to the
conditions of the teachers' schedule. Given, now in
anemic condition... Table 2: Summary of the Data from the Teachers
Question
Theme
Data or Information
1
Internal
Motivation
They are enthusiastic when being offered training o
scientific writing. However, there is a problem with the sc
2
Condition
of
the school
There is not yet a program that is planned. But, they wan
Yet, they want it. It seems that they cannot decide by them
3
Problem
with
scheduling
This is to confirm also that they have problem with the
and time. It seems that they just depend on their school pr
Question 1: What if we hold training in writing
scientific papers, namely popular scientific works? Teachers: Wow, please, sir. We want it. When is it? (With a smile and sitting with the researcher). Now it's
still Student Admission program, they are on holiday
today until July 18th. From the information on
the teachers, the answer was t
interested if a popular scientific
held in their school (Using Javan
Please do it). They said that they
time but it was adjusted to their te
not to disturb their teaching an Table 2: Summary of the Data from the Teachers
Question
Theme
Data or Information
1
Internal
Motivation
They are enthusiastic when being offered training on writing
scientific writing. However, there is a problem with the schedule. 2
Condition
of
the school
There is not yet a program that is planned. But, they want to get it,
Yet, they want it. It seems that they cannot decide by themselves
3
Problem
with
scheduling
This is to confirm also that they have problem with the schedule
and time. Second Visit Data It seems that they just depend on their school principal
Question 1: What if we hold training in writing
scientific papers, namely popular scientific works? Teachers: Wow, please, sir. We want it. When is it? (With a smile and sitting with the researcher). Now it's
still Student Admission program, they are on holiday
today until July 18th. From the information on the first qu
the teachers, the answer was that they w
interested if a popular scientific writing trai
held in their school (Using Javanese, Wah M
Please do it). They said that they were willin
time but it was adjusted to their teaching sched
not to disturb their teaching and learning Table 2: Summary of the Data from the Teachers
Question
Theme
Data or Information
1
Internal
Motivation
They are enthusiastic when being offered training on writing
scientific writing. However, there is a problem with the schedule. 2
Condition
of
the school
There is not yet a program that is planned. But, they want to get it,
Yet, they want it. It seems that they cannot decide by themselves
3
Problem
with
scheduling
This is to confirm also that they have problem with the schedule
and time. It seems that they just depend on their school principal
Question 1: What if we hold training in writing
From the information on the first qu Question 1: What if we hold training in writing
scientific papers, namely popular scientific works? Question 1: What if we hold training in writing
scientific papers, namely popular scientific works? From the information on the first question to
the teachers, the answer was that they were very
interested if a popular scientific writing training was
held in their school (Using Javanese, Wah Monggo /
Please do it). They said that they were willing at any
time but it was adjusted to their teaching schedule so as
not to disturb their teaching and learning process. Teachers: Wow, please, sir. We want it. When is it? (With a smile and sitting with the researcher). Now it's
still Student Admission program, they are on holiday
today until July 18th. *Corresponding Author: Djuwari 4 Djuwari et al.; Ind J Human Soc Sci; Vol-3, Iss-8 (Aug, 2022): 1-6 Second, there was information about obstacles at that
time, namely they were still busy with new student
registration or PMB. REFERENCE 1. Arvianto, F. (2018). Sumbangan Kecerdasan
Emosional
dan
Minat
Menulis
Terhadap
Kemampuan
Menulis
Argumentasi
Pada
Mahasiswa Fakultas Hukum, Universitas Sebelas
Maret, Surakarta. Metalingua: Jurnal Penelitian
Bahasa, 14(2), 151-165. 2. Akyol, H., & Aktaş, N. (2018). The Relationship
between Fourth-Grade Primary School Students’
xx Story-writing Skills and Their Motivation to
Write. Universal Journal of Educational Research. Question 3: How about scheduling the training
program on writing scientific articles? Question 3: How about scheduling the training
program on writing scientific articles? f
3. Boscolo, P., & Hidi, S. (2007). The multiple
meanings of motivation to write. Writing and
motivation, 19(1). 3. Boscolo, P., & Hidi, S. (2007). The multiple
meanings of motivation to write. Writing and
motivation, 19(1). Teachers: New are now busy with the new student
admission and we are also in holiday. Teachers: New are now busy with the new student
admission and we are also in holiday. 4. Kunjungan,
P. (2022). Wawancara
Pribadi
Kunjungan awal di Lokasi SMP PGRI 6 Surabaya,
pada 21 Mei 2022. In reference to the third question, it seems that
they have problem with their time. They said politely
and calmly that they are busy with the new students’
admission. Besides that, when visited at that time, it
was during the period of holiday. Therefore, they
cannot determine yet when the training program can be
started. It seems that they depend on the school
principal for the decision. 5. Kunnungan, P. (2022) Wawancara Kedua dengan
para Guru. 18 Juni 2022. SMP PGRI 6 Surabaya. 6. Djuwari, D. (2021). Synthesis and Novelty for
Developing
the
Framework
in
Academic
Writing. Philippine Social Science Journal, 4(2),
53-60. 7. Djuwari, D. (2013). Error Analysis of English
Sentences Written by Administrators at STIE
Perbanas
Surabaya,
Indonesia. IAMURE
International Journal of Education, 5(1), 1-1. Second Visit Data When they were interviewed, they
were on vacation which would end on July 18, 2022. During this period, they were busy with the admissions
program. school itself, both the principal and the teachers have a
high interest in holding scientific writing training. There
is synchronization of both the Principal and the
teachers. As for the conditions and policies that exist in
schools, So far, there has been no formal policy related
to scientific writing training for the teachers, both by
the school and the government at their school. On one hand, this is very important as in the
research conducted by Afianto (2018); & Jatnika
(2019). On the other hand, they had schedule problems
at that time because they were still busy with the new
student admissions program and the school holidays. So, there is high interest but constrained by their very
busy schedule, not only teaching but also being
involved in the process of finding new students, or
registering new students. In addition, there is an important factor to
consider, namely the high willingness of teachers and
school principal to the importance of popular scientific
writing skills. They also realize that scientific writing
skills are the same as increasing reasoning power. This
is at the same time improving the quality of teachers
and schools. The quality of schools is indicated by the
existence of school accreditation by the government. So, there is a relationship between the internal
motivation of teachers in scientific writing with external
motivation in the form of school policies and
government policies in writing. It needs a policy to
improve the scientific writing skills of teachers in
schools. Question 2: Is there any program related to writing
scientific papers here? Question 2: Is there any program related to writing
scientific papers here? Teachers: Not yet, sir. (While sitting relaxed, they
answered it.) Teachers: Not yet, sir. (While sitting relaxed, they
answered it.) From the second question, real information
was obtained about the condition of the school in
relation to school policies in the training program to
improve the writing skills of teachers in schools. This is
in line with the findings on the first visit with the
principal. It was stated explicitly, with a short answer
(Not yet, sir!). This means that there is no formal policy
on scientific writing training to improve teachers'
reasoning power and creative thinking. However, this is
a good asset in improving the quality of schools through
writing as well as reading or literacy activities (Prasti &
Suyatno, 2021). Here, there are indications of
similarities with the findings on the first visit with the
principal. It was stated that interest was present and
very high because they both realized the importance of
scientific writing skills. That is why; if there is a
program related to scientific writing training in their
school, then the program can be implemented properly. A suggestion for further research is the need to
search for more data, for example after scientific
writing training is held, it needs to find out if there is an
increase in scientific writing skills. This is also to
evaluate the scientific writing training program at the
school. In other words, there is a need for further
research related to the real skills of the teachers as well
as the effectiveness of the school's scientific writing
training program. *Corresponding Author: Djuwari *Corresponding Author: Djuwari CONCLUSION From exploratory data analysis with two field
visits, there are some basic conclusions. First, within the *Corresponding Author: Djuwari 5 Djuwari et al.; Ind J Human Soc Sci; Vol-3, Iss-8 (Aug, 2022): 1-6 8. Djuwari, D. (2010). Bahasa Indonesia untuk
Perguruan
Tinggi:
teori
dan
praktik \. Perpustakaan Nasional. Jakarta: STIE Perbanas
Pres. 9. Esra, M. E. Ş. E., & Sevilen, Ç. (2021). Factors
influencing EFL students’ motivation in online
learning: A qualitative case study. Journal of
Educational
Technology
and
Online
Learning, 4(1), 11-22. 10. Hildayani, R., Sugianto, M., Tarigan, R., &
Handayani, E. (2014). Psikologi perkembangan
anak. 11. Jatnika, S. A. (2019). Budaya Literasi untuk
Menumbuhkan
Minat
Membaca
dan
Menulis. Indonesian
Journal
of
Primary
Education, 3(2), 1-6. 12. Moeliono, A. M., Lapoliwa, H., Alwi, H., &
Sasangka, S. S. T. W. (2017). Tata bahasa baku
bahasa Indonesia. 13. Orel, M., & Mayerhoffer, M. (2021). Qualitative
research methods for the exploration of Coworking
environments. The Qualitative Report, 26(5), 1364-
1382 14. Priasti, S. N., & Suyatno, S. (2021). Penerapan
Pendidikan Karakter Gemar Membaca Melalui
Program
Literasi
di
Sekolah
Dasar. Jurnal
Kependidikan: Jurnal Hasil Penelitian dan Kajian
Kepustakaan di Bidang Pendidikan, Pengajaran
dan Pembelajaran, 7(2), 395-407. 15. Wandasari, Y., Kristiawan, M., & Arafat, Y. (2019). Policy evaluation of school’s literacy
movement on improving discipline of state high
school students. International Journal of Scientific
& Technology Research, 8(4), 190-198. 16. Williams, M., & Moser, T. (2019). The art of
coding and thematic exploration in qualitative
research. International Management Review, 15(1),
45-55. 6
|
https://openalex.org/W1590733440
|
http://revista.fct.unesp.br/index.php/pegada/article/download/938/1059
|
Portuguese
| null |
AS FACES DO PLANEJAMENTO URBANO
|
Revista Pegada Eletrônica/Pegada
| 2,012
|
cc-by
| 5,244
|
MUNDO DO TRABALHO MUNDO DO TRABALHO RESUMO As decisões ligadas ao planejamento urbano na cidade privilegiam determinadas classes
sociais em detrimento de outras, intensificando ainda mais o caráter desigual da cidade. Todavia, o planejamento deve ter como objetivo essencial em sua elaboração, amenizar as
desigualdades inerentes do próprio processo de produção do espaço urbano a fim de
alcançar a justiça social. No entanto, o planejamento possui faces distintas, as quais dão
origem a conflitos e disputas, e apontam novos caminhos para que ele - o planejamento –
seja posto em prática no intuito de minimizar as desigualdades existentes. PALAVRAS CHAVE Id
l
i
t pi
pl n j m nt
rb n j p
p
g
PALAVRAS-CHAVE: Ideologia; utopia; planejamento urbano. 1 Mestranda pelo Programa de Pós-Graduação em Geografia da Universidade Estadual Paulista “Júlio de
Mesquita Filho” (FCT/UNESP), campus de Presidente Prudente. Integrante do Grupo de Estudos em
Desenvolvimento Regional e Infraestrutura (GEDRI) e bolsista da Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP). FACES OF URBAN PLANNING Dayana Aparecida Marques de Oliveira Cruz1
d.dayana@hotmail.com Dayana Aparecida Marques de Oliveira Cruz1
d.dayana@hotmail.com INTRODUÇÃO O planejamento é um elemento fundamental para compreender a produção e
organização do espaço urbano, pois ele expressa os interesses dos segmentos coexistentes
na cidade e pode justificar ou não a ação deles. Tal contraditório como as ações dos agentes
sociais concretos (CORRÊA, 2005), o espaço e a legislação também o são. A legislação é a
síntese das intenções e interesses latentes no discurso hegemônico, pois é formulada
contendo elementos que facilitam certas iniciativas, sobretudo aquelas ligadas ao mercado
imobiliário referentes à valorização de localizações selecionadas pelos que o representam. O planejamento apesar de ser uma possibilidade de alcance da justiça social na
cidade é apresentado de forma a facilitar as ações que não contribuem com o alcance da
mesma, possibilitando práticas divergentes de algumas determinações presentes na redação
das leis que o regem. Prova disso, é a determinação presente no Estatuto da Cidade acerca
do cumprimento da função social da propriedade urbana, a qual não é posta em prática nas
cidades brasileiras. Diante disso, admitimos a caracterização de três faces ao planejamento urbano: o
planejamento ideológico, o planejamento utópico e o planejamento que é posto em prática. Objetivamos com essas reflexões, discutir acerca das características gerais do
planejamento realizado nas cidades brasileiras, a partir da reflexão teórica, tendo por base
autores que pesquisam acerca do planejamento urbano, e os que escreveram acerca dos
conceitos de ideologia e utopia. ABSTRACT Decisions relating to urban planning in the city, privilege certain classes others, further
intensifying the iniquity of the city. However, planning has different faces, which give rise
to conflicts and disputes, and indicate new avenues for him – the planning – is put in place
in order to minimize inequalities. KEY WORDS: Ideology, utopia, urban planning 1 Mestranda pelo Programa de Pós-Graduação em Geografia da Universidade Estadual Paulista “Júlio de
Mesquita Filho” (FCT/UNESP), campus de Presidente Prudente. Integrante do Grupo de Estudos em
Desenvolvimento Regional e Infraestrutura (GEDRI) e bolsista da Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP). Revista Pegada – vol. 12 n.2 dezembro/2011 81 MUNDO DO TRABALHO A IMPORTÂNCIA DO PLANEJAMENTO URBANO E A JUSTIÇA SOCIAL O planejamento é um dos elementos fundamentais para compreender a produção
do espaço urbano, pois ele expressa as ações e decisões dos agentes produtores do mesmo,
os quais se relacionam de forma contraditória e complexa. Na busca pela manutenção de
seus interesses, produzem o espaço de maneira desigual e contraditória, tal como a própria
sociedade capitalista em que os interesses individuais se sobressaem sobre o coletivo e o
público, o que pode ser visto na própria cidade, bem como nas leis que dela fazem parte, Revista Pegada – vol. 12 n.2 dezembro/2011 82 MUNDO DO TRABALHO dentre as quais destacamos neste trabalho, o Plano Diretor e a Lei de Uso e Ocupação do
Solo Urbano. dentre as quais destacamos neste trabalho, o Plano Diretor e a Lei de Uso e Ocupação do
Solo Urbano. Segundo SOUZA (2010) o planejamento remete ao futuro, significa tentar simular
os desdobramentos de um processo, no intuito de precaver-se dos problemas e de
aproveitar da melhor forma os benefícios, por isso é importante distinguir planejamento e
gestão2. Estas são ferramentas imprescindíveis para a promoção do desenvolvimento sócio-
espacial, cuja expressão se dá a partir da “(...) melhoria de vida e um aumento da justiça
social” (SOUZA, 2010, p. 75). Mas o que seria justiça social? Segundo HARVEY (1980, p. 82), a justiça social é
um princípio para resolver direitos conflitivos. “O princípio da justiça social, por isso,
destina-se à divisão dos benefícios e à alocação de danos surgidos no processo do
empreendimento conjunto do trabalho”, para isso, o autor assegura que deve haver dois
questionamentos: “O que estamos distribuindo? Entre quem ou que estamos distribuindo
renda?” (p. 83). Para tanto, a análise da justiça social está dividida em duas partes, que
compreendem o que é a distribuição justa e estabelece um parâmetro para que tal
distribuição seja realizada. Em função disso, são estabelecidos os seguintes critérios:
desigualdade intrínseca; valorização de serviços em termos de oferta e demanda;
necessidade; direitos herdados; mérito; contribuição ao bem comum; contribuição
produtiva atual; esforços e sacrifícios. Todavia, apesar de todos esses critérios serem
necessários, a essência da justiça social segue respectivamente três: necessidade;
contribuição ao bem comum e mérito (HARVEY, 1980). Para alcançar a justiça social é necessária uma forma de organização social, política
e econômica apropriada. 2 Planejamento e gestão são complementares, porém distintos. “O planejamento é a preparação para a
gestão futura (...) e a gestão a efetivação, ao menos em parte (...) das condições que o planejamento feito
no passado ajudou a construir” (SOUZA, 2010, p. 46). É importante enfatizar, portanto, que gestão
refere-se ao presente, tem haver com administrar uma situação com recursos disponíveis, tendo em vista
necessidades imediatas. A IMPORTÂNCIA DO PLANEJAMENTO URBANO E A JUSTIÇA SOCIAL No território uma das possibilidades seria encontrar uma forma
socialmente justa de determinar as fronteiras dos territórios e um modo justo de alocar os
recursos para ele. A partir do exame dos princípios de justiça social chega-se ao conceito de justiça
social territorial: a distribuição de renda deveria localizar dentro dos territórios a
necessidade da população e a mobilização de recursos necessários em diferentes esferas
para suprir as necessidades dos territórios. Revista Pegada – vol. 12 n.2 dezembro/2011 83 MUNDO DO TRABALHO No espaço intra-urbano a justiça social deve ser analisada tendo em vista as
desigualdades de renda expressas na própria paisagem urbana, cuja origem se dá na divisão
social do trabalho e que tem na renda um importante elemento de diferenciação, a qual
possibilita aos citadinos o consumo de determinadas mercadorias, equipamentos e do
próprio espaço. A nosso ver, o conceito de justiça social aplicado à cidade vem no sentido
de tentar contribuir com uma melhor distribuição de renda a partir da disposição mais
equitativa dos meios de consumo coletivos, de forma a proporcionar maior acessibilidade
aos citadinos, por meio de melhores condições de mobilidade através da reestruturação do
sistema de transporte coletivo. O planejamento urbano tem muito a contribuir para o entendimento dessas
questões, possui importante papel para o desenvolvimento sócio-espacial, como dito
anteriormente, em que a justiça social é um dos objetivos. No entanto, é mister ressaltar
que o planejamento (assim como a gestão) não é neutro, é a expressão da sociedade, cujas
marcas das desigualdades são visíveis perante o modo de produção vigente e, por isso é
carregado de ideologias e interesses. Interesses que os agentes produtores do espaço lutam
para assegurá-los. AS FACES DO PLANEJAMENTO URBANO SANTOS (2003) afirma que o planejamento possibilitou a entrada rápida e brutal
do capital internacionalizado nos países subdesenvolvidos, colaborando com “o
agravamento ou à exacerbação de disparidades sociais” (p. 13), por isso o planejamento é
visto como um instrumento do capital. SANTOS (2003) aponta três fases pelas quais
passou o planejamento no mundo, na primeira fase o planejamento substituiu a
colonização; na segunda, dedicou-se ao desenvolvimento dos monopólios na forma
transnacional; na terceira, o planejamento mostra-se em uma roupagem que dá a ilusão de
propiciar a superação da pobreza, o que na verdade, gera uma nova forma de pobreza, a
pobreza planejada. A experiência brasileira com o planejamento está diretamente vinculada à
estrutura política, econômica e social do país; os planos urbanísticos aparecem como
tentativas de solucionar determinados problemas da sociedade e envolvem a ideologia
dominante nas bases da sociedade hegemônica de cada período em questão. Revista Pegada – vol. 12 n.2 dezembro/2011 84 MUNDO DO TRABALHO O que previsto em lei é resultado e depende dos agentes produtores do espaço
urbano e de seus interesses para, de fato, ser implantado, além disso, a própria lei é passível
de diferentes interpretações e possui algumas brechas, o que facilita burlá-la. Fato que é
explícito no estudo de FIX (2001) que, ao analisar a ação do Estado e os interesses do setor
imobiliário nas edificações ao longo do Rio Pinheiros, na cidade de São Paulo fala da
construção do bairro “Panamby”, construído a partir de um planejamento com um Plano
Diretor próprio apenas para atender a classe alta. Para VILLAÇA (1999, p. 191), por estar atrelado aos interesses da classe
dominante, o planejamento urbano no Brasil é “(...) apenas discurso, o planejamento é uma
fachada ideológica, não legitimando ação concreta do Estado, mas, ao contrário,
procurando ocultá-la”. O autor justifica tal afirmação a partir da exposição dos períodos
pelo qual o planejamento passou no país, segundo ele, desde a ascensão dos planos de
melhoramentos embelezamentos até a formulação de “superplanos”, a classe dominante
fez sobressair seu discurso ideológico reformulando-o para garantir seus interesses. Fazendo um paralelo entre as afirmações de Villaça e a tipologia de SOUZA (2010),
observamos que, mesmo com a mudança das fortes tendências entre planejamento e
regulatório e perspectivas mercadófilas, um elemento permaneceu: a aliança entre
representantes da iniciativa privada e Estado, muitas vezes, havendo dificuldade de
diferenciação entre essas duas figuras. Para VILLAÇA (1999, p. AS FACES DO PLANEJAMENTO URBANO 180), só pode ser considerado da esfera do
planejamento urbano “(...) apenas aquelas ações do Estado sobre o urbano que tenham
sido objeto de algum plano (...)”. Mas, nesse sentido, cabe uma observação: se o
planejamento no Brasil é ideológico os planos são, na maior parte das vezes, formas de
esconder o planejamento que realmente se pratica, que é aquele pensado a fim de fazer
com que as decisões e ações atuais garantam também para o futuro o status quo. Por isso,
em nossa concepção essa forma extra-oficial de agir sobre o espaço também é planejada e,
por isso é considerada planejamento. Para avançar um pouco nessas questões nos atentaremos ao significado de
ideologia. Para Marx, ideologia é uma forma de falsa consciência que corresponde aos
interesses de classe, sendo: “(...) o conjunto de idéias especulativas e ilusórias (socialmente
determinadas) que os homens formam sobre a realidade, através da moral, da religião, da
metafísica, dos sistemas filosóficos, das doutrinas políticas e econômicas etc”. (LÖWY, Revista Pegada – vol. 12 n.2 dezembro/2011 85 MUNDO DO TRABALHO 1987, p. 10). A ideologia é um instrumento de dominação utilizado para que a dominação
seja mantida, mas que não seja percebida. CHAUÍ (1984, p. 92) resume ideologia como
“(...) o processo pelo qual as idéias da classe dominante se tornam idéias de todas as classes
sociais, se tornam idéias dominantes”. LÖWY (1987, p. 10 e 11) apresenta que a definição de ideologia é contraposta a
de utopia, sendo que a primeira é entendida como “(...) uma forma de pensamento
orientada para a reprodução da ordem estabelecida (...)”, e a segunda “(...) define as
representações, aspirações e imagens-de-desejo (Wunschbilder) que se orientam na ruptura da
ordem estabelecida e que exercem uma função subversiva (um wälzende Funktion)” [grifos do
autor]. O pensamento utópico é desacreditado, “(...) visto que no senso comum sempre
torna o substantivo „utopia‟ e o adjetivo „utópico‟ como sinônimos de, respectivamente,
fantasia inconseqüente e fantasista” (SOUZA, 2006, p. 517). A ideologia possui grande força através da dominação, que se fortalece a partir,
principalmente da manipulação por meio da mídia. Neste sentido, SOUZA (2006) busca
em Paulo Freire, a concepção de educação bancária para assimilar o educador ao planejador
(que chama de educador-social), cujo depósito de idéias e conhecimentos ocorre sem a
utilização do diálogo. AS FACES DO PLANEJAMENTO URBANO Souza propõe no âmbito do planejamento urbano o que Paulo Freire
propôs em relação à educação libertária, enfatizando a necessidade de um planejador que
estimule o diálogo entre ele e a sociedade a fim de construir coletivamente um
planejamento, que optamos chamar aqui de utópico. Neste sentido, deve haver maior comprometimento do Estado com as classes
sociais menos favorecidas, para que suas ações estejam pautadas no planejamento que
objetive promover maior desenvolvimento socioespacial, o que colaborará com a
transformação social, desde que não esteja apenas baseado na racionalidade técnica guiada
por critérios econômicos (VASCONCELLOS, 2001). O planejamento que não contempla as questões sociais aprofunda ainda mais as
desigualdades, gerando o que VILLAÇA (1998) interpretou como a existência de duas
cidades no mesmo espaço: a dos segregados involuntariamente e a dos auto-segregados, ou
seja, uma desigualdade social e espacial ao mesmo tempo. Embora o autor tenha pontuado
dois tipos de segregação, ele afirma que, na verdade, existe apenas uma, já que a voluntária
gera a involuntária, isso se deve ao fato de que a segregação se dá a partir de um
movimento dialético, seguindo a mesma lógica do escravo e do senhor. A estrutura social
no capitalismo demanda essas contradições, pois elas contribuem para o movimento Revista Pegada – vol. 12 n.2 dezembro/2011 86 MUNDO DO TRABALHO circulatório do capital sem o qual o capitalismo não existiria. LOJKINE (1997) apresenta
três níveis de segregação: o nível da habitação, o nível dos meios de consumo coletivos e o
nível do transporte domicílio-trabalho. circulatório do capital sem o qual o capitalismo não existiria. LOJKINE (1997) apresenta
três níveis de segregação: o nível da habitação, o nível dos meios de consumo coletivos e o
nível do transporte domicílio-trabalho. Para colocar o planejamento urbano utópico em prática não há apenas
dependência dos recursos técnicos e instrumentos legais, mas decisões não apenas na esfera
econômica como também e, principalmente política. Deve haver maior comprometimento com as classes menos favorecidas, para que,
se possa caminhar rumo à justiça social, a qual atualmente só existe no âmbito da utopia. AS FACES DO PLANEJAMENTO URBANO O
planejamento urbano é um importante elemento para discutir questões que se referem à
minimização das desigualdades na cidade, pois é a partir dele que as ações e decisões
tomadas no âmbito informal têm a possibilidade de serem regulamentadas, afinal é por
meio das leis que se viabilizam os projetos que colaboram com mudanças nas condições de
vida da população. O PLANEJAMENTO URBANO NO BRASIL Refletir acerca do planejamento urbano no Brasil é necessário, haja visto que as
mudanças sofridas por ele, denunciaram a ideologia existente em cada momento histórico
distinto. VILLAÇA (1999) ao tratar sobre a história do planejamento urbano, apresenta três
períodos, cuja importância se fez notável, a qual permite analisar o caráter do mesmo. Os
períodos destacados pelo autor compreendem: 1875 – 1930; 1930 a 1990; e de 1990 em
diante. O período compreendido entre 1875 e 1830, foi aquele em que as obras de
embelezamento e melhoramento tiveram destaque, sobretudo nas cidades do Rio de
Janeiro e de São Paulo. No Brasil, em meados na década de 1930 esse tipo de planejamento
representava a ação concreta do Estado, com objetivos que não condiziam com o discurso. Villaça destaca que “Foi sob a égide dos planos de embelezamento que nasceu o
planejamento urbano (lato sensu) brasileiro” (p. 193). Ao dar o exemplo do Rio de Janeiro,
o autor afirma que, já no início do planejamento no Brasil, constatou-se que “(...) os
interesses imobiliários estavam por detrás dos grandes projetos urbanos, os quais
patrocinavam, discutiam, defendiam ou atacavam” (p.195). Neste sentido a face ideológica Revista Pegada – vol. 12 n.2 dezembro/2011 87 MUNDO DO TRABALHO do planejamento urbano faz-se presente com a presença de um discurso baseado no
privilegiamento dos interesses da classe dominante. O discurso ideológico também se faz presente no segundo período que
VILLAÇA (1999) seleciona para caracterizar como um período importante na história do
planejamento urbano no Brasil, o qual vai de 1930 a 1990. O autor apresenta a tentativa de
esconder a origem dos problemas, atribuindo ao planejamento à função de solucioná-los,
os problemas eram, portanto, entendidos como fruto do crescimento caótico, para os quais
a solução estaria no planejamento racional com técnicas e métodos bem definidos. No período da ditadura militar o planejamento caracterizou-se como justificativa
para construção de obras, principalmente as de infraestruturas ligadas ao transporte. Para
SOUZA e RODRIGUES (2004), neste período o planejamento teve caráter conservador,
pois as decisões foram tomadas por um pequeno grupo para manutenção de uma ordem
vigente. O objetivo deste planejamento, durante a década de 1970 era (...) ordenar a cidade de forma que a ação dos diferentes tipos de
capitais (principalmente o imobiliário e o industrial) pudesse ser
facilitada, em outras palavras, buscava ordenar a cidade para permitir
maiores possibilidades de lucro. O PLANEJAMENTO URBANO NO BRASIL É nesse contexto extremamente
autoritário que vai acontecer uma série de intervenções e
transformações na cidade, intensificando e consolidando a segregação
residencial (SOUZA e RODRIGUES, 2004, p. 42 e 43) Assim, com o passar dos anos, o planejamento vai se tornando cada vez mais
próximo dos interesses dos agentes ligados à iniciativa privada, seus traços tomam forma a
cada adequação (necessária) no discurso dominante para manter o status quo, atribuindo ao
planejamento um caráter ideológico. Nessa perspectiva VILLAÇA (1999, p. 191) destaca
que o planejamento urbano na Europa e nos Estados Unidos, corresponde, em parte, a
ação concreta do Estado, diferentemente no Brasil que constitui-se “(...) apenas de
discurso, o planejamento é uma fachada ideológica, não legitimando ação concreta do
Estado, mas, ao contrário, procurando ocultá-la”. O que há de semelhante entre Brasil,
Europa e Estados Unidos é que atrás da fachada do planejamento existe a dominação, o
poder, “(...) a grande diferença está nos níveis de hegemonia, aceitação e credibilidade desse
„poder‟”. Com a construção dos planos, o discurso se tornou cada vez mais hegemônico,
todavia, com o processo de urbanização e a conseqüente intensificação da desigualdade,
sugiram movimentos urbanos que passaram a contestar esse discurso “(...) especial e Revista Pegada – vol. 12 n.2 dezembro/2011 88 MUNDO DO TRABALHO precisamente naqueles setores nos quais ela vinha tradicionalmente intervindo: nos setores
imobiliário e no do sistema viário e de transportes” (VILLAÇA, 1999, p. 204). Diante de tal
situação, a classe dominante adaptou seu discurso à nova realidade, essa iniciativa é
apontada por Villaça como a gênese planejamento integrado, o qual desencadeou os
superplanos. Surge, portanto, uma nova maneira de formular o planejamento em
substituição aos antigos planos de “melhoramento e embelezamento” no objetivo de
abranger os aspectos gerais da cidade e seus problemas, denominado “plano geral”, cujos
primeiros planos surgiram na década de 1930 com os planos Agache no Rio de Janeiro e
Prestes Maia em São Paulo, a principal novidade desses planos era o destaque para a infra-
estrutura, principalmente a de saneamento e transportes3. Mantem-se, entretanto, o interesse pelas oportunidades imobiliárias que
as remodelações urbanas oferecem, e nesse sentido o centro da cidade
ainda é o grande foco de atenção dos planos. No discurso, entretanto,
pretende-se abordar a cidade inteira. (VILLAÇA, 1999, p. 207). O plano Agache, do Rio de Janeiro é o primeiro a usar a expressão plano diretor. 3 O Plano Prestes Maia foi denominado apenas como “Estudo de um Plano de avenidas” porque várias
páginas para o sistema de transportes. O PLANEJAMENTO URBANO NO BRASIL No Brasil, o plano diretor se difunde a partir da década de 1940, é substituído por outros
nomes a partir de 1960, período que estendeu-se até o fim da década de 1980, passando
pelo período ditatorial em que foram associados à tecnocracia, volta a ter o nome original
com a Constituição de 1988. O discurso do plano diretor e do urbanismo multidisciplinar gera o “plano-
discurso”, assim chamado por VILLAÇA (1999). Para o autor, os planos representaram
três diferentes períodos no Brasil, entre os anos de 1930 a 1990: Urbanismo e plano diretor
(1930-1965 – apresentado anteriormente); do planejamento integrado e dos superplanos
(1965-1971) e dos planos sem mapa (1971-1992). O planejamento integrado trazia no discurso dos que o defendiam, a necessidade
de ver a cidade além de seus aspectos físicos, dessa forma o argumento era que “Os planos
não podem limitar-se a obras de modelagem urbana; eles devem ser integrados tanto do
ponto de vista interdisciplinar como do ponto de vista espacial, integrando a cidade em sua
região” (VILLAÇA, 1999, p. 212). O Planejamento integrado não se insere na esfera
política, mas na ideológica porque se limita ao discurso não atingindo a ação real do
Estado. Revista Pegada – vol. 12 n.2 dezembro/2011 89 MUNDO DO TRABALHO O apogeu técnico, no entanto, vem com os superplanos, que, devido à
abrangência aproximaram-se muito mais de recomendações para criação de outros planos,
distanciando o plano e suas propostas, da possibilidade de implantação. O auge foi atingido
na atuação do Serviço de habitação e Urbanismo (Serfhau), criado no início do regime
militar. Nessa época foi elaborado também o Plano Urbanístico Básico do município de
São Paulo (PUB), feito por um corpo técnico que, segundo Villaça após sua finalização
acabou indo direto para o arquivo da prefeitura. No início da década de 1970, mas especificamente no ano de 1971 é aprovada a lei
nº. O PLANEJAMENTO URBANO NO BRASIL 7.688 que instituía o Plano Diretor de Desenvolvimento Integrado do Município de São
Paulo (PDDI), esse plano caracteriza o terceiro período dos planos no Brasil, em que eles
passam de técnicos e sofisticados a simplórios, Villaça, apelida-os de planos sem mapa, essa
expressão adotada pelo autor busca designar o plano enquanto nova adaptação da ideologia
dominante em seu discurso, na tentativa de dar a impressão que o planejamento urbano
estava a seus cuidados e que, estava sendo constantemente aperfeiçoado, as características
desse novo tipo de plano são facilmente perceptíveis pois apresentam “(...) apenas
objetivos, políticas e diretrizes”, essa é uma estratégia interessante porque tenta esconder as
disputas e os conflitos, já que “a idéia do plano diretor de princípios e diretrizes está
associada à de „posterior detalhamento‟, e isso nunca ocorre”. (VILLAÇA, 1999, p. 221). Com tudo isso, o plano diretor passa a ser uma atividade intelectual, pautada apenas no
discurso que, cumpre sua missão ideológica ao maquiar os problemas da maioria e manter
os interesses dominantes. Para o autor, o Plano Diretor sobrevive porque está inserido na
ideologia dominante em relação aos problemas e suas causas e sobre a isenção e
objetividade da técnica. Nos anos oitenta devido à influência neoliberal, o endividamento e estagnação
econômica, o planejamento regulatório perde força. Este período, segundo SOUZA, (1988,
p. 70), caracterizou-se por “(...) tentativas formalistas e funcionalistas de planejamento,
traduzidas na filosofia dos planos diretores, com suas propostas de planejamento, e nos
múltiplos planos regionais e estaduais”. No entanto, no fim dos anos de 1980 novas possibilidades surgiram através da
Constituição de 1988 a qual representou um avanço na possibilidade do Plano Diretor ser
um instrumento que define se a propriedade está ou não cumprindo sua função social. Por
esse motivo, o último período que Villaça apresenta, sobre a história do planejamento no
Brasil, compreende a década de 1990 em diante e, para ele, representa uma reação às
formas anteriores de planejamento, pois trás uma nova visão baseada na retomada dos Revista Pegada – vol. 12 n.2 dezembro/2011 90 MUNDO DO TRABALHO planos diretores e da maior democratização da gestão urbana, sendo assim admite que o
processo de politização do planejamento no Brasil só tem início neste século. Para que pudessem ser implantados os princípios presentes na Constituição de
1988, eram necessários instrumentos que a regulamentassem e que incorporasse os
princípios constitucionais, previstos no capítulo que dispõe sobre a política urbana. O PLANEJAMENTO URBANO NO BRASIL Com a
aprovação do Estatuto da Cidade em 2001 (Projeto de Lei nº. 5.788/90), em combinação
com aquele capítulo e com o texto da Medida Provisória nº. 2.220/01 (...) a regulamentação urbanística passa a ser tratada como um processo,
com etapas sucessivas: a formulação de instrumentos urbanísticos que
serviam para realizar e implementar os princípios, objetivos e diretrizes
estabelecidos pelo Plano; sua aprovação na Câmara Municipal; sua
fiscalização e revisão periódica a partir do cotejamento entre a estratégia
proposta e os resultados alcançados. (AZEVEDO, 2004, p. 156) (...) a regulamentação urbanística passa a ser tratada como um processo,
com etapas sucessivas: a formulação de instrumentos urbanísticos que
serviam para realizar e implementar os princípios, objetivos e diretrizes
estabelecidos pelo Plano; sua aprovação na Câmara Municipal; sua
fiscalização e revisão periódica a partir do cotejamento entre a estratégia
proposta e os resultados alcançados. (AZEVEDO, 2004, p. 156) (...) a regulamentação urbanística passa a ser tratada como um processo,
com etapas sucessivas: a formulação de instrumentos urbanísticos que
serviam para realizar e implementar os princípios, objetivos e diretrizes
estabelecidos pelo Plano; sua aprovação na Câmara Municipal; sua
fiscalização e revisão periódica a partir do cotejamento entre a estratégia
proposta e os resultados alcançados. (AZEVEDO, 2004, p. 156) Foi a partir da implantação que os resultados causados pelo planejamento tiveram
a possibilidade de serem acompanhados de perto pelo poder público municipal. Com isso,
os municípios brasileiros passaram a ser vistos como unidades de planejamento no espaço,
sendo que ao poder público local foi dada maior autonomia em relação ao gerenciamento
de seu ordenamento territorial, por meio do estabelecimento do Plano Diretor4, o que
proporcionou responder de maneira mais efetiva às necessidades da população
(RODRIGUES, 2005). Todavia, existem obstáculos para a implantação dos princípios contidos no
Estatuto da Cidade, esses obstáculos são colocados, pois os interesses entre os agentes que
coexistem na cidade são distintos. SOUZA e RODRIGUES (2004) apontam os obstáculos
como sendo de natureza política, econômica e sociopolítica. SILVA (2003) acredita que o
Estatuto da Cidade alcançará a plenitude quando incorporar políticas de caráter distintos,
dentre muitas que menciona está à política de transporte. Apesar dessa nova direção que toma o planejamento no Brasil, a partir de 1990,
SOUZA e RODRIGUES (2004) afirmam que é nessa década, que o planejamento
consolida seu caráter mercadófilo, pautado na defesa dos interesses individuais em
detrimento do público e do coletivo. 4 O Plano Diretor normatiza os princípios presentes no Estatuto da Cidade, na escala municipal e, consiste
em uma lei obrigatória para municípios com mais de vinte e mil habitantes. O PLANEJAMENTO URBANO NO BRASIL Isso se deve a aproximação cada vez maior entre o
poder público e a iniciativa privada. Neste sentido, para respaldar algumas ações e decisões Revista Pegada – vol. 12 n.2 dezembro/2011 91 MUNDO DO TRABALHO o discurso ideológico se faz presente, justificando certas iniciativas como necessárias para a
população. o discurso ideológico se faz presente, justificando certas iniciativas como necessárias para a
população. CONSIDERAÇÕES FINAIS O planejamento que não contempla as questões sociais corrobora com o aumento
da injustiça social e da desigualdade. Apesar da elaboração das leis que regulamentam o
planejamento urbano estarem suscetíveis aos interesses da iniciativa privada, ainda assim,
há necessidade de um planejamento urbano elaborado de forma crítica, que contemple os
distintos âmbitos da vida social (CARNEIRO, 1998), tratando de maneira diferente, os
diferentes, no intuito de dispor de forma mais eqüitativa os investimentos na cidade. Para colocar o planejamento urbano utópico em prática não há apenas
dependência dos recursos técnicos e instrumentos legais, mas decisões não apenas na esfera
econômica como também e, principalmente política. A ideologia imposta ao planejamento
tem sua raiz no conflito das classes sociais, em que aquelas que possuem maior poder
aquisitivo influenciam de forma decisiva na elaboração e aprovação das leis que
regulamentam o planejamento urbano, visto que, é maior a quantidade de representantes
do poder público que fazem parte dessa classe, confundindo, portanto, os interesses do
Estado com os interesses hegemônicos, resultado do estabelecimento de alianças entre
iniciativa privada e poder público. O planejamento posto em prática se dá de forma perversa, pois está a serviço do
capital, e é maquiado pelas leis e planos que tentam justificar suas ações. Neste sentido,
emerge a cada momento histórico a necessidade de um planejamento que contemple as
necessidades sociais, o qual tem sido concebido atualmente apenas no âmbito da utopia. Revista Pegada – vol. 12 n.2 dezembro/2011 92 MUNDO DO TRABALHO REFERÊNCIAS BIBLIOGRÁFICAS AZEVEDO, Abílio Moacir de. Territorialidade e plano diretor em São José do Rio
Preto. 2004. 275 f. Dissertação (Mestrado em Geografia)- Instituto de Geociências e
Ciências Exatas, Universidade Estadual Paulista, Rio Claro, 2004. CARNEIRO, Ruy de Jesus Marçal. Organização da cidade: Planejamento municipal,
plano diretor, urbanificação. São Paulo: Max Limonad, 1998. CHAUÍ, M. de S. O que é ideologia. São Paulo: Brasiliense, 1984. CORRÊA, Roberto Lobato. O espaço urbano. 4. ed. São Paulo: Ática, 2005. CORRÊA, Roberto Lobato. O espaço urbano. 4. ed. São Paulo: Ática, 2005. FIX, Mariana. Parceiros da exclusão. São Paulo: Boitempo, 2001, p. 15-107. FIX, Mariana. Parceiros da exclusão. São Paulo: Boitempo, 2001, p. 15-107. HARVEY, David. A justiça social e a cidade. São Paulo: Hucitec, 1980. LOJKINE, Jean. O Estado Capitalista e a Questão Urbana. 2. ed. Tradução de Estrela
dos S. Abreu. São Paulo: Martins Fontes, 1997. LÖWY, Michel. As Aventuras de Karl Marx contra o Barão de Münchhausen. São
Paulo: Busca Vida, 1987. MARICATO, Ermínia. Brasil, cidades: alternativas para a crise urbana. 2.ed. Petrópolis: Vozes, 2002. RODRIGUES, Arlete Moysés. Direito à e o estatuto da cidade, In: CIDADES: Revista
científica/Grupo de estudos urbanos – v. 2, n. 3, Presidente Prudente, 2005, p. 85 –
110. SANTOS, Milton. Economia Espacial: Críticas e Alternativas. 2. ed. São Paulo: Edusp,
2003. SILVA, José Borzacchielo da. Estatuto da cidade versus Estatuto de cidade – eis a questão. In: CARLOS, Ana Fani Alessandri; LEMOS, Amália Inês Geraiges (orgs). Dilemas
urbanos: novas abordagens sobre a cidade. São Paulo: Contexto, 2003, p. 29 – 34. SOUZA, Marcelo Lopes de. Mudar a cidade: uma introdução crítica ao planejamento
e gestão urbanos. 6. ed. Rio de Janeiro: Bertrand Brasil, 2010. SOUZA, Marcelo Lopes de. A prisão e a agora: reflexões em torno da democratização Revista Pegada – vol. 12 n.2 dezembro/2011 93 MUNDO DO TRABALHO do planejamento e da gestão das cidades. Rio de Janeiro: Bertrand Brasil, 2006. do planejamento e da gestão das cidades. Rio de Janeiro: Bertrand Brasil, 2006. SOUZA, Marcelo Lopes de; RODRIGUES, Glauco Bruce. Planejamento urbano e
ativismos sociais. São Paulo: Unesp, 2004. SOUZA, Marcelo Lopes de; RODRIGUES, Glauco Bruce. Planejamento urbano e
ativismos sociais. São Paulo: Unesp, 2004. SOUZA, Maria Adélia de. Governo Urbano. São Paulo: Nobel, 1988. VASCONCELLOS, Eduardo Alcântara. Transporte urbano, espaço e equidade:
análise das políticas públicas. São Paulo: Annablume, 2001. VILLAÇA, Flávio. Espaço intra-urbano no Brasil. São Paulo: Studio Nobel/FAPESP
1998. VILLAÇA, Flávio. Espaço intra-urbano no Brasil. São Paulo: Studio Nobel/FAPESP,
1998. VILLAÇA, Flávio. Uma contribuição para a história do planejamento urbano no Brasil. In:
DEÁK, Csaba; SCHIFFER, Sueli Ramos. (orgs). O processo de urbanização no Brasil. São Paulo: Editora da Universidade de São Paulo, 1999, p. 169 – 243. VILLAÇA, Flávio. Uma contribuição para a história do planejamento urbano no Brasil. In:
DEÁK, Csaba; SCHIFFER, Sueli Ramos. (orgs). O processo de urbanização no Brasil. São Paulo: Editora da Universidade de São Paulo, 1999, p. 169 – 243. Revista Pegada – vol. 12 n.2 dezembro/2011 94
|
https://openalex.org/W4380537295
|
https://www.revistas.usp.br/gis/article/download/201955/194492
|
Portuguese
| null |
Olinda quer cantar: expressões carnavalescas de uma cidade sem carnaval
|
GIS - Gesto, Imagem e Som - Revista de Antropologia
| 2,023
|
cc-by
| 4,059
|
ORCID
http://orcid.org/0000-0002-4932-7894
DOI
10.11606/issn.2525-3123.
gis.2023.201955
OLINDA QUER
CANTAR: EXPRESSÕES
CARNAVALESCAS DE
UMA CIDADE SEM
CARNAVAL
DOSSIÊ MUNDOS EM PERFORMANCE: NAPEDRA
20 ANOS
FERNANDA DE CARVALHO AZEVEDO MELLO
Universidade Federal do Rio Grande do Norte, Rio Grande do Nor-
te, Natal, Brasil, 59075-000 – ppgas@cchla.ufrn.br
INTRODUÇÃO
A linguagem audiovisual é utilizada na disciplina
antropológica como um caminho possível de elabora-
ção e divulgação das pesquisas etnográficas. A inserção
do registro de imagens no fazer da Antropologia foi
concretizada com a perspectiva de que a cultura de um
FOTOGRAFIA 1 FOTOGRAFIA 1 ORCID
http://orcid.org/0000-0002-4932-7894
DOI
10.11606/issn.2525-3123.
gis.2023.201955
OLINDA QUER
CANTAR: EXPRESSÕES
CARNAVALESCAS DE
UMA CIDADE SEM
CARNAVAL
DOSSIÊ MUNDOS EM PERFORMANCE: NAPEDRA
20 ANOS
FERNANDA DE CARVALHO AZEVEDO MELLO
Universidade Federal do Rio Grande do Norte, Rio Grande do Nor-
te, Natal, Brasil, 59075-000 – ppgas@cchla.ufrn.br
INTRODUÇÃO
A linguagem audiovisual é utilizada na disciplina
antropológica como um caminho possível de elabora-
ção e divulgação das pesquisas etnográficas. A inserção
do registro de imagens no fazer da Antropologia foi
concretizada com a perspectiva de que a cultura de um
FOTOGRAFIA 1 ORCID
http://orcid.org/0000-0002-4932-7894
DOI
10.11606/issn.2525-3123. gis.2023.201955
OLINDA QUER
CANTAR: EXPRESSÕES
CARNAVALESCAS DE
UMA CIDADE SEM
CARNAVAL
DOSSIÊ MUNDOS EM PERFORMANCE: NAPEDRA
20 ANOS
FERNANDA DE CARVALHO AZEVEDO MELLO
Universidade Federal do Rio Grande do Norte, Rio Grande do Nor-
te, Natal, Brasil, 59075-000 – ppgas@cchla.ufrn.br
INTRODUÇÃO
A linguagem audiovisual é utilizada na disciplina
antropológica como um caminho possível de elabora-
ção e divulgação das pesquisas etnográficas. A inserção
do registro de imagens no fazer da Antropologia foi
concretizada com a perspectiva de que a cultura de um
FOTOGRAFIA 1 INTRODUÇÃO A linguagem audiovisual é utilizada na disciplina
antropológica como um caminho possível de elabora-
ção e divulgação das pesquisas etnográficas. A inserção
do registro de imagens no fazer da Antropologia foi
concretizada com a perspectiva de que a cultura de um 1
São Paulo, v. 8, e-201955, 2023 1
São Paulo, v. 8, e-201955, 2023 povo não está apenas em seus artefatos, mas em seus hábitos, valores e
comportamentos. Em suas primeiras décadas, essa aproximação esteve
pautada pelo entendimento racionalista de que a imagem poderia trazer
maior objetividade à coleta de dados. “Segundo essa perspectiva, gestos,
falas, movimentos e expressões poderiam ser conservados nos filmes
assim como se conservam potes de barro e máscaras” (Barbosa e Cunha
2006, 8). Acreditava-se que a utilização da imagem pela Antropologia
traduziria melhor que palavras a exatidão, a verdade, a realidade em
si. Contudo, neste trabalho, elas representam mais que isso. Para serem
apreendidas em sua totalidade, as imagens que trago contam com a
emoção dos amantes já há muito saudosos daquele Carnaval, ainda que
não se veja (quase) nenhuma folia nas proporções que a cidade costuma
ver nessa época. Neste trabalho, trago fotografias tiradas na cidade-alta de Olinda, em
Pernambuco, durante o período (que seria) de Carnaval, em 2022 cance-
lado pelo segundo ano consecutivo pela prefeitura da cidade por causa
da pandemia de Covid-19. O sentimento gerado ao percorrer ruas que
estariam em meu próprio roteiro dos quatro dias de festejos de momo
era o de nostalgia de coisa futura, saudade de algo que conheço e que
anseio re-conhecer todos os anos; presença e ausência ao mesmo tempo. O papel da fotografia, segundo Sylvia Caiuby Novaes (2014), é justamente
o de instigar o olhar, implicando-o em sua desnaturalização: as ladei-
ras vazias estão cheias de coisas diferentes das tradicionais multidões. A fotografia, dessa forma, não é um retrato fiel da realidade, uma vez
que seu sentido é construído no diálogo da forma (como se fotografa e
como estão organizadas as fotografias) e do conteúdo (o que se fotografa)
com as bagagens subjetivas de quem fotografa (a antropóloga-fotógrafa)
e quem interpreta (o destinatário). “É o silêncio eloquente das imagens
que podemos levar para nossa disciplina, com tudo que, a seu modo, as
fotografias tem (sic) a dizer” (Caiuby Novaes 2014, 64). INTRODUÇÃO Para David MacDougall (1997), os meios visuais diferem de um texto verbal,
pois este último é construído por meio de um processo de descrição, e os
primeiros permitem que o conhecimento aconteça por meio da familia-
rização. Em outras palavras, segundo Fabiene Gama (2016), as imagens
contam com quem as observará para conseguir comunicar suas verdades
e seus sentidos, apoiando-se na reflexão crescente dentro da Antropologia
sobre o espaço que têm a percepção, os afetos e os sentidos na produção
do conhecimento etnográfico: Há diferentes níveis de interpretação, que acontecem simul-
taneamente de forma denotativa – apontando para as infor-
mações presentes na imagem – e conotativa – apontando
para diversas outras informações implícitas e simbólicas –, 2
São Paulo, v. 8, e-201955, 2023 e que são de ordem perceptiva (imediatas), cognitiva (relacio-
nadas à bagagem cultural ou experiência pessoal) e ideoló-
gica ou ética (relacionadas a valores). Isso significa que uma
mesma imagem pode ser interpretada de forma diferente
por pessoas que possuem conhecimentos diferentes sobre
o assunto fotografado (Gama 2016, 119). e que são de ordem perceptiva (imediatas), cognitiva (relacio-
nadas à bagagem cultural ou experiência pessoal) e ideoló-
gica ou ética (relacionadas a valores). Isso significa que uma
mesma imagem pode ser interpretada de forma diferente
por pessoas que possuem conhecimentos diferentes sobre
o assunto fotografado (Gama 2016, 119). Antecipando algumas diferentes experiências de interpretação – daquelas
que conhecem e das que desconhecem o Carnaval nessa cidade –, divido
as fotografias em outras três seções, cada uma contando com textos ver-
bais que auxiliam as imagens a darem conta de transmitir a experiência
vivida. Busco, com isso, chamar a atenção para a montagem deste tra-
balho e expô-la como método de construção do relato. Segundo Samain
(2012), a imagem é uma “memória de memórias”, uma “forma que pensa”,
e neste texto, a montagem, as escolhas cromáticas e as ordenações foram
pensadas para enriquecer a percepção de uma experiência subjetiva. Em essência, cada seção cobre a maioria dos significados atribuídos aos
grupos de imagens. Ademais, as cores nas imagens estão ausentes ou
bastante desbotadas em direção ao preto, branco e cinza com os objetivos
de contrastar à experiência colorida, rica e multissensorial da prática
em primeira pessoa, e de deixar a melancolia do não Carnaval visível. O
critério por trás da diferença entre poucas cores ou nenhuma referencia
a intensidade da memória associada a cada conteúdo imagético. 1. A popularização do personagem de Zé Pereira, que dá nome ao sábado de Carnaval, datada
do século XIX, ainda é cercada de muitas incertezas e divergências entre os historiadores,
contudo, a maioria deles parece concordar tratar-se do português José Nogueira de Azevedo
Paredes, um sapateiro que decidiu sair às ruas durante os dias de Carnaval tocando um
surdo. Nos anos seguintes, outros instrumentos e seus tocadores foram se juntando ao tipo
de folia inaugurada por José Nogueira. Alguns defendem que foi a partir dessa manifestação
que surgiram os blocos de rua. Dos bonecos gigantes – figuras tradicionais do Carnaval
em Pernambuco –, o de Zé Pereira é o mais antigo do estado, atualmente com mais de 100
anos. É incerto sobre como ‘Zé’ Nogueira se tornou Zé Pereira. Para mais detalhes, acessar
o texto disponível em: <https://tribunadosertao.com.br/noticias/2016/02/06/26731-sabado-
-de-ze-pereira-misterios-sobre-o-dono-sabado-de-carnaval>
2. Sobre o assunto, consultar Franco e Souza Leão (2018). INTRODUÇÃO A ideia de um período festivo no ano marcado por “inversões” tem origem
no velho continente europeu e chegou ao Brasil a partir da colonização. Mas, aos poucos, a festa foi se adaptando à realidade social específica
daqui: o entrudo português, uma bagunça generalizada nas ruas onde
cada pessoa/grupo experimenta/faz a folia à sua própria moda, foi a
forma de festejar com maior participação de populares a tomar as ruas
e, após o surgimento de Zé Pereira1, contribuiu com a origem de nossos
blocos de rua (Baribiere, Gonçalves e Menezes Neto 2022, 16); e os bailes de
máscaras venezianos, por sua vez, foram importados para diferenciar as
festas da elite das festas de rua (Lopes Junior 2019), e podemos associá-los
à “camarotização”2 – a venda de espaços cercados, elitizados e climatizados
no meio da folia – que encontramos facilmente tanto no festejo em Olinda 3 São Paulo, v. 8, e-201955, 2023 quanto no Recife. Nessas cidades, o carnaval é tradicionalmente de rua,
ainda que conte com diversos outros modelos mais ou menos populares
para além dos camarotes. Cidadãos comuns formam os blocos de fan-
farra que tomam as ruas absolutamente lotadas de foliões, animando a
multidão ao embalar frevos3 conhecidos de todas/os/es, levando foliões
a uma inebriação compartilhada. quanto no Recife. Nessas cidades, o carnaval é tradicionalmente de rua,
ainda que conte com diversos outros modelos mais ou menos populares
para além dos camarotes. Cidadãos comuns formam os blocos de fan-
farra que tomam as ruas absolutamente lotadas de foliões, animando a
multidão ao embalar frevos3 conhecidos de todas/os/es, levando foliões
a uma inebriação compartilhada. Com a chegada da pandemia de COVID-19 ao Brasil, logo após o Carna-
val de 2020, e com a necessidade de isolamento social, passamos a nos
organizar de formas diferentes, evitando “aglomerações”. Lives, trans-
missões no YouTube e Facebook: nossas festas se tornaram digitais. Com
o cancelamento do carnaval de 2021, notamos em várias partes do país
que algumas das festas, iniciativas, mobilizações e celebrações de Momo
foram para as telas (Sá Gonçalves 2021). Dos blocos olindenses, alguns
dos tradicionais fizeram seus desfiles e comemorações de modo virtual. Normalmente contavam com bonecos gigantes tradicionais do Carnaval
do estado, orquestras de frevos, passista de frevo, anfitrião e estandartes
em um festival de cores. 3. Estilo musical com ritmo alucinante e passos de dança inspirados na capoeira que, desde
2012, é considerado Patrimônio Cultural Imaterial da Humanidade pela Unesco.
4. Foi o caso para os blocos O Homem da Meia Noite, Troça Carnavalesca Mista Cariri Olin-
dense, Boi da Macuca, Pitombeira dos Quatro Cantos. Notar Hiperlink para as transmissões
disponíveis no YouTube. INTRODUÇÃO Mas sem plateia presente, sem público ao vivo,
sem multidão se aglomerando.4 Em 2022, ano das imagens deste ensaio, ainda foi possível observar algu-
mas manifestações do chamado “Carnaval digital”, mas a volta às ruas já
se iniciava, ainda que timidamente, como veremos nas páginas seguintes. É nesse sentido que as fotografias dialogam com a memória do que deveria
ser, acarretando um deslocamento para outro momento além daquele
fotografado, especialmente entre aqueles que já viveram algum Carnaval
nas Ladeiras de Olinda, evento que causa um forte impacto na vida de todos
que o conheceram, gostem ou não da folia aglomerada sob o fervor do sol. Para as pessoas não familiarizadas com esse Carnaval e essas ruas, os
textos que antecedem cada conjunto de fotografias ajudarão a reconhecer
os elementos em falta e instigar, por meio das imagens, a vontade de (re)
visitar aquelas ruas quando finalmente estiverem propriamente cheias
de novo. A sequência das fotos que veremos foi pensada com o objetivo
de apresentar a presença permanente da população olindense em seu
Carnaval através de intervenções artísticas que fazem alusão ao carna-
val; remeter aos sentimentos – similares, mas não iguais – de saudade e
nostalgia que ocupam aquelas ruas vazias; e, por fim, apontar algumas
persistências carnavalescas, sejam elas sanitariamente responsáveis no
contexto de pandemia ou aglomerações que trazem risco de contaminação, São Paulo, v. 8, e-201955, 2023 ainda que estejam em tamanho reduzido em relação ao original e restritos
a alguns dos locais mais populares das prévias carnavalescas ainda que estejam em tamanho reduzido em relação ao original e restritos
a alguns dos locais mais populares das prévias carnavalescas. Antes de seguirmos, trago abaixo um mapa de parte da Cidade Alta com
a identificação do local onde cada foto, identificada por sua ordem de
apresentação, foi tirada: ainda que estejam em tamanho reduzido em relação ao original e restritos
a alguns dos locais mais populares das prévias carnavalescas. Antes de seguirmos, trago abaixo um mapa de parte da Cidade Alta com
a identificação do local onde cada foto, identificada por sua ordem de
apresentação, foi tirada: Antes de seguirmos, trago abaixo um mapa de parte da Cidade Alta com
a identificação do local onde cada foto, identificada por sua ordem de
apresentação, foi tirada: IMAGEM 1
Mapa das
fotografias na
Cidade Alta/
Olinda (PE). Fonte: Google
Maps. 7
São Paulo, v. 8, e-201955, 2023 HINO DO ELEFANTE: O CONSTANTE DIÁLOGO ENTRE OLINDA E
SEU CARNAVAL Minha primeira parada foi a Praça do Carmo. Estaria cheia de barracas de
comidas, ambulantes vendendo bombons, água, refrigerantes e cervejas,
fitas e adereços colorindo toda a cidade, frevo em alto e bom som sendo
dançado magnificamente em um palco montado na praça. É o mesmo
palco da abertura e do encerramento oficiais dos festejos de Momo em
Olinda. Naquele último sábado de Zé Pereira, porém, poucas coisas naquela
localidade indicavam que estaríamos em festa. A saudade, contudo, pare-
cia se fazer ouvir no vento, que, sem a multidão aglomerada, batia mais
frequente e fortemente em meu rosto. Era possível ouvir o frevo de outrora em minha imaginação se eu fechasse
meus olhos. O título de cada seção a partir desta leva o nome de um famoso
frevo e aposta numa experiência de leitura multissensorial, buscando
responder à pergunta levantada por Alexsânder Nakaóka Elias (2019, 268) 5
São Paulo, v. 8, e-201955, 2023 sobre a possibilidade de incluir no texto etnográfico “outros elementos
culturais importantes relativos ao grupo e/ou comunidade”. Minha pro-
posta é, dessa forma, a de uma experiência sinestésica, assim como fez
Elias em sua tese. Mesmo vazia, era possível observar uma cidade que
ama o carnaval. As manifestações de intimidade de Olinda com o festejo
estavam tatuadas, assimiladas, incorporadas no dia a dia da cidade. Os
títulos contêm um hiperlink para ouvir os respectivos frevos no Youtube,
artifício utilizado já no título desta seção. FOTOGRAFIA 2 6
São Paulo, v. 8, e-201955, 2023 FOTOGRAFIA 3 FOTOGRAFIA 3
FOTOGRAFIA 4 FOTOGRAFIA 3 FOTOGRAFIA 4 FOTOGRAFIA 4 FOTOGRAFIA 4 7
São Paulo, v. 8, e-201955, 2023 9
São Paulo, v. 8, e-201955, 2023 ÚLTIMO REGRESSO: O SENTIDO DO VAZIO Logo minhas pernas me levariam ao lugar em que eu passo a maior parte
das minhas horas de folia. A rua 13 de Maio – mais facilmente reconhecida
como a “rua dos frangos”, reduto de lésbicas, gays, bissexuais, travestis,
transsexuais e pessoas sexualmente curiosas durante o carnaval – está
no fim de uma considerável subida desde a perimetral que passa ao lado
da Cidade Alta. Vê-la significa que estamos perto da festa e do sucesso. Quando chegamos, meu grupo e eu, normalmente dobramos à direita
e nos esprememos até um espaço onde as correntes de pessoas não nos
carreguem em direções não intencionadas. Poder transitar livremente,
conseguir observar os degraus da escadaria do Museu de Arte Contempo-
rânea (MAC), observar a ausência de barracas competindo espaço com as
pessoas, a ausência de pessoas se agarrando, não havia ninguém bêbado
ou caindo. Faltava algo. Faltava tudo. Naquele dia, a “13” era a rua resi-
dencial que normalmente é fora dos dias de festa. Em rápida conversa com a recepcionista da Pousada Auto-Astral, Hoseana,
soube que a música era desestimulada – ou proibida, segundo sua con-
cepção – nas tradicionais áreas do Carnaval de Olinda. Segundo ela, o
sertanejo na praia estava liberado. A medida buscava evitar empolgar
transeuntes a se aglomerarem nas ladeiras, haja vista o perigo que a
pandemia ainda representava. Carros da Polícia Militar da Companhia
Independente de Apoio ao Turista (CIATur) e da fiscalização municipal
podiam ser vistos frequentemente, prontos para dispersar qualquer início
de aglomeração e intenção de festa. O silêncio ecoava e fazia doer a sau-
dade. As imagens que seguem ardem, como propõe Didi-Huberman (2012,
16), por brincarem com sentidos, por transportarem pra outro momento
no espaço/tempo, por fazerem sentir a urgência, […] por seu[s] intempestivo[s] movimento[s], incapaz[es] como
é[são] de deter[em]-se no caminho (como se costuma dizer
“queimar etapas”), capaz[es] como é[são] de bifurcar[em]
sempre, de ir[em] bruscamente a outra parte (como se cos-
tuma dizer “queimar a cortesia”; despedir-se à francesa). 8
São Paulo, v. 8, e-201955, 2023 FOTOGRAFIA 5
FOTOGRAFIA 6 FOTOGRAFIA 5 FOTOGRAFIA 6 FOTOGRAFIA 6 FOTOGRAFIA 6 9
São Paulo, v. 8, e-201955, 2023 FOTOGRAFIA 7
FOTOGRAFIA 8 FOTOGRAFIA 7 FOTOGRAFIA 7 FOTOGRAFIA 8 10
São Paulo, v. 8, e-201955, 2023 MADEIRA DE LEI: “SE TIVESSE, EU NÃO RESISTIRIA” Por mais que a prefeitura e o governo do estado tivessem montado uma
estrutura de desestímulo à festa, entre muitos a vontade de brincar falou
mais alto. Em conversas com os moradores do entorno, descobri que tanto
na sexta quanto no sábado (26 e 27 de fevereiro de 2022) as ladeiras esti-
veram cheias desde o fim da tarde adentrando a noite. Adaptando-me a
essa informação, no domingo, cheguei à Rua 13 de Maio no fim da tarde
e pude presenciar o início da aglomeração que viria a ser noticiada nos
jornais locais. Nem de longe é possível comparar o que se viu apenas durante o fim de
semana com o Carnaval a que aquelas ladeiras estão acostumadas. A
multidão que presenciei se concentrou nos Quatro Cantos, conforme o
Desenho 1. Caminhar mantendo o distanciamento social era difícil, mas
longe de ser impossível. Enquanto isso, policiais militares podiam ser
vistos fazendo o que apenas posso descrever como segurança pública, pois
o efetivo de quatro pessoas era insuficiente para sequer tentar dispersar
o grupo grande de pessoas que ocupavam o espaço. Contudo, não se pode dizer que o carnaval passou sem festa nas ladeiras
de Olinda ou sem que manifestações carnavalescas pudessem ser vistas
em formas de fantasias, adereços e glitter. Enquanto caminhava para
fotografar bonecos gigantes que estavam “sentados” numa caminhonete,
ouvi um grupo de três pessoas que conversam casualmente na frente de
uma casa: Senhora: … porque se tivesse, eu não resistiria. Morador: Eu só não fui pra praia porque não ia ter nada,
porque eu também não ia resistir. É gostoso demais! (DIÁRIO
DE CAMPO, 27 fev. 22). Ainda que em menores proporções, o frevo, ao entardecer, podia ser ouvido
sem fechar os olhos: uma das moradoras colocou uma enorme caixa de
som na janela de sua casa na “13” enquanto tomava uma cerveja com
duas outras pessoas, numa mesa colocada por ela mesma do outro lado
da rua. “A polícia até passa, olha, mas como não tem muita gente, eles
não fazem nada”, me explicou Rafael. Ele, sua mãe e a amiga/moradora
brindavam ao Carnaval em um local de Carnaval, ainda que não hou-
vesse propriamente um Carnaval. Uma criança que passava com a mãe
ensaiou alguns passos de frevo, e Margarida, mãe de Rafael, declarou: “É
isto, minha filha! Está no sangue de todo olindense”. 11
São Paulo, v. MADEIRA DE LEI: “SE TIVESSE, EU NÃO RESISTIRIA” 8, e-201955, 2023 DESENHO 1
Localizando a
aglomeração. Fonte: autoria
própria. Material
utilizado: iPad e
Pencil iPad WB. FOTOGRAFIA 9 DESENHO 1
Localizando a
aglomeração. Fonte: autoria
própria. Material
utilizado: iPad e
Pencil iPad WB. DESENHO 1
Localizando a
aglomeração. Fonte: autoria
própria. Material
utilizado: iPad e
Pencil iPad WB. FOTOGRAFIA 9 FOTOGRAFIA 9 FOTOGRAFIA 9 12
São Paulo, v. 8, e-201955, 2023 FOTOGRAFIA 10
FOTOGRAFIA 11 FOTOGRAFIA 10 FOTOGRAFIA 10 FOTOGRAFIA 11 FOTOGRAFIA 11 13
São Paulo, v. 8, e-201955, 2023 FOTOGRAFIA 12
FOTOGRAFIA 13 FOTOGRAFIA 12 FOTOGRAFIA 13 14
São Paulo, v. 8, e-201955, 2023 FOTOGRAFIA 14 FOTOGRAFIA 14 KEYWORDS
Anthropology and
image; Carnival;
Pandemics;
Olinda-PE. REFERÊNCIAS BIBLIOGRÁFICAS Baribiere, Ricardo José, Renata de Sá Gonçalves e Hugo Menezes Neto. 2022. O carnaval
e a pesquisa universitária: antropologia, artes e letras em diálogo. Entrevista
com Maria Laura Cavalcanti e Felipe Ferreira e Fred Góes. Sociol. Antropol, vol. 12,
no 03: e210070. Barbosa, Andréa e Edgar Teodoro da Cunha. 2006. Antropologia e imagem. Rio de Janeiro:
Zahar. Caiuby Novaes, Sylvia. 2014. O silêncio eloquente das imagens fotográficas e sua impor-
tância na etnografia. Cadernos de Arte e Antropologia, vol. 3, no. 2: 57-67. Didi-Huberman, Georges. 2012. Quando as imagens tocam o real. Tradução de Patrícia
Carmello e Vera Casa Nova. PÓS: Revista do Programa de Pós-graduação em Artes
da EBA/UFMG, vol. 2, no. 4: 206-219. Elias, Alexsander Nakaoka. 2019. Por uma etnografia multissensorial. TESSITURAS | Revista
de Antropologia e Arqueologia, vol. 7, no. 2: 266-293. Franco, Suelen Matozo e André Luiz Maranhão de Souza Leão. 2018. Lógica de mercado
como medida de eficiência da organização do Carnaval de Olinda. Rev. Adm. Con-
temp, vol. 22, no 5: 661-682. Gama, Fabiene. 2016. Sobre emoções, imagens e os sentidos: estratégias para experi-
mentar, documentar e expressar dados etnográficos. RBSE – Revista Brasileira de
Sociologia da Emoção, vol. 15, no. 45: 116-130. 5
São Paulo, v. 8, e-201955, 2023 Lopes Junior, Rubens. 2019. O carnaval como manifestação popular: um paralelo entre a
concepção beltraniana de carnaval em Recife e Olinda e o surgimento do carnaval
carioca. RIF, vol. 17, no. 39: 181-196. Lopes Junior, Rubens. 2019. O carnaval como manifestação popular: um paralelo entre a
concepção beltraniana de carnaval em Recife e Olinda e o surgimento do carnaval
carioca. RIF, vol. 17, no. 39: 181-196. Lopes Junior, Rubens. 2019. O carnaval como manifestação popular: um paralelo entre a
concepção beltraniana de carnaval em Recife e Olinda e o surgimento do carnaval
carioca. RIF, vol. 17, no. 39: 181-196. MacDougall, David. 1997. “The visual in anthropology”. In Rethinking visual anthropology,
ed. Marcus Banks e Howard Morphy. New Haven, London: Yale University Press:
276-295. Sá Gonçalves, Renata de. 2021. Descontinuidades e estratégias da nobre dança do carna-
val. In A falta que a festa faz: celebrações populares e antropologia na pandemia, org. Maria Laura Cavalcanti e Renata de Sá Gonçalves. Rio de Janeiro: Museu Nacional,
Universidade Federal do Rio de Janeiro: 302-316. Samain, Etienne. (org.). 2012. Como pensam as imagens. Campinas: Unicamp. FOTOGRAFIA 15 16
São Paulo, v. 8, e-201955, 2023 RESUMO Em Olinda (PE), durante o mês de momo, as foliãs tomam as ruas com
muito glitter, brilho e corpos à mostra. Não há um centímetro sequer
de distância entre ninguém, e é muito comum observarmos, de alguma
forma, como os apaixonados beijos rápidos que são trocados entre desco-
nhecidas conseguem diminuir ainda mais a lonjura. A montagem desse
trabalho teve como duplo objetivo mostrar as ruas do trajeto imaginado
para o Carnaval de Olinda no ano de 2022; e, a partir da ausência de corpos
e da fanfarra, cancelada pelo segundo ano consecutivo pela prefeitura da
cidade, refletir sobre os sentidos da festa para a cidade. Em outras pala-
vras, o presente trabalho expõe os registros, por meio de fotografias, do
não Carnaval, do não beijo, da não multidão, assim como as resistências
e insistências em manter acesa a chama da folia, por meio de fotografias
do percurso evocado pela memória de carnavais passados. PALAVRAS-CHAVE
Antropologia e
imagem; Carnaval;
Pandemia;
Olinda-PE. Recebido: 07/09/2022
Reapresentado: 10/01/2023
Aprovado: 14/02/2023 ABSTRACT In Olinda (PE), during its most festive month, the revelers take to the
streets with lots of glitter and bodies on display. There is not even an inch
of distance between anyone and it is very common to observe, in some
way, how the passionate quick kisses that are exchanged between stran-
gers manage to further diminish the distance. The assembly of this paper
has as a double objective to show the streets of the imagined route for the
Carnival of Olinda in the year 2022; and, from the absence of bodies and
fanfare, canceled for the second consecutive year by the city hall, reflect
on the meanings of the party for the city. In other words, the present arti-
cle exposes the records, through photographs, of the non-carnival, of the
non-kiss, of the non-crowd, as well as the resistances and insistences on
keeping the flame of the revelry alive, through photographs of the route
evoked by the memory of past carnivals. Fernanda de Carvalho Azevedo Mello é doutoranda em Antropologia Social pela Universidade
Federal do Rio Grande do Norte (UFRN) e mestra em Antropologia pela Universidade Federal
de Pernambuco (UFPE). Cursou graduação em Relações Internacionais na Faculdade Integrada
do Recife (FIR) e Ciências Sociais na Universidade Federal Rural de Pernambuco (UFRPE). Atualmente, desenvolve pesquisa no Carnaval de Olinda (PE), etnografando a eroticidade e
a paquera entre mulheres. Integra o grupo de estudos Gênero, Corpo e Sexualidade (GCS)
da UFRN. E-mail: nandacmello@hotmail.com Licença de uso. Este artigo está licenciado sob a Licença Creative Commons CC-BY. Com
essa licença você pode compartilhar, adaptar, criar para qualquer fim, desde que atribua a
autoria da obra. Recebido: 07/09/2022
Reapresentado: 10/01/2023
Aprovado: 14/02/2023 17
São Paulo, v. 8, e-201955, 2023
|
https://openalex.org/W2056981524
|
https://www.frontiersin.org/articles/10.3389/fpsyg.2012.00224/pdf
|
English
| null |
Training Visual Imagery: Improvements of Metacognition, but not Imagery Strength
|
Frontiers in psychology
| 2,012
|
cc-by
| 12,062
|
INTRODUCTION Sasaki et al., 2010); can imagery also improve with daily prac-
tice? There is some evidence to suggest that perceptual learning
can occur from training without physical stimulation. Repeti-
tively imagining the crucial part of a visual bisection stimulus
(visual spatial judgment) or imagining a low-contrast Gabor
pattern (contrast judgment) can improve performance on sub-
sequent perceptual tasks (Tartaglia et al., 2009). Similarly, imag-
ining motor-acts facilitates performance on corresponding tasks
by training relevant parts of motor cortex, and by strengthen-
ing associations between processes and actions (Driskell et al.,
1994; Weiss et al., 1994; Feltz and Landers, 2007). To date,
research has mainly focused on the effects imagery training
has on subsequent perceptual tasks. Here, we look directly at
the influence of imagery training on the strength of imagery
itself. Mental imagery can be described as the retrieval of perceptual
information from memory, and the subsequent examination of
this information in the “minds eye.” Research has provided a
growing body of behavioral and neuroimaging evidence that
there is considerable overlap between the “minds eye” and actual
perception (Chen et al., 1998; Kreiman et al., 2000; O’Craven
and Kanwisher, 2000; Zatorre and Halpern, 2005). For exam-
ple, behavioral studies have demonstrated that imagery content
can selectively influence perception (Perky, 1910; McDermott and
Roediger, 1994; Pearson et al., 2008). Imagery has been shown to
affect visual detection thresholds (Ishai and Sagi, 1997), perfor-
mance on a visual acuity task (Craver-Lemley and Reeves, 1992),
and to induce negative aftereffects (Gilden et al.,1995) in much the
same way as a sensory stimulus. Recent neuroimaging studies show
that there is considerable spatial overlap between activated areas
of the brain during both visual perception and visual imagery, for
example information about a pattern held in mind during work-
ing memory or imagery can be present in visual sensory cortex
(Kosslyn et al., 1995; Slotnick et al., 2005; Harrison and Tong,
2009; Serences et al., 2009; Stokes et al., 2009). Like perception,
visual imagery is impaired when visual cortical activity is dis-
turbed by means of transcranial magnetic stimulation (Kosslyn
et al., 1999). One of the hallmarks of mental imagery is the considerable
difference in reported imagery strength and vividness observed
across individuals (Galton, 1883; McKellar, 1965; Marks, 1973;
Amedi et al., 2005; Cui et al., 2007). Some individuals claim
veridical, vivid imagery, while others doubt its entire existence
(McKellar, 1965). ORIGINAL RESEARCH ARTICLE
published: 10 July 2012 Training visual imagery: improvements of metacognition,
but not imagery strength Rosanne L. Rademaker 1,2,3* and Joel Pearson2,3,4
1 Cognitive Neuroscience Department, Maastricht University, Maastricht, Netherlands
2 Psychology Department, Vanderbilt University, Nashville, TN, USA
3 Vanderbilt Vision Research Center, Vanderbilt University, Nashville, TN, USA
4 School of Psychology, The University of New South Wales, Sydney, NSW, Australia Rosanne L. Rademaker 1,2,3* and Joel Pearson2,3,4
1 Cognitive Neuroscience Department, Maastricht University, Maastricht, Netherlands
2 Psychology Department, Vanderbilt University, Nashville, TN, USA
3 Vanderbilt Vision Research Center, Vanderbilt University, Nashville, TN, USA
4 School of Psychology, The University of New South Wales, Sydney, NSW, Australia *Correspondence: Correspondence:
Rosanne L. Rademaker, Cognitive
Neuroscience Department,
Maastricht University,
Universiteitssingel 40, 6229 ER
Maastricht, Netherlands. e-mail: rosanne.rademaker@
maastrichtuniversity.nl Keywords: visual imagery, training, learning, metacognition, introspection, binocular rivalry, consciousness Keywords: visual imagery, training, learning, metacognition, introspection, binocular rivalry, consciousness Edited by: Visual imagery has been closely linked to brain mechanisms involved in perception. Can
visual imagery, like visual perception, improve by means of training? Previous research has
demonstrated that people can reliably evaluate the vividness of single episodes of imag-
ination – might the metacognition of imagery also improve over the course of training? We had participants imagine colored Gabor patterns for an hour a day, over the course of
five consecutive days, and again 2 weeks after training. Participants rated the subjective
vividness and effort of their mental imagery on each trial. The influence of imagery on
subsequent binocular rivalry dominance was taken as our measure of imagery strength. We found no overall effect of training on imagery strength. Training did, however, improve
participant’s metacognition of imagery. Trial-by-trial ratings of vividness gained predictive
power on subsequent rivalry dominance as a function of training. These data suggest
that, while imagery strength might be immune to training in the current context, people’s
metacognitive understanding of mental imagery can improve with practice. Stephen Michael Kosslyn, Stanford
University, USA
Reviewed by:
Stephen Michael Kosslyn, Stanford
University, USA
Gregoire Borst, Université Paris
Descartes, France
*Correspondence:
Rosanne L. Rademaker, Cognitive
Neuroscience Department,
Maastricht University,
Universiteitssingel 40, 6229 ER
Maastricht, Netherlands. e-mail: rosanne.rademaker@
maastrichtuniversity.nl Stephen Michael Kosslyn, Stanford
University, USA INTRODUCTION To assess metacognition we use a method derived from signal
detection theory (Swets, 1986; Macmillan and Creelman, 1991;
Galvin et al., 2003; Kornbrot, 2006) that has been successfully
employed in a variety of recent metacognition studies (Fleming
et al., 2010; Song et al., 2011). Using this method, we looked at the
likelihood that imagery biased subsequent rivalry, given a certain
level of imagery vividness. Signal detection allows us to estimate a
single quantitative “sensitivity” measure of metacognitive ability,
derived from these objective (amount of perceptual bias) and sub-
jective (ratings of vividness) variables. This measure of sensitivity
is criterion free, which means that it is not prone to changes in cri-
terion (rating-magnitude), and it is not affected by irregular use
of the rating scale (which generally results in unequal numbers of
observations across the various conditions). We have previously demonstrated that imagery can alter future
competitive visual interactions in favor of the imagined stimulus
on a large percentage of trials (Pearson et al., 2008, 2011), while
catch-trial presentations of mock rivalry stimuli do not reveal such
bias,rulingoutthepossibilityof demandcharacteristics(Pylyshyn,
2003). Indeed, scores on offline imagery questionnaires predict
imagery strength measured using rivalry (Pearson et al., 2011) and
rivalry has been utilized to examine the role of imagery during
visual working memory (Keogh and Pearson, 2011). Thus, there is
compellingevidencethatrivalrybias(or“perceptualbias”)isause-
ful way to measure imagery strength in general (e.g.,encompassing
perceptual elements and sensations of vividness). In the current
study, imagery strength is the underlying construct of interest,
and the extent to which imagery biases perception is taken as a
reliable measure of imagery strength. The subjective experiences
associatedwithimagerystrengthareprobedbyhavingparticipants
report the “vividness” of their mental images. By way of preview, here we report that imagery strength – mea-
sured as the extent to which imagery biases perception during
binocular rivalry – did not increase over the 5-day training period. Interestingly, participant’s metacognition of imagery did signifi-
cantly improve over the training period. This dissociation between
imagery strength and metacognitive ability suggests a degree of
independence between the two processes. Can people evaluate the phenomenal qualities of internally
generated experiences, such as whether a mental image is vivid
or detailed? Recently, an attempt was made to answer the ques-
tion of knowing ones own thoughts (exemplifying the problem
of “metacognition;” Flavell, 1979) in relation to mental imagery
(Pearson et al., 2011). INTRODUCTION The factors causing such differences in imagery
strength remain largely unknown. One hypothesis is that individ-
uals who actively practice, or whose everyday activities involve
strong use of imagery, might have strengthened their imagery
through training and practice (Sacks, 2010). We sought to exam-
ine such a proposal in the lab by engaging individuals in
an imagery task daily, over a period of 5 days. Can repeated
instances of forming visual imagery lead to improved imagery
strength? If visual imagery can indeed be defined as the recreation of
a perceptual representation in the absence of retinal input (Ishai
and Sagi, 1995), one may wonder exactly how similar imagery is
to perception. Specifically, prolonged visual practice can improve
perceptual skill (Fahle and Poggio, 2002; Fine and Jacobs, 2002; July 2012 | Volume 3 | Article 224 | 1 www.frontiersin.org www.frontiersin.org The training of imagery and metacognition Rademaker and Pearson be stable and task independent, suggesting a common cognitive
process (Song et al., 2011). To address this question researchers must be able to reli-
ably measure imagery strength from 1 day to the next. Previous
work demonstrated that sustained imagery has a pronounced
and visually specific impact on subsequent perception (Pearson
et al., 2008, 2011). These studies utilized a visual phenomenon
called binocular rivalry; when two different patterns are pre-
sented one to each eye, only one of the patterns is consciously
perceived. Subtle experimental manipulations, such as attention
(Meng and Tong, 2004; Mitchell et al., 2004; Chong and Blake,
2006; Kamphuisen et al., 2007), sensory memory (Pearson and
Brascamp,2008),or imagery (Pearsonet al.,2008),can bring about
a slight imbalance in the neural states, creating a bias that helps
one pattern win the race for dominance at the expense of the
other. Little is known regarding the stability and independence of
metacognition of mental imagery. If metacognition for percep-
tual tasks originates from a common cognitive process, might
a similar process allow people to have metacognition of mental
imagery? Despite the highly subjective and volitional nature of
imagery, people are reasonably good at imagery metacognition
(Pearson et al., 2011). Is this ability stable, or might metacognition
of imagery improve with repeated practice? Here, we also investi-
gated the degree of imagery metacognition as a function of daily
training. PARTICIPANTS Nine observers (six female) participated in the experiment.All had
normal or corrected-to-normal visual acuity and normal stere-
ovision, and all provided written informed consent. Observers
received payment for their participation ($10 per hour, plus a $5
per hour bonus upon completion) with the exception of a partici-
pating author (RR) and participant BW. The study was carried out
with the approval of the Institutional Review Board at Vanderbilt
University. Frontiers in Psychology | Perception Science INTRODUCTION This study provided compelling new evi-
dence that people have accurate metacognitive knowledge at
fine-grained scale, regarding specific instances of imagery: On
individual trials, higher ratings of imagery vividness predicted a
greater likelihood that the imagined pattern would appear domi-
nant during subsequent rivalry (Pearson et al.,2011). Interestingly,
repeated attempts to form a particular visual image can lead to dif-
ferent degrees of success with each try, causing imagery strength
to fluctuate from one moment to the next. Despite this variance in
imagery strength, people demonstrate good metacognitive under-
standing of their imagery, and can readily evaluate how vivid their
mental images are on a particular occasion. MATERIALS
b Observers viewed the stimuli on a luminance-calibrated CRT
monitor with 1152 × 870 resolution and a 75-Hz refresh rate in
an otherwise darkened room. Visual stimuli were generated with
Matlab 7.5.0 (R2007b) and the Psychophysics toolbox (Brainard,
1997; Pelli, 1997) under Mac OSX. Observers sat at a viewing
distance of 56 cm, and used a chinrest to maintain a stable head
position. A mirror stereoscope was used to present a different pat-
tern to each eye, and binocular convergence of the two images was
aided by a white bull’s eye fixation dot (0.95˚) at the center of each
monocular half-image. Participants were instructed to maintain
steady fixation throughout all experimental trials. At a general level, there has been a growing interest in metacog-
nitive judgments of memory and sensory decision-making (Kiani
and Shadlen, 2009; Fleming et al., 2010; Rounis et al., 2010; Song
et al., 2011). Frontal brain regions are important for introspec-
tive or metacognitive ability (Kepecs et al., 2008; Fleming et al.,
2010), which suggests that the neural substrates of metacogni-
tive ability are distinct from those supporting primary perception. Although the ability to introspect varies substantially across indi-
viduals, within a single individual metacognitive ability seems to July 2012 | Volume 3 | Article 224 | 2 The training of imagery and metacognition Rademaker and Pearson Rivalry stimuli consisted of a green and a red grating (spatial
frequency = 1.23 c/˚) surrounding a central fixation point, pre-
sented against a black background with a mean luminance of
0.09 cd/m2. CIE color values of the stimuli were as follows – green:
x = 0.293, y = 0.572; red: x = 0.602, y = 0.353. Gratings were pre-
sented at 75% contrast and had a Gaussian-shaped luminance
profile (mean luminance = 6.95 cd/m2) that faded to black at the
stimulus edge (Gaussian σ = 4.29˚). Five observers were trained
with a green grating of orientation 112.5˚ and a red grating
of orientation 22.5˚, while on generalization blocks they were
presented with 67.5˚ green, and 157.5˚ red gratings. The oppo-
site was true for the remaining four observers, meaning that
we counterbalanced which grating-pairs were used for training
and generalization between participants. On catch trials, a mock
rivalry stimulus was presented consisting of a physical blend
of the green and red rivalry patterns. This stimulus was pre-
sented to both eyes simultaneously in order to avoid interocular
competition. MATERIALS
b Presentation of the mock-stimulus allowed us to
test for decisional bias and demand characteristics (Landsberger,
1958). into the lab at or around the same time on each day of training,
and were dark-adapted for a couple of minutes before the start of
each experimental session. Duringtheexperiment,participantswerebrieflypresentedwith
a randomly chosen (equal number of both) central cue (“G” for
green, or “R” for red) at the beginning of each imagery-trial
(Figure 1A). Subsequently, participants would engage in visual
imagery of the cued pattern for an 8-s period. After completing
this imagery period, the word “vividness?” cued participants to
first report the quality of their imagery by means of left-handed
button presses (1 = almost no imagery, 2 = some weak imagery,
3 = moderate imagery, 4 = strong imagery almost like perception),
after which they were cued by the word “effort?” to report the
amount of vigor with which they had tried to imagine the pattern
(1 = almost no effort, 2 = some effort, 3 = moderate effort, 4 = tried
very hard to form a mental image). Observers were instructed to
use the full range of the rating scale to the best of their abilities. As soon as a participant had responded to both questions, a
rivalry display (90% of trials) or a mock display (10% of trials)
was presented for 750 ms. On rivalry trials, the green grating was
presented to the left eye, and the red grating to the right eye. On
mock trials, the plaid-stimulus was presented to both eyes simul-
taneously. Participants reported which image had appeared most
dominant, by pressing one of three buttons (1 = green, 2 = mixed,
3 = red). For this response, the right hand was used in order to
minimize potential response conflict between the two hands. A
“mixed” response could be made on all trials (rivalry and mock
trials). On rivalry trials, the observer could give a mixed response
in case he or she was unable to distinguish which grating had
appeared more dominant due to binocular combination or piece-
meal rivalry. This type of mixed percept was reported on 6.49% of
rivalry trials (SEM = 2.49%). The dominant eye plays a key role in determining which of
two monocular images is likely to be perceived at the onset of
binocular rivalry. MATERIALS
b Therefore, individual fine-tuning of stimulus
contrast was done before the start of the experiment, and before
each daily session, to control for differences in ocular dominance
between observers. We used the same procedure as in previous
research (Pearson et al., 2008, 2011; Keogh and Pearson, 2011),
matching the relative strength of the rivalry gratings to the point
at which perceptual competition is most balanced, and thus most
susceptible to disruption. PROCEDURE To investigate whether visual imagery can be improved by means
of training,and to see how this relates to metacognition of imagery
over time, we had observers perform a visual imagery task on five
consecutive days, for about an hour a day. A sixth follow-up ses-
sion was conducted 2–3 weeks after training. Participants came A single training session consisted of two blocks of 70 trials
each. Within each block, seven catch trials were randomly inter-
leaved between the rivalry trials.We tested potential generalization
of learning to non-trained orientations on day 1 and 5 of train-
ing, and during follow-up. On these days, observers performed 1s
8s
Imagery
Cue
Subjective ratings
1s
R
vivid? effort? ~1s
~1s
0.75s
Rivalry
A
B
p(self-report | no perceptual bias)
p(self-report | perceptual bias)
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
left eye
right eye
FIGURE 1 | Sample trial sequence and example ROC II curve. (A)
Participants were presented with a random cue (“G” or “R”) after which they
formed a mental image of the green or red grating over an 8-s period. Participants were then cued to report the vividness of the imagined item, and
the effort exerted while imagining the item, on an absolute scale from 1 to 4. After a brief flash of the rivalry display, participants reported which grating had
appeared perceptually dominant, or whether their percept was an equal mix
of the two. On 10% of the trials – instead of the rivalry display – a
mock-stimulus was presented to both eyes simultaneously, consisting of a
physical combination of both the green and red grating. (B) To determine how
well subjective ratings predict perceptual bias, type II ROC sensitivity was
calculated by taking the area under the ROC curve (Aroc). This area is the sum
of the area of the half-square triangle (dark-gray shaded region) and the area
between the diagonal and the ROC function (light-gray shaded region). 1s
8s
Imagery
Cue
Subjective ratings
1s
R
vivid? effort? ~1s
~1s
0.75s
Rivalry
A
B
p(self-repor
p(self-report | perceptual bias)
0
0.2
0
0.2
0.4
0.6
0.8
1
left eye
right eye B
p(self-report | no perceptual bias)
p(self-report | perceptual bias)
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1 A of the two. PROCEDURE On 10% of the trials – instead of the rivalry display – a
mock-stimulus was presented to both eyes simultaneously, consisting of a
physical combination of both the green and red grating. (B) To determine how
well subjective ratings predict perceptual bias, type II ROC sensitivity was
calculated by taking the area under the ROC curve (Aroc). This area is the sum
of the area of the half-square triangle (dark-gray shaded region) and the area
between the diagonal and the ROC function (light-gray shaded region). FIGURE 1 | Sample trial sequence and example ROC II curve. (A)
Participants were presented with a random cue (“G” or “R”) after which they
formed a mental image of the green or red grating over an 8-s period. Participants were then cued to report the vividness of the imagined item, and
the effort exerted while imagining the item, on an absolute scale from 1 to 4. After a brief flash of the rivalry display, participants reported which grating had
appeared perceptually dominant, or whether their percept was an equal mix of the two. On 10% of the trials – instead of the rivalry display – a
mock-stimulus was presented to both eyes simultaneously, consisting of a
physical combination of both the green and red grating. (B) To determine how
well subjective ratings predict perceptual bias, type II ROC sensitivity was
calculated by taking the area under the ROC curve (Aroc). This area is the sum
of the area of the half-square triangle (dark-gray shaded region) and the area
between the diagonal and the ROC function (light-gray shaded region). FIGURE 1 | Sample trial sequence and example ROC II curve. (A) of the two. On 10% of the trials – instead of the rivalry display – a
mock-stimulus was presented to both eyes simultaneously, consisting of a
physical combination of both the green and red grating. (B) To determine how
well subjective ratings predict perceptual bias, type II ROC sensitivity was
calculated by taking the area under the ROC curve (Aroc). This area is the sum
of the area of the half-square triangle (dark-gray shaded region) and the area
between the diagonal and the ROC function (light-gray shaded region). FIGURE 1 | Sample trial sequence and example ROC II curve. ANALYSES To assess the strength of visual imagery, we looked at the per-
ceptual facilitation (or bias) of imagery on rivalry. This was
calculated as the percentage of trials in which the imagined grat-
ing matched subsequent perception during rivalry (Pearson et al.,
2008), excluding trials on which a mixed percept was reported. A
perceptual bias greater than 50% (chance) on the rivalry trials but
not on the catch trials suggests facilitation due to imagery con-
tent. Due to experimenter error, a small number of runs (7 out
of 108) were missing from the data. Where necessary, we used tri-
linear interpolation to infer the mean percentage of bias. For the
day-by-day analysis (Figure 2) only one data point was interpo-
lated (percentage perceptual bias for participant CB on day 4); the
session-by-session analysis of the same data required interpolation
of all seven missing runs. Aroc = 0.25
4
X
i=1
h hi+1 −fi
2 −
hi −fi+1
2i
+ 0.5
(1) (1) The bias of the ROC II curve (Broc) was defined as the ratio
K B/K A, where K B is the area between the ROC curve and the
major diagonal (dashed line in Figure 1B) to the right of the
minor diagonal (dotted line in Figure 1B), and K A is the area
between the ROC curve and the major diagonal to the left of the
minor diagonal. A neutral bias would give Broc equal to zero, while
a negative or positive Broc indicates a bias toward lower or higher
ratings respectively. Broc = ln
0.25
2P
i=1
h hi+1 −fi
2 −
hi −fi+1
2i
0.25
4P
i=3
h hi+1 −fi
2 −
hi −fi+1
2i
(2) Data obtained from subjective ratings of vividness (and effort)
were analyzed by constructing type II receiver operating charac-
teristic (ROC) curves for each participant on each day of training. This method of assessing metacognitive ability is derived from
signal detection methods (Swets, 1986; Macmillan and Creelman,
1991; Galvin et al., 2003; Kornbrot, 2006) and has been success-
fully employed in a variety of recent inquiries about metacognition
(Fleming et al., 2010; Song et al., 2011). PROCEDURE (A)
Participants were presented with a random cue (“G” or “R”) after which they
formed a mental image of the green or red grating over an 8-s period. Participants were then cued to report the vividness of the imagined item, and
the effort exerted while imagining the item, on an absolute scale from 1 to 4. After a brief flash of the rivalry display, participants reported which grating had
appeared perceptually dominant, or whether their percept was an equal mix July 2012 | Volume 3 | Article 224 | 3 www.frontiersin.org The training of imagery and metacognition Rademaker and Pearson twice the amount of trials, with training and generalization blocks
presented separately and in a randomized order. twice the amount of trials, with training and generalization blocks
presented separately and in a randomized order. is the sum of the area of the half-square triangle (dark-gray shaded
region in Figure 1B) and the area between the diagonal and the
ROC function (light-gray shaded region in Figure 1B): Frontiers in Psychology | Perception Science ANALYSES Essentially, the only differ-
ence between type I and type II tasks lies in the event that is being
discriminated: Type I decisions are about the occurrence of events
independent of the observer (so,distinguishing between signal and
noise), whereas type II decisions are about whether a decision was
correct or incorrect (so, making a metacognitive judgment). (2) Previous research has shown that subjective ratings of vivid-
ness – but not effort – are predictive of how much perceptual bias
someone experiences (Pearson et al., 2011). To confirm this, we
also applied the ROC II methods described above to participant’s
ratings of exerted effort. This effort-based ROC thus characterizes
the probability of a participant being perceptually biased given a
certain level of self-reported effort. Finally, to determine whether
the ROC II model did a good job accounting for our metacognitive
data, we fit a linear regression model: Since it is not possible to be “correct” or “incorrect” about
an internally generated image, we adapted the definition of the
type II decision to include judgments about the vividness of
single episodes of mental imagery and its effect on the per-
ceptual outcome during brief instances of subsequent binocular
rivalry. Applying the signal detection logic, we categorized trials
where participants reported high vividness and where imagery
subsequently biased perception as “hits.” Trials where partici-
pants reported high vividness but perception was not subsequently
biased were categorized as “false alarms.” Here, the ROC II char-
acterizes the probability of a participant being perceptually biased
during rivalry, given a certain level of self-reported vividness. z (h) = β0 + β1z
f
+ ε
(3) (3) Where z is the inverse of the cumulative normal distribu-
tion function. The ROC II model provided a good fit to the
self-reported vividness (mean R2 = 0.976 ± 0.004) and effort data
(mean R2 = 0.981 ± 0.007). IMAGERY TRAINING Sustained mental imagery can bias the perception of an ambigu-
ous display, resulting in a reliable measure of imagery strength on
a trial-to-trial basis (Pearson et al., 2008, 2011). When people rate
their imagery as more vivid, the likelihood that imagery influences
perception is larger (Pearson et al., 2011). Thus, if training men-
tal imagery would result in more vivid images, one would expect
to see an increase of perceptual bias over time. Figure 2 shows
the mean imagery strength (or “perceptual bias”) as a function of
days of training and again 2 weeks later. A within-subjects ANOVA
revealed that training did not increase the amount of perceptual
bias over time [F(5,40) < 1]. To construct the ROC II curves, we calculated p(rating = i |
perceptual bias) and p(rating = i | no perceptual bias) for all i,
and transformed these into cumulative probabilities before plot-
ting them against each other (anchored at [0,0] and [1,1]). Distribution-free methods were employed to characterize type
II ROC sensitivity by calculating the area under the ROC curve
(Aroc), and type II ROC bias (Broc). These parameters are derived
from simple geometry and do not make assumptions about the
shape of the distribution (Kornbrot, 2006). The area under the
ROC curve (Aroc) quantifies the extent to which metacogni-
tive judgments are predictive of perceptual bias during rivalry
(Figure 1B); a diagonally flat ROC function indicates little pre-
dictive value of the metacognitive judgment on the subsequent
perceptual outcome during rivalry. The area under the ROC curve Mental imagery did bias perception in favor of the imagined
grating [F(5,40) = 8.861; p = 0.018] which is consistent with pre-
vious work demonstrating the effect of mental imagery on rivalry
(Pearson et al., 2008, 2011). Unsurprisingly – considering the
lack of a training effect – gratings of both trained and untrained July 2012 | Volume 3 | Article 224 | 4 The training of imagery and metacognition Rademaker and Pearson 1
2
3
4
5
2-3 weeks
45
50
55
60
65
70
Day of training
Perceptual bias (% of trials)
training
generalization
FIGURE 2 | Imagery biased perception during rivalry in favor of the
previously imagined grating, but this perceptual bias did not
significantly change over the course of a 5-day training. The blue line
represents the mean training data, and the blue shaded regions represent
±1 SEM. METACOGNITIVE JUDGMENTS To assess whether people’s metacognitive insights about imagery
strength improve over the course of training, we constructed ROC
II curves for each individual observer, on each day of training
(Figure 4; Materials and Methods). The extent to which metacog-
nitive judgments of vividness predict perceptual bias was quan-
tified as the area under each ROC II curve. Data presented in
Figure 4 demonstrate that on earlier days of training (darker green
lines) the area under the curve is smaller than on later days of
training (lighter green lines). The upward bowing profile of the
curves observable in over half of our participants demonstrates
that vividness judgments indeed predict perceptual bias. There is a clear trend toward more metacognitive abil-
ity over time for both the trained [F(5,40) = 1.742; p = 0.147]
and
untrained
(generalization)
orientation
[F(2,16) = 7.416;
p = 0.005]. Trained and untrained orientations do not statisti-
cally differ [F(1,8) < 1]. This lack of orientation specificity may
not be surprising considering that metacognition for perception
is something presumably supported by higher-level frontal areas
of the brain (Fleming et al., 2010), where responses are invariant
to fine-grained orientation information. Hence, we simplified our
analysis by collapsing the data from all orientations before con-
structing the ROC II curves as displayed in Figure 4. Estimates of
the type II ROC sensitivity Aroc are therefore slightly more reliable
on day 1, day 5, and during follow-up, since they are constructed
based on more data. FIGURE 2 | Imagery biased perception during rivalry in favor of the
previously imagined grating, but this perceptual bias did not
significantly change over the course of a 5-day training. The blue line
represents the mean training data, and the blue shaded regions represent
±1 SEM. The parts of the plot depicted in red represent data from blocks
where we tested training generalization to other orientations. (generalization) orientations yielded similar perceptual biases: a
within-subjects ANOVA for training days 1, 5, and follow-up
revealed no main effect of orientation [F(1,8) < 1]. Analyzing the
data by session did not unveil any hidden differences in perceptual
bias over time [F(11,88) = 1.106; p = 0.366], which excludes the
possibility that most learning took place between the first couple
of sessions. IMAGERY TRAINING The parts of the plot depicted in red represent data from blocks
where we tested training generalization to other orientations. negligible influence on participant’s reports of rivalry dominance,
as previously documented (Pearson et al., 2011). 1
2
3
4
5
2-3 weeks
45
50
55
60
65
70
Day of training
Perceptual bias (% of trials)
training
generalization METACOGNITIVE JUDGMENTS The information from Figure 4 is summarized in Figure 5,
showing the main effect of training: vividness judgments pre-
dict perceptual bias increasingly better over time [F(5,40) = 3.075;
p = 0.019]. This trend is linear when only looking at training
days 1–5 [F(1,8) = 5.846; p = 0.042] but becomes quadratic when
follow-up is included [F(1,8) = 8.778; p = 0.018],indicating a drop
of the proportion Aroc at follow-up. Nevertheless, planned com-
parisons (uncorrected t-tests) show that – with the exception of
day 1 – the predictive value of self-reported vividness on the per-
ceptual outcome is larger than would be expected by chance (one-
tailed one-sample t-test day 1: p = 0.243; all others: p < 0.029),
and this ability is still present 2–3 weeks after training (p = 0.021). Additional evidence that mental imagery was not improved
by training comes from participant’s introspective judgments
of imagery vividness. Mean self-reported vividness of men-
tal imagery was statistically the same on all days of training
[F(5,40) = 1.224; p = 0.316]. Self-reports of exerted effort did not
change over the course of training either [F(5,40) < 1]. In summary,
neither the perceptual measure of imagery strength (“perceptual
bias”) nor ratings of vividness showed any significant change over
the 5-days of training. Thus, it appears that training in this study
was unable to increase imagery strength over time. CATCH TRIALS In previous work we demonstrated that people can reliably
evaluate the vividness of their mental imagery from one trial
to the next (Pearson et al., 2011). Participants in that previous
study were tested only once. Thus, one might expect to find evi-
dence for metacognition of imagery strength on day 1 of training
in the current experiment. However, the difference in analyses
used to determine metacognition in our previous and current
work (within-subjects analysis of variance, and area under ROC II
curve respectively), make it hard to directly compare the findings. A within-subjects analysis of variance performed on the current
data shows that on day 1 of training, participants marginally (but
not significantly) showed a main effect of vividness on perceptual
bias [F(3,15) = 2.83; p = 0.074]. However, a lack of observed power
(0.558) indicates that at this sample size there is only a small (44%)
chance of finding a significant effect (at α = 0.05) when assuming
that people have metacognitive insights into their own imagery Catch trials were presented in a randomly interleaved fashion on
10% of all experimental trials, to determine whether observers
showed response bias in favor of the imagined grating. On these
trials,a mock rivalry display was presented consisting of a balanced
physical combination of the green and red gratings shown to both
eyes simultaneously. If the effects observed during rivalry were due
to decisional bias or demand characteristics, we expect to find the
same degree of response bias on catch trials. We analyzed bias by
coding veridical“mixed”responses to the catch trials as 50%,while
responses that matched the cued pattern were coded as 100%, and
responses opposite to the cued grating were coded as 0%. The
percentage of catch trials during which participant’s responses
were biased in favor of the cued grating are shown in Figure 3
(for all days of training). On average, this bias was 50.79%. CATCH TRIALS This
indicates that demand characteristics and decisional bias have a July 2012 | Volume 3 | Article 224 | 5 www.frontiersin.org The training of imagery and metacognition Rademaker and Pearson 0
10
20
30
40
50
60
70
80
90
100
day 1
day 2
day 3
day 4
day 5
2−3 weeks
Perceptual bias (% of trials)
MM
AA
EY
DB
CB
EE
RS
BW
RR
FIGURE 3 | Mean percentage of catch trials on which participant’s response to the fake-rivalry display was biased in favor of the imagined grating. A
score of 50% indicates a lack of bias. Gray shaded areas show each individual participant, the colored bars represent the 5 days of training plus follow-up
2–3 weeks later. FIGURE 3 | Mean percentage of catch trials on which participant’s response to the fake-rivalry display was biased in favor of the imagined grating. A
score of 50% indicates a lack of bias. Gray shaded areas show each individual participant, the colored bars represent the 5 days of training plus follow-up
2–3 weeks later. day 1
day 2
day 3
day 4
day 5
2−3 weeks
RR
BW
RS
CB
DB
EY
MM
AA
p(vividness | no perceptual bias)
p(vividness | perceptual bias)
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
EE
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
FIGURE 4 | Participant’s metacognition of imagery over the course of training. ROC II curves based on the vividness ratings reported by our participants
(collapsed over Gabor orientations). Each plot represents an individual participant; the colored lines represent the ROC curves for the different days of training. FIGURE 4 | Participant’s metacognition of imagery over the course of training. ROC II curves based on the vividness ratings reported by our participants
(collapsed over Gabor orientations). Frontiers in Psychology | Perception Science CATCH TRIALS Each plot represents an individual participant; the colored lines represent the ROC curves for the different days of training. Frontiers in Psychology | Perception Science Frontiers in Psychology | Perception Science July 2012 | Volume 3 | Article 224 | 6 The training of imagery and metacognition Rademaker and Pearson strength at the population level. An a priori power analysis indi-
cates that, assuming a medium effect size, 21 subjects would be
required to obtain a power of 0.95. that,as expected,effort did not predict perceptual bias [F(1,8) < 1]. The area under the ROC II curve (Aroc), which quantifies the
degreetowhichself-reportedeffortpredictsperceptualbiasduring
rivalry,did not differ from chance on any of the training days (two-
tailed one-sample t-tests all p > 0.081). Neither did we observe a
change over time for the trained [F(5,40) < 1], untrained (general-
ized) [F(2,16) = 2.711; p = 0.097],or collapsed [F(5,40) < 1] grating
orientations. Vividness ratings are predictive of the efficacy that mental
imagery has at biasing the perception of rivaling stimuli. By
contrast, self-reported effort for imagery was not hypothesized to
predict perceptual bias.Attempts to exert greater effort do not nec-
essarily result in highly effective imagery, as demonstrated by pre-
vious work (Pearson et al., 2011). To ensure that our findings were
specific to introspective vividness – and not effort – we constructed
ROC II curves (as in Figure 4; Materials and Methods) based on
the effort ratings reported by our participants. The pooled (across
participants) curves per day are shown in Figure6A;the diagonally
flat function indicates a weak link between self-reported effort rat-
ings and perceptual bias during rivalry. Figure 6B demonstrates Vividness appears to predict perceptual bias more strongly over
the course of training, whereas effort does not predict perceptual
bias at all. Can this finding be explained by the way participants
used the rating scales? Participant’s average reported vividness
(2.57 ± 0.21) and effort (2.86 ± 0.14) did not significantly dif-
fer (p = 0.261). In other words, subjective vividness – but not
effort – is predictive of how well something was imagined inde-
pendent of rating-magnitude. Signal detection theory considers
metacognitive ability (sensitivity) and rating-magnitude (bias) as
two independent properties (Galvin et al., 2003). In accordance
with this notion,we find that individuals with higher self-reported
vividness were not better at evaluating their imagery strength
and vice versa. CATCH TRIALS FIGURE 5 | Imagery metacognition improves over the course of FIGURE 5 | Imagery metacognition improves over the course of
training. The extent to which self-reported vividness predicts perceptual
bias (quantified as the area under the ROC II curve after collapsing all FIGURE 5 | Imagery metacognition improves over the course of
training. The extent to which self-reported vividness predicts perceptual
bias (quantified as the area under the ROC II curve after collapsing all
grating orientations, as also shown in Figure 4) plotted against day of
training. Shaded areas represent ±1 SEM. training. The extent to which self reported vividness predicts perceptual
bias (quantified as the area under the ROC II curve after collapsing all
grating orientations, as also shown in Figure 4) plotted against day of
training. Shaded areas represent ±1 SEM. p(effort | no perceptual bias)
p(effort | perceptual bias)
day 1
day 2
day 3
day 4
day 5
2−3 weeks
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
B
Proportion A effort
1
2
3
4
5
2-3 weeks
Day of training
roc
0.4
0.5
0.6
0.7
0.65
0.55
0.45
A
FIGURE 6 | Participant’s self-reported effort with which the
gratings were imagined does not predict perceptual bias. (A) ROC
II curves based on the effort ratings pooled across participants. Green
colored lines represent the different days of training. The diagonally flat
functions indicate a weak link between self-reported effort ratings and
perceptual bias during rivalry. (B) The extent to which self-reported
effort predicts perceptual bias (quantified as the area under the ROC II
curve) plotted against each day of training. Self-reported effort does not
predict perceptual bias, and this does not change over time. Data was
collapsed over Gabor orientations, since outcomes did not differ
between the two sets of orientations [F (1,8) < 1]. Shaded areas
represent ±1 SEM. www.frontiersin.org
July 2012 | Volume 3 | Article 224 | 7 B
Proportion A effort
1
2
3
4
5
2-3 weeks
Day of training
roc
0.4
0.5
0.6
0.7
0.65
0.55
0.45
effort predicts perceptual bias (quantified as the area under the ROC II
curve) plotted against each day of training. Self-reported effort does not
predict perceptual bias, and this does not change over time. Data was
collapsed over Gabor orientations, since outcomes did not differ
between the two sets of orientations [F (1,8) < 1]. CATCH TRIALS (A) ROC
II curves based on the effort ratings pooled across participants. Green
colored lines represent the different days of training. The diagonally flat
functions indicate a weak link between self-reported effort ratings and
perceptual bias during rivalry. (B) The extent to which self-reported
effort predicts perceptual bias (quantified as the area under the ROC II
curve) plotted against each day of training. Self-reported effort does not
predict perceptual bias, and this does not change over time. Data was
collapsed over Gabor orientations, since outcomes did not differ
between the two sets of orientations [F (1,8) < 1]. Shaded areas
represent ±1 SEM. 0.4
0.5
0.6
0.7
1
2
3
4
5
2-3 weeks
Day of training
Proportion A vividness
roc
0.65
0.55
0.45
FIGURE 5 | Imagery metacognition improves over the course of
training. The extent to which self-reported vividness predicts perceptual
bias (quantified as the area under the ROC II curve after collapsing all
grating orientations, as also shown in Figure 4) plotted against day of
training. Shaded areas represent ±1 SEM. 0.4
0.5
0.6
0.7
1
2
3
4
5
2-3 weeks
Day of training
Proportion A vividness
roc
0.65
0.55
0.45
FIGURE 5 | Imagery metacognition improves over the course of
training. The extent to which self-reported vividness predicts perceptual
bias (quantified as the area under the ROC II curve after collapsing all
grating orientations, as also shown in Figure 4) plotted against day of
training. Shaded areas represent ±1 SEM. 0.4
0.5
0.6
0.7
1
2
3
4
5
2-3 weeks
Day of training
Proportion A vividness
roc
0.65
0.55
0.45 The type II bias of the ROC curve (Broc) provides us with a
distribution-free estimate of the criterion used by participants to
provide their subjective ratings. A neutral bias would give Broc
equal to zero, while a negative or positive Broc indicates a bias
toward lower or higher ratings respectively. This estimate corre-
sponded very well with the actual rating-magnitude collected dur-
ing the experiment: self-reported vividness ratings and estimated
vividness bias (Broc vividness) were highly correlated (r = 0.913;
p < 0.001), as were self-reported effort and estimated effort bias
(r = 0.889; p = 0.001). The close resemblance between these two
variables – both measures of participant’s criterion – helps validate
the distribution-free approach used to determine ROC estimates
in the current paradigm. CATCH TRIALS Specifically, participant’s ability to make accurate
metacognitive judgments of their mental imagery (Aroc vivid-
ness) and participant’s mean vividness ratings were not correlated
(r = 0.063; p = 0.873). ings and perceptual bias during rivalry. Figure 6B demonstrates
0.4
0.5
0.6
0.7
1
2
3
4
5
2-3 weeks
Day of training
Proportion A vividness
roc
0.65
0.55
0.45
FIGURE 5 | Imagery metacognition improves over the course of
training. The extent to which self-reported vividness predicts perceptual
bias (quantified as the area under the ROC II curve after collapsing all
grating orientations, as also shown in Figure 4) plotted against day of
training. Shaded areas represent ±1 SEM. effort
is predictive of how well something was imagined inde
pendent of rating-magnitude. Signal detection theory considers
metacognitive ability (sensitivity) and rating-magnitude (bias) as
two independent properties (Galvin et al., 2003). In accordance
with this notion,we find that individuals with higher self-reported
vividness were not better at evaluating their imagery strength
and vice versa. Specifically, participant’s ability to make accurate
metacognitive judgments of their mental imagery (Aroc vivid-
ness) and participant’s mean vividness ratings were not correlated
(r = 0.063; p = 0.873). The type II bias of the ROC curve (Broc) provides us with a
distribution-free estimate of the criterion used by participants to
provide their subjective ratings. A neutral bias would give Broc
equal to zero, while a negative or positive Broc indicates a bias
toward lower or higher ratings respectively. This estimate corre-
sponded very well with the actual rating-magnitude collected dur-
ing the experiment: self-reported vividness ratings and estimated
vividness bias (Broc vividness) were highly correlated (r = 0.913;
p < 0.001), as were self-reported effort and estimated effort bias
(r = 0.889; p = 0.001). The close resemblance between these two
variables – both measures of participant’s criterion – helps validate
the distribution-free approach used to determine ROC estimates
in the current paradigm. p(effort | no perceptual bias)
p(effort | perceptual bias)
day 1
day 2
day 3
day 4
day 5
2−3 weeks
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
B
Proportion A effort
1
2
3
4
5
2-3 weeks
Day of training
roc
0.4
0.5
0.6
0.7
0.65
0.55
0.45
A
FIGURE 6 | Participant’s self-reported effort with which the
gratings were imagined does not predict perceptual bias. DISCUSSION The research presented here suggests that mental imagery strength
does not improve over the course of our 5-day training regime. Over the 5-days, no changes were observed relating to imagery
strength as measured by rivalry, nor were there any changes in
the average introspective judgments of imagery vividness. We fur-
ther demonstrated that self-reported vividness of mental imagery
predicts the perceptual consequences of single epochs of imagery. More importantly, this prediction becomes stronger with practice,
implying increased metacognition of imagery over the course of
training. Self-reported effort of mental imagery on the other hand,
did not predict perceptual outcomes. There have been reports of visual imagery increasing perfor-
mance on subsequent perceptual tasks (Tartaglia et al., 2009). Yet
we were unable to find an increase in facilitation of rivalry domi-
nance after 5 days of training. The question is of course, why? The
emphasis of the research presented here was on improving imagery
strength over time. This is a notably different emphasis from stud-
ies that have investigated how imagery training changes perceptual
skills (Tartaglia et al., 2009). One obvious explanation for the lack
of an imagery training effect in this study is that imagery strength
simply cannot improve with practice. This idea is corroborated
by the fact that neither imagery bias, nor subjective ratings of
imagery strength showed a significant increase as a function of
training. Introspective ratings of imagery strength are reflected
in the perceptual outcomes during rivalry, and the close relation-
ship between the two implies they measure the same underlying
construct (Pearson et al., 2011). Thus, several aspects of the data
support the hypothesis that it is not possible – or very difficult – to
improve imagery strength by means of training. One could hypothesize that imagery strength is liable to
improvement, but we simply failed to find any in this study due to
the configuration of our task. Research into the process of improv-
ing perceptual skill – or perceptual learning – provides useful con-
text in support of this hypothesis. One influential view known as
the reverse hierarchy theory (Ahissar and Hochstein, 2004), states
that learning is gated by top-down, task-related factors: Learning
begins at high-level areas of the brain, after which it trickles down
the hierarchy, fine-tuning the read out from lower level areas. CATCH TRIALS Shaded areas
represent ±1 SEM. B
Proportion A effort
1
2
3
4
5
2-3 weeks
Day of training
roc
0.4
0.5
0.6
0.7
0.65
0.55
0.45 p(effort | no perceptual bias)
p(effort | perceptual bias)
day 1
day 2
day 3
day 4
day 5
2−3 weeks
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
A p(effort | no perceptual bias)
0
0.2
0.4
0.6
0.8
1 effort predicts perceptual bias (quantified as the area under the ROC II
curve) plotted against each day of training. Self-reported effort does not
predict perceptual bias, and this does not change over time. Data was
collapsed over Gabor orientations, since outcomes did not differ
between the two sets of orientations [F (1,8) < 1]. Shaded areas
represent ±1 SEM. effort predicts perceptual bias (quantified as the area under the ROC II
curve) plotted against each day of training. Self-reported effort does not
predict perceptual bias, and this does not change over time. Data was
collapsed over Gabor orientations, since outcomes did not differ
between the two sets of orientations [F (1,8) < 1]. Shaded areas
represent ±1 SEM. FIGURE 6 | Participant’s self-reported effort with which the
gratings were imagined does not predict perceptual bias. (A) ROC
II curves based on the effort ratings pooled across participants. Green
colored lines represent the different days of training. The diagonally flat
functions indicate a weak link between self-reported effort ratings and
perceptual bias during rivalry. (B) The extent to which self-reported FIGURE 6 | Participant’s self-reported effort with which the
i
i
i
d d
di
l bi
(A) ROC GURE 6 | Participant’s self-reported effort with which the July 2012 | Volume 3 | Article 224 | 7 www.frontiersin.org The training of imagery and metacognition Rademaker and Pearson through imagery training, yet, changes at this cortical level may
not be sufficient to improve imagery strength itself. Strikingly, Figure 4 shows large differences between individu-
als: the degree to which metacognitive vividness judgments predict
perceptual bias varies quite a bit from one person to the next
(Aroc = 0.48–0.71). This type of variability is not uncommon, and
previous studies have reported similarly large individual differ-
ences in metacognitive ability for perceptual tasks (Song et al.,
2011). Besides large differences related to metacognition, also the
overall amount of perceptual bias experienced by our partici-
pants varied widely (46–76%). CATCH TRIALS Nonetheless, participant’s overall
metacognitive ability and the percent perceptual bias they experi-
enced throughout the experiment,were uncorrelated (r = −0.027;
p = 0.945). This suggests that participant’s metacognitive ability
in this task is independent of imagery strength, as measured by
binocular rivalry. Historically,mental imagery has been considered a fainter form
of perception (Hume, 1739). Evidence to support this notion
comes from functional magnetic resonance imaging (fMRI) stud-
ies demonstrating that the magnitude of brain activity is lower
during imagery than during bottom-up perception (Goebel et al.,
1998; O’Craven and Kanwisher, 2000). Likewise, single neuron
recordings in the medial temporal lobe of humans found fewer
neurons that were recruited during imagery than during per-
ception, and that the firing rate of these cells was lower during
imagery compared to perception (Kreiman et al., 2000). In the
case of perceptual bias during rivalry, imagery is presumed to
influence or boost the memory trace that exists between one
rivalry presentation and the next, and the location and orienta-
tion specificity of this memory trace implies that it is composed
of primarily low-level characteristics (Ishai and Sagi, 1995; Pear-
son et al., 2008; Slotnick, 2008). Mechanisms such as a gain in
sensitivity for the imagined pattern, or the strengthening of sen-
sory traces, would be needed to modify population activity in
lower visual areas mediating alternations of conscious percep-
tion during rivalry. Imagery may simply lack sufficient impact
to induce permanent plastic changes at these lowest sensory levels. Future research directions aiming to improve imagery strength
could investigate the necessity of bottom-up information for
learning. Specifically, it would be interesting to see if there is a
transfer from improving visual perception by means of prolonged
training with actual sensory stimuli, to improvements of imagery
strength. Frontiers in Psychology | Perception Science DISCUSSION This
theory invokes a number of detailed predictions, namely, early
(fast) learning should be related to high-level changes, whereas
asymptotic (slow) learning should involve plasticity in low-level
sensory areas – if required by the task. There is considerable evi-
dence supporting this view (Ahissar and Hochstein, 1993, 1997;
Dosher and Lu, 1998; Dupuis-Roy and Gosselin, 2007). In light of the reverse hierarchy framework,our training regime
issuspecttoacriticalvulnerability.Namely:trainingduration.Five
days may have been insufficient time to reach the asymptotic learn-
ing phase. The Tartaglia study previously mentioned (Tartaglia
et al., 2009) trained participant’s imagery for 10 days, twice as long
as in our study, and found an improvement on perceptual tasks. Assuming that specific cellular plastic processes at the hierarchical
level of ocular dominance columns can only occur during asymp-
totic learning, longer training might be necessary when aiming to
influence rivalry perception. The idea that training cannot easily change imagery strength
might be explained by the manner in which imagery strength is
linked to brain anatomy. The Tartaglia study (Tartaglia et al.,2009)
had participants repetitively imagine the crucial part of a bisection
stimulus (spatial judgment) or a low-contrast Gabor pattern (con-
trast judgment). They found improved perceptual performance
on a subsequent perceptual bisection task and a Gabor detection
task after imagery training, and this improvement generalized to
untrained orientations. This lack of orientation specificity implies
that learning through imagery did not involve plastic changes in
early visual cortex, but probably involved higher-level extra-striate
areas. Higher-level changes may boost perceptual performance Recent research has demonstrated that perceptual learning can
also occur without a specific task and outside of awareness, as
long as the information of interest is paired with feedback or July 2012 | Volume 3 | Article 224 | 8 The training of imagery and metacognition Rademaker and Pearson a reward signal (Seitz and Watanabe, 2003, 2005; Seitz et al.,
2009) or with online-feedback via decoded fMRI signals (Shi-
bata et al., 2011). Our experimental design lacked a direct reward
signal. Perhaps if successful epochs of imagery were paired
with a reward signal, this could facilitate learning. In practice
the implementation of a reward may prove difficult to realize. Often, measures of imagery strength are dependent on subjec-
tive reports, and offering rewards based on only self-reports
could induce strong response and observer biases. DISCUSSION Considering the
orthogonal training orientations of our experiment, disruption of
consolidation seems an unlikely explanation for the ineffectiveness
of imagery training. Introspective or “metacognitive” sensitivity is important to
guide actions and to make decisions (Vickers, 1979; Daw et al.,
2005; Dayan and Daw, 2008) and being able to adequately esti-
mate ones confidence can help drive adaptive behavior (Kepecs
et al., 2008). In its simplest form, low confidence that a recent
decision was correct may prompt reexamination of the evidence,
or seeking a second-opinion. In the event of internally gener-
ated experiences such as mental imagery, low confidence that an
image was veridical and life-like may lead someone to recon-
sider such an experience. A better metacognitive understanding
may help the imaginer bridge the gap between first and third-
person perspective. For example, people can resolve potential
ambiguities about perception by comparing their own percep-
tual experience with the subjective experience of another person
(Bahrami et al., 2010). Similarly, when the imaginer has a better
understanding of the authenticity of his or her mental image, it
will be easier to communicate its content to another person. In
sum, increasing the efficiency with which people introspect the
quality of their mental images can prove a novel and important
finding. Can people become better at knowing their own thoughts? We
were able to improve subject’s ability to judge the vividness of their
imagery. This improvement was still present during a follow-up
test, implying a long lasting effect of training on metacognitive
evaluation of mental imagery. Furthermore, training of metacog-
nition was not orientation specific: metacognition was improved
for both trained and untrained sets of orientations. It is likely
that the improvement of metacognition reported here originates
from higher-level brain areas. This is in concordance with the
suspected high-level neural locus of metacognitive ability for per-
ception (Kepecs et al., 2008; Kiani and Shadlen, 2009; Fleming
et al., 2010) as well as the idea that networks in high-level cortical
regions orchestrate strategic choices during early learning, allocat-
ing attention and motivation in response to specific task demands
(Willingham, 1999; Hochstein and Ahissar, 2002; Doyon et al.,
2003). In conclusion, we discussed a variety of reasons why train-
ing did not lead to an improvement of imagery strength in the
current study. DISCUSSION Neverthe-
less, it is possible that our training was insufficient to obtain an
effect, and providing feedback, rewards, or some manner of get-
ting participants to intentionally try and increase their imagery
strength, could have been a more effective way to train mental
imagery. (Fleming et al., 2010), and there is a marked dissociation between
metacognitive ability and performance on visual perceptual tasks
(Lau and Passingham, 2006; Lau, 2008; Rounis et al., 2010; Song
et al., 2011). Thus, metacognitive ability can be viewed as a stable
and task independent cognitive process that can be improved with
practice, independent of performance on other tasks. Changes
in high-level neuronal populations are likely candidates for this
learning. The ability to introspect on private thoughts is key to human
subjective experience. Yet, people’s ability to evaluate internally
generated experiences – such as imagery – is not as self-evident as it
may appear. Although a large number of studies now demonstrate
that something as private as a mental image can be successfully
studied from a third-person perspective (Ishai and Sagi, 1995,
1997; Kosslyn et al., 2001; Pearson et al., 2008; Tartaglia et al.,
2009), research has only recently begun to tackle issues related
to the first-person perspective (Pearson et al., 2011). The core
problem from the first-person perspective of the imaginer is that
self-generated instances of imagery, unlike perception, cannot be
directly compared with a perceptual template. Nevertheless, peo-
ple seem quite capable of knowing if a mental image is accurate,
vivid, or detailed. And practice further improves this first-person
introspective ability. Why might such metacognitive knowledge be
important? During memory consolidation, initially fragile memory traces
become stabilized due to practice-induced plasticity in task rele-
vantbrainareas(Karni,1996;Dudai,2004).Cantheineffectiveness
of imagery training be due to somehow disrupted memory consol-
idation? Classically, consolidation has been defined as a time lim-
ited process directly following learning (Dudai, 2004). However,
recent studies indicate that interference is rather time indepen-
dent, and can occur at long intervals after training (Goedert and
Willingham,2002; Caithness et al.,2004; Zhang et al.,2008). Inter-
ference can be considered strongly stimulus dependent, resulting
from similarity between the learned and interfering stimulus, and
the corresponding neuronal populations recruited by these stimuli
(Seitz et al., 2005; Been et al., 2011). Specifically, for Gabor pat-
terns most interference occurs when interfering stimuli differ from
the learned orientation by 30˚, while no interference is observed
from orthogonal orientations (Been et al., 2011). REFERENCES correlates, computation and behav-
ioural impact of decision confi-
dence. Nature 455, 227–231. in the theory of signal detectability:
discrimination between correct and
incorrect decisions. Psychon. Bull. Rev. 10, 843–876. Daw, N. D., Niv, Y., and Dayan, P. (2005). Uncertainty-based competi-
tion between prefrontal and dorso-
lateral striatal systems for behavioral
control. Nat. Neurosci. 8,1704–1711. in the theory of signal detectability:
discrimination between correct and
incorrect decisions. Psychon. Bull. Rev. 10, 843–876. Ahissar, M., and Hochstein, S. (1993). Attentional control of early percep-
tual learning. Proc. Natl. Acad. Sci. U.S.A. 90, 5718–5722. Kiani, R., and Shadlen, M. N. (2009). Representation of confidence asso-
ciated with a decision by neurons
in the parietal cortex. Science 324,
759–764. Gilden, D., Blake, R., and Hurst, G. (1995). Neural adaptation of imag-
inary visual motion. Cogn. Psychol. 28, 1–16. Ahissar, M., and Hochstein, S. (1997). Task difficulty and the specificity
of perceptual learning. Nature 387,
401–406. Dayan, P., and Daw, N. D. (2008). Deci-
sion theory, reinforcement learning,
and the brain. Cogn. Affect. Behav. Neurosci. 8, 429–453. and the brain. Cogn. Affect. Behav. Neurosci. 8, 429–453. Kornbrot, D. E. (2006). Signal detec-
tion theory, the approach of choice:
model-based and distribution-free
measures and evaluation. Percept. Psychophys. 68, 393–414. Goebel, R., Khorram-Sefat, D., Muckli,
L., Hacker, H., and Singer, W. (1998). The constructive nature of
vision: direct evidence from func-
tional magnetic resonance imag-
ing studies of apparent motion and
motion imagery. Eur. J. Neurosci. 10,
1563–1573. Dosher, B. A., and Lu, Z. L. (1998). Perceptual learning reflects exter-
nal
noise
filtering
and
internal
noise reduction through channel
reweighting. Proc. Natl. Acad. Sci. U.S.A. 95, 13988–13993. Ahissar, M., and Hochstein, S. (2004). The
reverse
hierarchy
theory
of
visual
perceptual
learning. Trends Cogn. Sci. (Regul. Ed.) 8,
457–464. Kosslyn, S. M., Ganis, G., and Thomp-
son, W. L. (2001). Neural founda-
tions of imagery. Nat. Rev. Neurosci. 2, 635–642. Amedi, A., Malach, R., and Pascual-
Leone, A. (2005). Negative BOLD
differentiates visual imagery and
perception. Neuron 48, 859–872. Doyon, J., Penhune, V., and Ungerlei-
der, L. G. (2003). Distinct contri-
bution of the cortico-striatal and
cortico-cerebellar systems to motor
skill learning. Neuropsychologia 41,
252–262. Goedert, K. M., and Willingham, D. B. (2002). Patterns of interference in
sequence learning and prism adap-
tation inconsistent with the consol-
idation hypothesis. Learn. Mem. 9,
279–292. Kosslyn, S. M., Pascual-Leone, A., Feli-
cian, O., Camposano, S., Keenan,
J. REFERENCES P., Thompson, W. L., Ganis, G.,
Sukel, K. E., and Alpert, N. M. (1999). The role of area 17 in
visual imagery: convergent evidence
from PET and rTMS. Science 284,
167–170. Bahrami, B., Olsen, K., Latham, P. E.,
Roepstorff, A., Rees, G., and Frith,
C. D. (2010). Optimally interacting
minds. Science 329, 1081–1085. Driskell, J., Copper, C., and Moran,
A. (1994). Does mental practice
enhance performance? J. of Appl. Psychol. 79, 481–491. Harrison, S., and Tong, F. (2009). Decoding reveals the contents of
visual working memory in early
visual areas. Nature 458, 632–635. Been, M., Jans, B., and De Weerd, P. (2011). Time-limited consolidation
and task interference: no direct link. J. Neurosci. 31, 14944–14951. Kosslyn, S. M., Thompson, W. L., Kim,
I. J., and Alpert, N. M. (1995). Topo-
graphical representations of men-
tal images in primary visual cortex. Nature 378, 496–498. Dudai, Y. (2004). The neurobiology
of consolidations, or, how stable is
the engram? Annu. Rev. Psychol. 55,
51–86. Brainard, D. H. (1997). The psy-
chophysics toolbox. Spat. Vis. 10,
433–436. Hochstein, S., and Ahissar, M. (2002). View from the top: hierarchies and
reverse hierarchies in the visual sys-
tem. Neuron 36, 791–804. Dupuis-Roy,N.,and Gosselin,F. (2007). Perceptual learning without signal. Vision Res. 47, 349–356. Caithness, G., Osu, R., Bays, P., Chase,
H., Klassen, J., Kawato, M., Wolpert,
D. M., and Flanagan, J. R. (2004). Failure to consolidate the consolida-
tion theory of learning for sensori-
motor adaptation tasks. J. Neurosci. 24, 8662–8671. Kreiman, G., Koch, C., and Fried, I. (2000). Imagery neurons in the
human brain. Nature 408, 357–361. Hume, D. (1739). A Treatise on Human
Nature, Vol. 1. London: John Noon. Fahle, M., and Poggio, T. (2002). Percep-
tual Learning. Cambridge, MA: MIT
Press. Ishai, A., and Sagi, D. (1995). Common
mechanisms
of
visual
imagery
and
perception. Science
268,
1772–1774. Landsberger, H. A. (1958). Hawthorne
Revisited. Ithaca, NY: Cornell Uni-
versity. Feltz, D. L., and Landers, D. M. (2007). The Effects of Mental Practice on
Motor Skill Learning and Perfor-
mance:AMeta-analysis.Champaign,
IL: Human Kinetics. Chen,W.,Kato,T.,Zhu,X. H.,Ogawa,S.,
Tank, D. W., and Ugurbil, K. (1998). Human primary visual cortex and
lateral geniculate nucleus activation
during visual imagery. Neuroreport
9, 3669–3674. Ishai, A., and Sagi, D. (1997). Visual
imagery: effects of short-and long-
term memory. J. Cogn. Neurosci. 9,
734–742. Lau, H. C. (2008). A higher order
Bayesian decision theory of con-
sciousness. Prog. Brain Res. 168,
35–48. Lau, H. DISCUSSION Such an improvement may simply be very dif-
ficult to document, or our task may not have been optimally
suited to detect improvements of imagery strength. Neverthe-
less, we demonstrated that people’s ability to introspect their
own imagery strength does improve with training, which sug-
gests distinct mechanisms underlying imagery and metacognition. Being able to improve metacognition by means of practice can
have important implications for real-life situations. It would be
interesting to know if training metacognition could help people
improve certain cognitive functions, such as decision-making or Our study demonstrated improvements of the metacogni-
tion of imagery, whereas no changes in imagery strength itself
were observed. This dissociation suggests distinct brain mech-
anisms underlying metacognition and visual imagery respec-
tively. Similar distinctions have been made regarding metacogni-
tion of perception: Neuroanatomical substrates of introspective
ability are distinct from those supporting primary perception July 2012 | Volume 3 | Article 224 | 9 www.frontiersin.org www.frontiersin.org The training of imagery and metacognition Rademaker and Pearson ACKNOWLEDGMENTS ACKNOWLEDGMENTS planning actions. If so,this may prove especially helpful for specific
patient populations. Finally, future investigations of prolonged
training of imagery can prove advantageous in outlining the over-
lap between mechanisms of perception and imagery. Imagery as
defined here is a highly voluntary process that allows introspec-
tion in the absence of direct perceptual input. As such, imagery
can provide a unique gateway to understanding how perceptual
and introspective processes are represented in the brain. We thank Frank Tong for discussions and helpful input with design
and execution of this work. We also thank Jan Brascamp, Elias
Cohen, Janneke Jehee, Jan Schepers, and Jascha Swisher for helpful
comments and discussions regarding this research. This work was
funded by grants from the National Health and Medical Research
Council of Australia CJ Martin Fellowship 457146 and project
grant APP1024800 to Joel Pearson. REFERENCES in the theory of signal detectability:
discrimination between correct and
incorrect decisions. Psychon. Bull.
Rev. 10, 843–876. REFERENCES C., and Passingham, R. E. (2006).Relativeblindsightinnormal
observers and the neural correlate
of visual consciousness. Proc. Natl. Acad. Sci. U.S.A. 103, 18763–18768. Kamphuisen, A., Van Wezel, R., and
VanEe,R.(2007).Inter-oculartrans-
fer of stimulus cueing in dom-
inance selection at the onset of
binocular rivalry. Vision Res. 47,
1142–1144. Fine, I., and Jacobs, R. A. (2002). Com-
paring perceptual learning tasks: a
review. J. Vis. 2, 190–203. Chong, S., and Blake, R. (2006). Exoge-
nous
attention
and
endogenous
attention influence initial domi-
nance in binocular rivalry. Vision
Res. 46, 1794–1803. Flavell, J. H. (1979). Metacognition and
cognitive monitoring: a new area
of cognitive-developmental inquiry. Am. Psychol. 34, 906–911. Macmillan, N. A., and Creelman, C. D. (1991). Detection Theory: A User’s
Guide. Cambridge: Cambridge Uni-
versity Press. Karni,A. (1996). The acquisition of per-
ceptual and motor skills: a mem-
ory system in the adult human cor-
tex. Brain Res. Cogn. Brain Res. 5,
39–48. Craver-Lemley,
C.,
and
Reeves, A. (1992). How visual imagery inter-
feres with vision. Psychol. Rev. 99,
633–649. Fleming, S. M., Weil, R. S., Nagy, Z.,
Dolan, R. J., and Rees, G. (2010). Relating introspective accuracy to
individual differences in brain struc-
ture. Science 329, 1541–1543. Marks, D. F. (1973). Visual imagery dif-
ferences in the recall of pictures. Br. J. Psychol. 64, 17–24. Cui, X., Jeter, C. B., Yang, D., Mon-
tague, P. R., and Eagleman, D. M. (2007).Vividnessof mentalimagery:
individual variability can be mea-
sured objectively. Vision Res. 47,
474–478. Keogh, R., and Pearson, J. (2011). Mental imagery and visual work-
ing memory. PLoS ONE 6, e29221. doi:10.1371/journal.pone.0029221 Galton, F. (1883). Inquiries into Human
Faculty and its Development. Lon-
don: Macmillan. McDermott, K. B., and Roediger, H. L. (1994). Effects of imagery on
perceptual implicit memory tests. J. Exp. Psychol. Learn Mem. Cogn. 20,
1379–1390. g
y
,
doi:10.1371/journal.pone.0029221 Galvin, S. J., Podd, J. V., Drga, V., and
Whitmore, J. (2003). Type 2 tasks Kepecs, A., Uchida, N., Zariwala, H. A.,
and Mainen, Z. F. (2008). Neural Frontiers in Psychology | Perception Science July 2012 | Volume 3 | Article 224 | 10 The training of imagery and metacognition Rademaker and Pearson Willingham, D. B. (1999). Implicit
motor sequence learning is not
purely perceptual. Mem. Cognit. 27,
561–572. Slotnick, S. D. (2008). Imagery: mental
pictures disrupt perceptual rivalry. Curr. Biol. 18, R603–R605. McKellar, P. (1965). The Investigation
of Mental Images. Harmondsworth:
Penguin Books. Rounis, E., Maniscalco, B., Rothwell,
J. REFERENCES C., Passingham, R. E., and Lau,
H. (2010). Theta-burst transcranial
magnetic stimulation to the pre-
frontal cortex impairs metacognitive
visual awareness. Cogn. Neurosci. 1,
165–175. Meng, M., and Tong, F. (2004). Can
attention selectively bias bistable
perception? Differences
between
binocular rivalry and ambiguous
figures. J. Vis. 4, 539–551. Slotnick, S. D., Thompson, W. L.,
and Kosslyn, S. M. (2005). Visual
mental
imagery
induces
retino-
topically organized activation of
early visual areas. Cereb. Cortex 15,
1570–1583. Zatorre,R. J.,and Halpern,A. R. (2005). Mental concerts: musical imagery
and auditory cortex. Neuron 47,
9–12. Sacks, O. (2010). The Mind’s Eye. New
York: Alfred A. Knopf. Mitchell, J., Stoner, G., and Reynolds,
J. (2004). Object-based attention
determines dominance in binocular
rivalry. Nature 429, 410–413. Zhang, J.-Y., Kuai, S.-G., Xiao, L.-
Q., Klein, S. A., Levi, D. M., and
Yu, C. (2008). Stimulus coding
rules for perceptual learning. PLoS
Biol. 6, e197. doi:10.1371/journal. pbio.0060197 Song, C., Kanai, R., Fleming, S. M.,Weil,
R. S., Schwarzkopf, D. S., and Rees,
G. (2011). Relating inter-individual
differences in metacognitive perfor-
mance on different perceptual tasks. Conscious. Cogn. 20, 1787–1792. Sasaki,Y., Nanez, J. E., and Watanabe, T. (2010). Advances in visual percep-
tual learning and plasticity. Nat. Rev. Neurosci. 11, 53–60. O’Craven,K.,and Kanwisher,N. (2000). Mental imagery of faces and places
activates corresponding stimulus-
specific brain regions. J. Cogn. Neu-
rosci. 12, 1013–1023. Seitz, A., and Watanabe, T. (2005). A
unified model for perceptual learn-
ing. Trends Cogn. Sci. (Regul. Ed.) 9,
329–334. Conflict of Interest Statement: The
authors declare that the research was
conducted in the absence of any com-
mercial or financial relationships that
could be construed as a potential con-
flict of interest. Stokes, M., Thompson, R., Cusack, R.,
and Duncan, J. (2009). Top-down
activation of shape-specific popu-
lation codes in visual cortex dur-
ing mental imagery. J. Neurosci. 29,
1565–1572. Pearson, J., and Brascamp, J. (2008). Sensory memory for ambiguous
vision. Trends Cogn. Sci. (Regul. Ed.)
12, 334–341. Seitz, A. R., Kim, D., and Watanabe,
T. (2009). Rewards evoke learning
of unconsciously processed visual
stimuli in adult humans. Neuron 61,
700–707. Pearson, J., Clifford, C., and Tong, F. (2008). The
functional
impact
of
mental
imagery
on
con-
scious perception. Curr. Biol. 18,
982–986. Swets, J. A. (1986). Form of empirical
ROCs in discrimination and diag-
nostic tasks: implications for theory
and measurement of performance. Psychol. Bull. 99, 181–198. July 2012 | Volume 3 | Article 224 | 11 REFERENCES Received: 27 April 2012; accepted: 16 June
2012; published online: 10 July 2012. Seitz, A. R., and Watanabe, T. (2003). Psychophysics: is subliminal learn-
ing really passive? Nature 422, 36. Received: 27 April 2012; accepted: 16 June
2012; published online: 10 July 2012. Citation: Rademaker RL and Pearson J
(2012)Training visual imagery: improve-
ments of metacognition, but not imagery
strength. Front. Psychology 3:224. doi:
10.3389/fpsyg.2012.00224 Citation: Rademaker RL and Pearson J
(2012)Training visual imagery: improve-
ments of metacognition, but not imagery
strength. Front. Psychology 3:224. doi:
10.3389/fpsyg.2012.00224 Seitz, A. R., Yamagishi, N., Werner,
B., Goda, N., Kawato, M., and
Watanabe, T. (2005). Task-specific
disruption of perceptual learning. Proc. Natl. Acad. Sci. U.S.A. 102,
14895–14900. Pearson, J., Rademaker, R. L., and Tong,
F. (2011). Evaluating the mind’s eye:
the metacognition of visual imagery. Psychol. Sci. 22, 1535–1542. Tartaglia, E. M., Bamert, L., Mast, F. W., and Herzog, M. H. (2009). Human
perceptual
learning
by
mental imagery. Curr. Biol. 19,
2081–2085. This article was submitted to Frontiers in
PerceptionScience,aspecialtyof Frontiers
in Psychology. Pelli, D. G. (1997). The VideoToolbox
software for visual psychophysics:
transforming numbers into movies. Spat. Vis. 10, 437–442. Vickers, D. (1979). Decision Processes
in Visual Perception. New York, NY:
Academic Press. Serences, J. T., Ester, E. F., Vogel, E. K., and Awh, E. (2009). Stimulus-
specific delay activity in human pri-
mary visual cortex. Psychol. Sci. 20,
207–214. Copyright © 2012 Rademaker and Pear-
son. This is an open-access article distrib-
utedunderthetermsof theCreativeCom-
mons Attribution License, which per-
mits use, distribution and reproduction
in other forums, provided the original
authors and source are credited and sub-
ject to any copyright notices concerning
any third-party graphics etc. Perky, C. (1910). An experimental study
of imagination?. Am. J. Psychol. 21,
422–452. Weiss, T., Hansen, E., Rost, R., Beyer,
L., Merten, F., Nichelmann, C., and
Zippel, C. (1994). Mental practice of
motor skills used in poststroke reha-
bilitation has own effects on central
nervous activation. Int. J. Neurosci. 78, 157–166. Shibata, K., Watanabe, T., Sasaki,Y., and
Kawato,M. (2011). Perceptual learn-
ing incepted by decoded fMRI neu-
rofeedbackwithoutstimuluspresen-
tation. Science 334, 1413–1415. Pylyshyn, Z. (2003). Return of the men-
tal image: are there really pictures in
the brain? Trends Cogn. Sci. (Regul. Ed.) 7, 113–118. July 2012 | Volume 3 | Article 224 | 11 www.frontiersin.org www.frontiersin.org
|
https://openalex.org/W4285039724
|
https://iris.unige.it/bitstream/11567/1093673/1/%5bJ11%5dIEEEACCESS2022.pdf
|
English
| null |
Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks
|
IEEE access
| 2,022
|
cc-by
| 16,158
|
Two-Stage Multiclass Modeling Approach for
Intermodal Rail-Road Transport Networks CECILIA PASQUALE
, (Member, IEEE), ENRICO SIRI
, (Member, IEEE),
SILVIA SIRI
, (Member, IEEE), AND SIMONA SACONE, (Member, IEEE)
Department of Informatics, Bioengineering, Robotics and Systems Engineering, University of Genoa, 16145 Genoa, Italy
Corresponding author: Cecilia Pasquale (cecilia.pasquale@edu.unige.it) CECILIA PASQUALE
, (Member, IEEE), ENRICO SIRI
, (Member, IEEE),
SILVIA SIRI
, (Member, IEEE), AND SIMONA SACONE, (Member, IEEE)
Department of Informatics, Bioengineering, Robotics and Systems Engineering, University of Genoa, 16145 Genoa, Italy
Corresponding author: Cecilia Pasquale (cecilia.pasquale@edu.unige.it) This work was supported by the European Union via the Structural Reform Support Program and implemented by the Centro Italiano di
Eccellenza sulla Logistica i Trasporti e le Infrastrutture (CIELI)—University of Genoa (UniGe), in cooperation with the European
Commission, under Project ‘‘New Economic Regulation for Transport in Case of Emergency Events.’’ ABSTRACT In this paper, a two-stage modeling framework is proposed to represent the route choices
and dynamics of two classes of traffic flows, namely passengers and freight, in a large-scale intermodal
transportation network. More in details, the transport modes considered in this model are road transport,
represented through a highway network, and rail transport. The methodology adopted in this work relies on
a multi-class intermodal assignment model combined with a multi-class intermodal dynamic model. The
proposed modeling scheme can be adopted for different purposes, such as to support decision makers who
intend to utilize the full mobility capacity of a geographical area by allocating the mobility demand on
different modes or by suggesting intermodal itineraries also when the network is affected by disruptive
events. The proposed framework has been tested on a benchmark network with the aim of showing the
mutual relations that occur between different modes of transport in case a connection of the intermodal
network fails. Specifically, the analysis reported in the paper shows that the collapse of a railway arc can
cause a high increase in travel times for some highway arcs, which result 30-40% higher than in the pre-
disruption scenario, and an even greater growth in the average occupancy of many arcs. INDEX TERMS Assignment model, dynamic discrete-time model, intermodal rail-road transport network,
freight transport. INDEX TERMS Assignment model, dynamic discrete-time model, intermodal rail-road transport network,
freight transport. is work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLUME 10, 2022
This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons Received 28 June 2022, accepted 6 July 2022, date of publication 11 July 2022, date of current version 18 July 2022. Received 28 June 2022, accepted 6 July 2022, date of publication 11 July 2022, date of current version 18 July 2022. Digital Object Identifier 10.1109/ACCESS.2022.3189775 I. INTRODUCTION of the infrastructure network layout due to the verification of
disruptive events. The need to move passengers and freight is becoming increas-
ingly pervasive in conducting daily human activities and
more generally in the social and economic advancement of
countries. As a result, the availability and accessibility of
transport infrastructures and mobility services allow this need
to be met and, at the same time, increase the attractiveness of
an area. In this regard, it is of utmost importance to quantify
the capacity necessary to satisfy the transport demand of a
geographical area and to try to predict how the transport sys-
tems included in it will react to unforeseen events. These latter
may appear in different forms as changes in demand, e.g. the
transfer of passengers from public to private transport that
have occurred due to the ongoing pandemic, or modification Model-based approaches are the foremost methodologies
for evaluating the performance of transportation networks
because they allow: • to quantify the efficiency of transport networks subject
to different scenarios through the calculation of perfor-
mance indexes (e.g., average travel times, fuel consump-
tion, pollutant emissions, etc.); • to evaluate the effects produced by the occurrence of
critical events (e.g., infrastructure collapse, natural dis-
asters limiting the functionality of transport systems,
terrorist attacks, etc.); • to evaluate the effects produced by the introduction of
new systems (e.g., new infrastructure, modification of
existing layout, etc.); The associate editor coordinating the review of this manuscript and
approving it for publication was Wen-Sheng Zhao
. • to develop or test regulation policies. 73583 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks dynamic model is improved. As a consequence, the results
reported and discussed in this paper are completely new. Our work is a model-based approach which relies on
the combination of a traffic assignment model with a
discrete-time dynamic model used for simulation. In par-
ticular, the assignment model is adopted to represent the
route choices of users and to evaluate, in a static way, the
distribution of flows on the network. The simulative model,
instead, is used to represent the impact of the route choices
on the transport network by describing the flow evolution
over time. I. INTRODUCTION By analyzing the literature on these topics, it is
possible to observe that most of the researches’ efforts have
been focused so far on the definition of assignment and sim-
ulative models on mono-modal transport networks, i.e. road
networks or railway networks only (see for instance [1]–[3]). However, if the goal is to analyze the accessibility of a given
geographic area or to give better information to users, it is
necessary to adopt more extensive models which consider
the intermodal transport network as a whole, by properly
modeling the different modes of transport and the possibility
of transfer among them. To summarize, the modeling framework proposed in this
work is composed of two stages (see Fig. 1): • a multi-class intermodal rail-road assignment model; • a multi-class intermodal rail-road assignment model; • a multi-class intermodal rail-road discrete-time dynamic
model; and has the following goals: • to represent the route choices and the dynamic behavior
of traffic flows in an intermodal rail-road network; • to explicitly capture the behavior of passengers and
freight in the network; • to allow performance analysis of the intermodal network
in different scenarios. The present paper is organized as follows. A literature
review on intermodal transport network models is presented
in Section II. In Section III the general features of the pro-
posed modeling framework and the basic notation are intro-
duced. Then, the proposed two-stage modeling approach is
presented: the assignment model is outlined in Section IV,
while the discrete-time dynamic model is described in
Section V. The application of the proposed methodology to a
test network is shown in Section VI, while some conclusive
remarks are gathered in Section VII. Another aspect that cannot be overlooked in the analysis
of transport networks is that, as in all critical infrastruc-
ture systems, the interdependencies increase the potential for
cascading failures [4]. Referring specifically to intermodal
transport networks [5], the interdependence among different
transport modes is particularly relevant. It can be related
to pursue the transport activity itself, in this case several
transport services operate synergistically to allow the sat-
isfaction of the mobility demand of users and the efficient
distribution of goods and services. Interdependence can be
related to physical reasons such as the overlap of different
routes, even belonging to different transport modes, through
bridges or tunnels, or to hidden relationships, such as those
analyzed in [6]. I. INTRODUCTION Regardless of how these interdependencies
occur, they imply an increased vulnerability of the transport
network as a whole. In fact, it is possible that critical events
affecting even one mode of transport could cause a ripple
effect involving other transport modalities or, in the worst
case, the entire transport network. For these reasons, the
development of intermodal models represents an important
step in the analysis of complex and highly interdependent
systems such as transport networks. II. LITERATURE REVIEW II. LITERATURE REVIEW Sketch of the two-stage modeling framework. the network, the time-dependent transport times on roads, and
the time schedules for trains and barges. When dealing with
freight intermodal networks, it is worth noting that the system
performance must include different factors, such as costs,
times, flexibility, reliability, quality and sustainability [32]
and, then, multi-criteria analysis can be effectively applied
in this context (see e.g. [33], [34]). freight flows. In particular, intermodal traffic assignment has
been studied for some decades [20]–[23], by
considering
the so-called hypernetworks, which allow to include both
links representing a portion of the real intermodal transport
network (road, rail, private or public transport) and links
associated with users’ decisions. Intermodal traffic assign-
ment approaches have also been studied more recently, e.g. in [24] the intermodal transport network of an urban area
is represented as a graph composed of a number of sub-
networks, each of which is associated with a transportation
mode. In that work, the intermodal network is translated
into an augmented-state network in which transfer rules and
transfer probabilities between the different transport modes
are defined. In [25], a stochastic model is proposed in order
to account for the reliability of the chosen transport modes
and paths, while in [26] different modes are considered, only-
driving, carpooling, ride-hailing, public transit and park-and-
ride, for a urban transportation system including private cars,
freight trucks, buses, and so on. freight flows. In particular, intermodal traffic assignment has
been studied for some decades [20]–[23], by
considering
the so-called hypernetworks, which allow to include both
links representing a portion of the real intermodal transport
network (road, rail, private or public transport) and links
associated with users’ decisions. Intermodal traffic assign-
ment approaches have also been studied more recently, e.g. in [24] the intermodal transport network of an urban area
is represented as a graph composed of a number of sub-
networks, each of which is associated with a transportation
mode. In that work, the intermodal network is translated
into an augmented-state network in which transfer rules and
transfer probabilities between the different transport modes
are defined. In [25], a stochastic model is proposed in order
to account for the reliability of the chosen transport modes
and paths, while in [26] different modes are considered, only-
driving, carpooling, ride-hailing, public transit and park-and-
ride, for a urban transportation system including private cars,
freight trucks, buses, and so on. II. LITERATURE REVIEW On the basis of the related literature review reported above,
we can highlight that the main novelties and contributions of
this work stand in: • combining a traffic assignment model with a simulative
model; • capturing the travel choice and dynamic behavior of
passengers and freight jointly in an intermodal network. • representing a large-scale transport network covering a
regional territory. III. GENERAL FEATURES AND BASIC NOTATION As shown in Fig. 1, the proposed modeling scheme consists
of two stages: an assignment model defined to allocate the
demand of passengers and freight on a intermodal transport
network, and a discrete-time dynamic model that allows to
replicate the evolution of the system over time. It is worth
noting that the discrete-time dynamic model receives as input
the mobility demand and the path choices (route and modal
choices) defined through the intermodal assignment model. Simulation-based approaches are either referred to passen-
gers or freight separately. For instance, [27]–[29] provide
simulative approaches for representing the choices of passen-
gers in urban intermodal networks. In [27], three transport
modes are considered, i.e. private vehicle transport, public
vehicle transport and pedestrian transport, all represented
through a three-dimensional macroscopic fundamental dia-
gram (MFD) that allows to evaluate the accumulation of
private and public vehicles according to different traffic
scenarios. In [28], the considered transport modes are private
vehicle transport and public road transport: private trans-
portation is represented through the MFD, while constant
transfer speeds are considered for public transportation. The
derived intermodal model is used for the definition of adap-
tive pricing policies. The work presented in [29] proposes
an agent-based simulation tool to test innovative planning
methods involving pedestrians and autonomous shuttles in
large urban areas. Regarding instead freight logistics, [30]
reports a very detailed survey about simulation approaches
for intermodal freight transportation systems, used to repre-
sent stakeholders, decision makers, operations, and planning
activities. In [31], a dynamic model for an intermodal freight
transport network is proposed to represent the mode changes
at intermodal terminals, the physical capacity constraints of Both the assignment model and the dynamic model are
based on a regional intermodal transport network in which the
considered transport modes are road transport, represented
by a highway network, and rail transport. The two modes
of transport are connected with some intermodal arcs distin-
guished depending on the flow class, i.e., passengers or cargo
units, which they can receive. The rail-road intermodal trans-
port network is represented by means of an oriented graph,
as depicted in Fig. 2, denoted with G = (N, A), in which N
indicates the set of nodes, whereas A = AH ∪AR∪AIp∪AIf
represents the set of arcs. II. LITERATURE REVIEW Intermodal transport networks have been studied extensively
by operations researchers with the purpose of taking deci-
sions referred to freight transport operations. The related
planning problems are normally distinguished in strategic,
tactical and operational ones, according to the length of the
planning horizon (more details and classifications can be
found in the review papers [9]–[11]). Location decisions are
probably the most representative examples of strategic plan-
ning problems [12] and regard the definition of the optimal
positioning of nodes of the intermodal network (see e.g. [13]–[15]). Tactical and operational decisions in intermodal
networks regard medium-short term horizons and address
different types of problems, such as, for example, the ser-
vice network design [16], [17] or the selection of the best
routes [18], [19]. This work, based on preliminary papers [7] and [8], falls in
this field of research by representing large-scale transporta-
tion networks in which the considered transport modes are
road and rail connected to each other through appropriate
intermodal connections. Another peculiarity of this modeling
framework, both in the assignment and in the dynamic model,
is that the user demand is multi-class, hence it is distinguished
into passengers and cargo units (e.g., containers). This dis-
tinction is of particular importance because passenger and
freight flows may be characterized by different behaviors
and may be subject to different restrictions, such as on route
choices. Note that, compared with [7] and [8], in the present
work the assignment model is multi-class (while in the previ-
ous versions there was no distinction of user classes) and the Differently from these latter papers, in which the goal is
to design and organize activities in an intermodal freight net-
work, in this work we aim at defining a modeling framework
for representing the spontaneous behavior of both freight and
passengers in an intermodal network, not only in terms of
alternative path choices but also including their dynamics. Hence, the scientific literature considered as a reference for
this work is associated, firstly, with traffic assignment models
and, secondly, with simulation-based approaches for inter-
modal networks. The majority of the works addressing assignment mod-
els for intermodal transport networks are referred to urban
areas and consider the choices of passengers rather than 73584 VOLUME 10, 2022 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks FIGURE 1. Sketch of the two-stage modeling framework. FIGURE 1. IV. THE INTERMODAL ASSIGNMENT MODEL FOR
PASSENGER AND FREIGHT FLOWS The purpose of the intermodal assignment model is to rep-
resent the spontaneous decisions of users about their routes. The purpose of the intermodal assignment model is to rep-
resent the spontaneous decisions of users about their routes. Before providing the details of the proposed approach, it is
important to emphasize that the assignment model is a static
approach in which all the mobility demand, estimated for a
specific time window, is assumed to use the transport network
simultaneously. As shown in Fig. 1, the result of this model is
the redistribution of flows on the paths, i.e., f od,1
l
for passen-
gers and f od,2
l
for freight, which are obtained by the solution
of some optimization problems. The flows thus obtained will
be suitably transformed into splitting rates, see Section V, and
used as input data for the dynamic model. FIGURE 2. Sketch of the intermodal transport network. Intermodal arcs are distinguished for passengers and for
freight because modal shifts for passengers and freight typi-
cally occur at different locations and in different ways. Let P(i) indicate the set of nodes preceding node i and S(i)
the set of nodes succeeding node i, i ∈N. The network
is defined in an origin-destination-oriented mode in which
JO ⊆N represents the set of all possible origin nodes,
JD ⊆N represents the set of all possible destination nodes. The nodes which are not origins nor destinations simply allow
the transit between successive arcs. The intermodal arcs, for
both flow classes, are considered as fictitious arcs that allow
modal transfers between road transport and rail transport and
vice versa. For this reason, an origin node o ∈JO cannot be
followed by intermodal arcs, and similarly, a destination node
d ∈JD cannot be preceded by intermodal arcs. The passenger and freight assignment models are treated
separately, firstly introducing the intermodal assignment
model for passengers and, then, applying the intermodal
assignment model for freight, since the latter uses the results
of the passenger assignment to define the freight route
choices. In particular, for the passenger assignment procedure
we assume that the marginal impact on the network due to
the presence of freight is sufficiently small so that it does
not decisively influence the behavior of passengers. IV. THE INTERMODAL ASSIGNMENT MODEL FOR
PASSENGER AND FREIGHT FLOWS This
assumption is quite reasonable since, referring to the overall
flows on a transport network, the freight component typically
constitutes a rather low percentage compared with the flow
of passengers. In the proposed approach the flows of users are distin-
guished in passengers and freight, with superscript c denoting
the class of users. In particular we define with c = 1 the
passenger flow and with c = 2 the freight flow. Then, Dod,c
indicates the demand of class c that originates at node o ∈
JO and has destination at node d ∈JD: this is the total
demand associated with the od pair for the whole simulation
horizon. This demand defined for all od pairs represents the
so-called ‘‘origin-destination matrix’’. Let us also specify that
the passenger demand, i.e., the demand for class c = 1,
is expressed in number of passengers, while the demand of
freight, corresponding to c = 2, is expressed in number of
cargo units. The assignment procedure presented below is conducted
in an iterative manner in which at the first step the inter-
modal assignment model for passenger flows described in
Section IV-A is run. Then the results of this assignment are
used to estimate the average passenger travel times on the
network using the dynamic model described in Section V. Finally, the intermodal freight assignment model, presented
in Section IV-B, is run using the average travel times defined
in the previous step. The main parameters and variables of the proposed
approach are summarized in Tables 1-3. Specifically, Table 1
collects all the parameters that are used in both the assignment
model and the dynamic simulation model. Table 2 collects the
parameters and variables used in the assignment model only,
while Table 3 reports parameters and variables used in the
dynamic simulation model only. III. GENERAL FEATURES AND BASIC NOTATION Each subset of arcs is defined as
follows: • AH is the set of highway connections; • AR is the set of railway connections; • AIp is the set of intermodal arcs for passengers;
If • AIf is the set of intermodal arcs for freight. 73585 73585 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks FIGURE 2. Sketch of the intermodal transport network. (i, j) ∈AH ∪AR, the following condition: (i, j) ∈AH ∪AR, the following condition: (i, j) ∈AH ∪AR, the following condition: T ≤min
1i,j
vH
i,j
, 1i,j
vR
i,j
(1) (1) A. THE INTERMODAL ASSIGNMENT MODEL FOR
PASSENGER FLOWS Modeling the behavior and therefore the mobility choices of
users is a challenging problem for which several approaches
have been developed by researchers. One of the most
accepted, and widely adopted, methodologies concerns the
use of traffic assignment models. Given an origin-destination
matrix, representing the users’ demand, and knowing the
functional characteristics of the network infrastructure, a traf-
fic assignment model allows to estimate how users will
be dispersed in the network, taking into account the rela-
tion between the infrastructure supply and the interaction of
users mutual choices. The criteria underlying the mathemat-
ical representation of such behaviors can vary considerably As it will be described in Section V, the dynamic behavior
of the whole network is represented with a discrete-time
model in which the time horizon is divided in K time steps,
where k = 1, . . . , K indicates the temporal stage, and T [h]
represents the sample time interval. In order to ensure a cor-
rect time discretization, the length of the time interval T must
allow a proper dynamic evolution of the system, therefore the
length of the time step is chosen in order to fulfil, for any arc 73586 VOLUME 10, 2022 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks TABLE 1. Parameters common to the two models in the two-stage modeling framework. TABLE 1. Parameters common to the two models in the two-stage modeling framework. TABLE 2. Parameters and variables present only in the assignment model described in Section IV. TABLE 2. Parameters and variables present only in the assignment model described in Section IV. depending on the assignment model used. A comprehensive
review of traffic assignment models is given in [35]. at a given time minimizes their own travel time’’. A way
to compute the arc flows corresponding to such equilibrium
involves finding the optimum solution of the Beckmann’s
Transformation [37]. The N-Path Restricted variant of the
aforementioned model [38] is obtained by constraining the
assignment to a priori defined sets of admissible paths, which
do not necessarily include all the possible ones for each
origin-destination pair. This allows to avoid all the paths that
are theoretically possible but quite implausible in practice. In this paper, all the paths involving more than one modal depending on the assignment model used. A comprehensive
review of traffic assignment models is given in [35]. A. THE INTERMODAL ASSIGNMENT MODEL FOR
PASSENGER FLOWS In
the
present
work,
the
N-Path
Restricted
User-
Equilibrium traffic assignment model has been applied. Generally speaking, the User Equilibrium traffic assignment
model aims to estimate the network equilibrium state such
that no user has unilaterally any interest in choosing an
alternative path, since no other path would guarantee lower
travel times. Such equilibrium is achieved if Wardrop’s first
principle [36] is met, whereby ‘‘users choose the path that In
the
present
work,
the
N-Path
Restricted
User-
Equilibrium traffic assignment model has been applied. Generally speaking, the User Equilibrium traffic assignment
model aims to estimate the network equilibrium state such
that no user has unilaterally any interest in choosing an
alternative path, since no other path would guarantee lower
travel times. Such equilibrium is achieved if Wardrop’s first
principle [36] is met, whereby ‘‘users choose the path that VOLUME 10, 2022 73587 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks ABLE 3. Parameters and variables present only in the dynamic model described in Section V. TABLE 3. Parameters and variables present only in the dynamic model described in Section V. flows. Moreover, constraints (5) define the relation among
f od,1
l
and x1
i,j by means of the path-arc incidence matrix whose
elements are defined as follows: shift have been excluded. Then, denoted with l, l = 1, . . . , L,
a generic path existing in the network and with Lod the set of
all possible paths connecting the od pair, let Pod ⊆Lod be
the set of admissible paths from origin node o to destination
node d. The resulting optimization problem for the traffic
assignment of passengers demand in the intermodal transport
network is the following. δod,1
i,j,l =
(
1
if (i, j) belongs to path l from o to d
0
otherwise
(6) δod,1
i,j,l =
(
1
if (i, j) belongs to path l from o to d
0
otherwise
(6) (6) In (2), the terms τ 1
i,j(·) are the performance functions of
arcs related to class c = 1, i.e., passengers. for (i, j) ∈AR. for (i, j) ∈AR. for (i, j) ∈AR. min y(x) =
X
(i,j)∈A
x2
i,j · ci,j(x2
i,j)
(13) (13) Finally, as mentioned above, the performance functions
of intermodal arcs are constant functions, but it has been
necessary to make them strictly increasing by introducing
a (although very small) relation of direct proportionality
between travel time and number of users on the arc in order to
guarantee the uniqueness of the solution of the optimization
problem, as detailed further on. subject to subject to jX
l∈Pod
f od,2
l
= Dod,2
o ∈JO, d ∈JD
(14)
f od,2
l
≥0
o ∈JO, d ∈JD, l ∈Pod
(15)
x2
i,j =
X
o∈JO
X
d∈JD
X
l∈Pod,2
f od,2
l
· δod,2
i,j,l
(i, j) ∈A
(16) (14) The performance functions for intermodal arcs are then
defined as follows τ 1
i,j(x1
i,j) = αi,j · T + 1
M x1
i,j
(9) (9) where f od,2
l
and x2
i,j are, respectively, the flow of class c =
2 of od pair using path l and the total flow of freight on
arc (i, j). Constraints (14)-(16) are analogous to constraints
(3)-(5) included in Problem 1, and where δod,2
i,j,l is defined as
follows for (i, j) ∈AIp, where αi,j ≥1 and M is a positive coefficient
sufficiently big such that the impact of the number of users on
the performance of an intermodal arc is negligible compared
to the other types of arcs. δod,2
i,j,l =
(
1
if (i, j) belongs to path l from o to d
0
otherwise
(17) By defining the performance functions of the arcs as above,
it can be proven that Problem 1 is strictly convex on a convex
domain and therefore admits a unique optimal solution with
respect to variables x1
i,j but not for variables f od,1
l
. Since
f od,1
l
, properly converted into splitting rates, represent the
input of the dynamic model, their uniqueness is an essen-
tial requirement in this work. Several methodologies have
been developed to overcome this issue associated with the
User-Equilibrium model [39]. A. THE INTERMODAL ASSIGNMENT MODEL FOR
PASSENGER FLOWS Problem 2: Problem 2: min h(f ) =
X
o∈JO
X
d∈JD
X
l∈Pod
f od,1
l
· ln(f od,1
l
)
(10)
subject to
X
l∈Pod
f od,1
l
= Dod,1
o ∈JO, d ∈JD
(11)
x1,UE
i,j
=
X
o∈JO
X
d∈JD
X
l∈Pod
f od,1
l
· δod,1
i,j,l
(i, j) ∈A
(12) min h(f ) =
X
o∈JO
X
d∈JD
X
l∈Pod
f od,1
l
· ln(f od,1
l
)
(10) (10) In order to reduce the computational effort, appropriate
linear versions of the same functions have been used within
the assignment process. In the case of highway arcs, the
resulting linear functions are obtained by interpolating two
points: the first is obtained using the free-flow travel time
corresponding to an arc completely empty, while the other
uses the value assumed by the hyperbolic function when the
number of users is equal to φ · nmax
i,j , where φ ∈[0, 1). The linear performance functions in the highway case are
therefore defined as follows subject to subject to jX
l∈Pod
f od,1
l
= Dod,1
o ∈JO, d ∈JD
(11)
x1,UE
i,j
=
X
o∈JO
X
d∈JD
X
l∈Pod
f od,1
l
· δod,1
i,j,l
(i, j) ∈A
(12) (11) Equations (11) – (12) convey the same constraints of
Problem 1 with the only but fundamental difference that the
flows on the arcs are now fixed and equal to x1,UE
i,j
, obtained
as solution of Problem 1. Also, the non-negativity constraint
(4) is now implicit in the fact that f od,1
l
appear in (10) as the
argument of a logarithm. τ 1
i,j(x1
i,j) =
1i,j
ωi,jnmax
i,j (1 −φ) · x1
i,j
1
η + 1i,j
vH
i,j
(7) (7) for (i, j) ∈AH. Similarly, for the railway case, the second interpolating
point is associated with the value assumed by the hyperbolic
function when the number of users reaches the technical limit
Cp1i,j
smin
i,j
(see (39)). The following linear performance function
for the railway case is therefore obtained: B. THE INTERMODAL ASSIGNMENT MODEL FOR
FREIGHT FLOWS In this section, the intermodal assignment model for freight
flows is presented. The purpose of this assignment model is to
replicate the average freight route choices by considering as
objective the minimization of the total travel costs needed to
satisfy a given demand. Given these premises, the intermodal
assignment model for freight is given as follows τ 1
i,j(x1
i,j) =
hi,jsmin
i,j
(smin
i,j −L) ·
x1
i,j
Cp + 1i,j
vR
i,j
(8) (8) g
g
g
Problem 3: Problem 3: Problem 3: A. THE INTERMODAL ASSIGNMENT MODEL FOR
PASSENGER FLOWS These functions,
representative of the functional characteristics of the network
arcs, express the relation between the travel time spent by
passengers traveling through an arc and the amount of con-
gestion on the same arc, which is expected to perform worse
(and thus resulting in increased travel times) if the number
of users traveling on it increases. The performance functions
adopted for this model are derived from (37), with c = 1,
introduced in Section V, considering the definitions provided
in (38) and (39). For highway and railway arcs, this equation
computes the transit time as a function of the estimated
speed. The average speed on these arcs, in turn, depends on
the number of users who are using those arcs. Regarding
intermodal passenger arcs, on the other hand, the transfer time
is considered constant and independent of the number of users
present on it. For further information the reader is referred to Problem 1: Problem 1: min z(x) =
X
(i,j)∈A
x1
i,j
Z
0
τ 1
i,j(ω)dω
(2) (2) subject to subject to jX
l∈Pod
f od,1
l
= Dod,1
o ∈JO, d ∈JD
(3)
f od,1
l
≥0
o ∈JO, d ∈JD, l ∈Pod
(4)
x1
i,j =
X
o∈JO
X
d∈JD
X
l∈Pod
f od,1
l
· δod,1
i,j,l
(i, j) ∈A
(5) (3) where f od,1
l
and x1
i,j are, respectively, the flow of class 1 of od
pair using path l and the total flow of class 1 on arc (i, j). Con-
straints (3) imply passengers’ demand satisfaction for each od
pair, while (4) are non-negativity constraints regarding traffic 73588 VOLUME 10, 2022 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks at the equilibrium, maximizing an entropy function and for
this reason more likely to occur [40]. the description of equations (37), (38) and (39) in Section V. It is worth noting that, with the exception of the intermodal
case, the link performance functions are strictly increasing
hyperbolic functions and therefore diverging in proximity to
the arc theoretical maximum capacity. The resulting optimization problem is as follows. V. THE INTERMODAL DYNAMIC MODEL FOR
PASSENGER AND FREIGHT FLOWS The complete list
of parameters and variables adopted in the dynamic model is
given in Tables 1 and 3; for further clarity, the main aggregate
variables used to describe the dynamics of the intermodal
road-rail system are recalled below: ,j
The performance functions for freight intermodal arcs
(i, j) ∈AIf are defined as follows: τ 2
i,j(x2
i,j) = γi,j · T + 1
M x2
i,j
(20) • nod,c
i,j (k) is the number of units of class c in arc (i, j)
associated with the od pair at time step k;
od c (20) where γi,j ≥1 and M is a positive coefficient large enough so
that the impact due to the presence of cargo units on the arc is
insignificant. Given the performance functions for the freight
transport of each arc, the cost function in (13) associated with
each arc of the network is defined as • Iod,c
i,j
(k) is the number of units of class c entering arc (i, j)
associated with the od pair at time step k;
od c • Ood,c
i,j (k) is the number of units of class c exiting arc (i, j)
associated with the od pair at time step k;
d • βod,c
i,j
(k) are the splitting rates of class c in arc (i, j)
associated with the od pair at time step k; note that the
condition P
j∈S(i) βod,c
i,j
(k) = 1 must be verified ∀i, ∀o,
∀d, ∀c, ∀k. ci,j(x2
i,j) = τ 2
i,j(x2
i,j)Ctime
i,j
+ 1i,jCspace
i,j
+ Cfix
i,j
(21) (21) where Ctime
i,j , Cspace
i,j
and Cfix
i,j are the cost per time unit, cost
per space unit, and the fixed cost of arc (i, j), respectively. Depending on the type of arc, the contribution made by each
of the three parameters can change significantly. For example,
in the case of an intermodal freight arc, it is reasonable to
assume Cspace
i,j
= 0. It is worth clarifying that different units are adopted in
the model, depending on the arc type and the flow class. V. THE INTERMODAL DYNAMIC MODEL FOR
PASSENGER AND FREIGHT FLOWS The dynamic model adopted in this work is used to cap-
ture the dynamic features of the overall system through a
set of discrete-time equations. Aggregate discrete-time mod-
els have already been used for performance evaluation and
optimization of specific intermodal transportation processes. In [41], [42], for instance, discrete-time models for freight
movements by rail in maritime terminals are described. The
model presented in this section is much more extensive
considering the movement not only of freight but also of
passengers and considering a more general applicative con-
text, that is an intermodal rail-road multi-class transport net-
work. Specifically, this dynamic model allows to evaluate the
impact of the users’ route choices and to analyze the behavior
of the intermodal system at a more detailed level than the one
provided by the assignment model. p
i,j
g
p
g
In the case of railway arcs, a similar approach is adopted
and the relative performance function is defined as described
below τ 2
i,j(x2
i,j) =
hi,jsmin
i,j
(smin
i,j −L) ·
x2
i,j
Cf + t1
i,j
(19) (19) which estimates the marginal contribution to the travel time
of the arc due to the presence of freight, summed to the travel
time due exclusively to the presence of passengers t1
i,j. The
variable x2
i,j expresses the amount of cargo units present on
the arc. Therefore, it follows that
x2
i,j
Cf represents the equivalent
number of freight trains. The free-flow travel time 1i,j
vR
i,j is
implicitly contained in t1
i,j. The system evolution in time and in space is described by
means of aggregate variables defined for each class c = 1, 2,
for each arc (i, j) ∈A, for each od pair, with o ∈JO, d ∈JD,
and for each time step k, k = 0, . . . , K. for (i, j) ∈AR. A possible solution is looking
for a pattern of flows f , coherent with the distribution of users (17) As done for the passenger assignment, the performance
functions that estimate the level of congestion and the travel
time on arcs are defined by linearizing (37), with (38) and
(39), where c = 2. Therefore, for highway arcs, the perfor-
mance function for freight is defined as follows τ 2
i,j(x2
i,j) =
1i,j
ωi,jnmax
i,j (1 −φ) · ςx2
i,j + t1
i,j
(18) (18) 73589 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks the routes most consistent with the routing problem described
in Problem 3. where ς is the conversion factor adopted to express the
number of trucks x2
i,j in the arcs (i, j)
∈
AH in terms
of passenger car equivalents. Equation (18) estimates the
marginal contribution on travel times due to the presence of
freight vehicles, which is summed to t1
i,j, that is the average
travel time over arc (i, j) due exclusively to the presence of
passengers, as calculated by the dynamic model. The term
1i,j
vH
i,j does not appear explicitly, since the free-flow travel time
is contained in t1
i,j. In fact, it is assumed that the maximum
speed vH
i,j is the same for both freight and passenger vehicles. V. THE INTERMODAL DYNAMIC MODEL FOR
PASSENGER AND FREIGHT FLOWS These splitting rates βod,c
i,j
(k), for each
user class c, are obtained from the flows f od,c
l
resulting from
the application of the intermodal and multi-class assignment
procedure i.e., ϵ1
n,i =
1
if (n, i) ∈AH ∪AR
η
if (n, i) ∈AIp and (i, j) ∈AR
1
η
if (n, i) ∈AIp and (i, j) ∈AH
(25) (25) βod,c
i,j
(k) =
P
l∈Pod,c f od,c
l
· δod,c
i,j,l
P
p∈P(i)
P
l∈Pod,c f od,c
l
· δod,c
p,i,l
(22) where η is the average number of passengers per car, used to
translate the number of vehicles in passengers and viceversa. (22) p
g
The conversion factor ξ1
i,j is instead defined considering
that the passenger demand is given in terms of number of
passengers and that an origin node cannot be followed by an
intermodal arc, therefore for all k, with k = 0 . . . , K. Note that βod,c
i,j
(k) are constant
along the simulation horizon since, in this work, Dod,c is the
total demand of the whole horizon. In case the simulation
horizon is divided in different time intervals, each one char-
acterized by a different demand, the intermodal assignment
model described in Section IV is applied for each time inter-
val, resulting in different splitting rates βod,c
i,j
(k). ξ1
i,j =
1
if (i, j) ∈AR
1
η
if (i, j) ∈AH
(26) (26) As for class c = 2, i.e., the class referring to freight, both
conversion factors ϵ2
n,i and ξ2
i,j are set equal to 1 because,
as mentioned above, a cargo unit is assumed to correspond
to one truck and to one rail wagon. i,j
Virtual queues at the origin nodes are considered in order
to model the presence of flows that have to wait before
entering the network. At this purpose, for each time step k,
k = 0, . . . , K, the following variables are introduced: Note that the flows that actually enter an arc (i, j) depend
on the capability of the arc to receive flows, i.e., the residual
capacity qres
i,j (k), calculated based on the total number of units
in the arc (i, j) at the time step k. 1) ENTERING FLOWS N tot
i,j (k) =
X
o∈JO
X
d∈JD
nod,1
i,j
(k)
Cp
+
X
o∈JO
X
d∈JD
nod,2
i,j
(k)
Cf
(28) )
The entering flows Iod,c
i,j
used in (23) are given by (28) Iod,c
i,j
=βod,c
i,j
(k)
X
n∈P(i)
ϵc
n,i · Ood,c
n,i (k)+ξc
i,j·qod,c(k)
(24) (24) for all (i, j) ∈AR. The total number of vehicles and the total
number of trains present in each arc at each time step must
ensure the following conditions: 0 ≤ntot
i,j (k) ≤nmax
i,j
and 0 ≤
N tot
i,j (k) ≤N max
i,j
. for all (i, j) ∈AR. The total number of vehicles and the total
number of trains present in each arc at each time step must
ensure the following conditions: 0 ≤ntot
i,j (k) ≤nmax
i,j
and 0 ≤
N tot
i,j (k) ≤N max
i,j
. meaning that the flows of each user class c that enter a generic
arc (i, j) of the network are the flows that actually succeed
in exiting from the previous arcs plus the flows that actually
manage to enter from node i if this is also an origin node o. These flows are then multiplied for βod,c
i,j
(k), which indicates
the portion that decides to use arc (i, j) to reach destination d. j
j
The residual capacity of highway and railway arcs qres
i,j (k)
is given by qres
i,j (k) =
(
nmax
i,j
−ntot
i,j (k)
if (i, j) ∈AH
N max
i,j
−N tot
i,j (k)
if (i, j) ∈AR
(29) (29) Since we are describing the behavior of two class of users
in an intermodal transport network, some parameters neces-
sary to quantify the effective traffic load in the network have
to be introduced, i.e., the two conversion factors ϵc
n,i and ξc
i,j
used in (24). Starting by class c = 1, i.e., passengers, and
considering that the transition between two different modes of
transport can only occur in an intermodal arc, the conversion As for the intermodal arcs (i, j) ∈AIp∪AIf , being fictitious
arcs, we chose to not impose bounds on the capacity. As for the intermodal arcs (i, j) ∈AIp∪AIf , being fictitious
arcs, we chose to not impose bounds on the capacity. V. THE INTERMODAL DYNAMIC MODEL FOR
PASSENGER AND FREIGHT FLOWS To this end let us define
with ntot
i,j (k) the total number of vehicles (expressed in terms
of passenger car equivalents) present in arc (i, j) ∈AH
at time step k and with N tot
i,j (k) the total number of trains
in arc (i, j) ∈AR at time step k, which are computed as
follows • qod,c(k) is the number of units of class c associated with
the od pair, that can actually enter the network from
node o ∈JO; • qod,c(k) is the number of units of class c associated with
the od pair, that can actually enter the network from
node o ∈JO;
d • lod,c(k) is the queue length of class c, associated with the
od pair, which waits at the origin node o ∈JO. • lod,c(k) is the queue length of class c, associated with the
od pair, which waits at the origin node o ∈JO. The dynamic evolution of the system is described, for each
class c and for each k, with k = 0, . . . , K, by the following
dynamic equation nod,c
i,j (k + 1) = nod,c
i,j (k) + Iod,c
i,j
(k) −Ood,c
i,j (k)
(23) (23) ntot
i,j (k) =
X
o∈JO
X
d∈JD
nod,1
i,j
(k) +
X
o∈JO
X
d∈JD
ςnod,2
i,j
(k) (27) (27) for all (i, j)
∈
A, o
∈
JO, d
∈
JD. Let us now
describe separately the entering flows Iod,c
i,j
(k) and the exiting
flows Ood,c
i,j (k). for all (i, j) ∈AH, where ς is a coefficient introduced in order
to translate the trucks in an equivalent number of cars, and V. THE INTERMODAL DYNAMIC MODEL FOR
PASSENGER AND FREIGHT FLOWS Specifically: • for class c = 1, i.e., passengers, the unit considered in
railway arcs is the number of passengers, while in high-
way arcs is the number of vehicles; in intermodal arcs
the units can be either passengers or vehicles depending
on the type of arc preceding the intermodal one; ,j
Being the performance functions (18)-(20) monotonically
increasing with respect to the freight flows and being the cost
function (21) linear with respect to the travel times, it fol-
lows that also the cost function of the arcs is monotonically
increasing with respect to the freight flows. This makes the
function y(·) strictly convex and defined on a convex set
(14)-(16) admitting a single optimal solution with respect
to the variables x2
i,j. However, also for the route choices for
freight flows, the uniqueness of the solution is not guaranteed
with respect to the flows on paths f od,2
l
. • for class c = 2, i.e., freight, the unit considered in
railway arcs is the number of railway wagons, while
in highway arcs is the number of trucks; in intermodal
arcs, again, the units depend on the type of preceding
arc. In this paper a single cargo unit corresponds to one
rail wagon and to one truck. Extending this model for
considering different load capacities in road and rail
modes is straightforward and omitted here only for the
sake of simplicity. To overcome this issue, a problem analogous to Problem 2
can be formulated to obtain the pattern of freight flows on 73590 73590 VOLUME 10, 2022 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks factor ϵ1
n,i is given by
ϵ1
n,i =
1
if (n, i) ∈AH ∪AR
η
if (n, i) ∈AIp and (i, j) ∈AR
1
η
if (n, i) ∈AIp and (i, j) ∈AH
(25) factor ϵ1
n,i is given by As mentioned in Section III, the dynamic model receives
as inputs the route choices of passengers and freight, i.e., the
splitting rates βod,c
i,j
(k). 1) ENTERING FLOWS The
potential outflow Sod,c
i,j
(k) is computed as W tot
i,j (k)=
X
o∈JO
X
d∈JD
wod,1
i,j
(k)+ςwod,2
i,j
(k)
if (i, j)∈AH
X
o∈JO
X
d∈JD
wod,1
i,j
(k)
Cp
+
wod,2
i,j
(k)
Cf
if (i, j)∈AR Sod,c
i,j
(k) =
T
ti,j(k)nod,c
i,j (k)
(36) (36) (31) (31) where ti,j(k) is the transfer time required to cover arc (i, j)
defined according to the following relation Hence, thanks to the residual capacity defined in (29)
we can determine the percentage of excess units πi,j(k) that
cannot enter at a given time step k in the arc (i, j) ti,j(k) =
1i,j
Vi,j(ntot
i,j (k))
if (i, j) ∈AH
1i,j
Vi,j(N tot
i,j (k))
if (i, j) ∈AR
αi,j · T
if (i, j) ∈AIp
γi,j · T
if (i, j) ∈AIf
(37) πi,j(k) =
max{0, W tot
i,j (k) −qres(k)}
W tot
i,j (k)
if W tot
i,j (k) > 0
0
otherwise (37) otherwise (32)
(32) where πi,j(k) ∈[0, 1]. Specifically, πi,j(k) is equal to 0 when
W tot
i,j < qres(k), i.e., when the residual capacity is sufficient to
host all units that want to enter arc (i, j), while πi,j(k) is equal
to 1 when qres(k) = 0, i.e., when the residual capacity is zero
and all units that want to enter arc (i, j) cannot do so. For intermodal connections allowing the modal change
from rail to road, the transfer time ti,j(k) is considered con-
stant and equal to αi,j · T, with αi,j ≥1 for each (i, j) ∈AIp
and equal to γi,j · T, with γi,j ≥1 for each arc (i, j) ∈AIf . Instead, in intermodal connections allowing the road-to-rail
modal change, the transfer time is not fixed and depends on
the possibility to fill trains, i.e., on (35). Having said that, the units that can effectively enter from
an origin node is computed as follow qod,c(k) =
Dod,c
K
+lod,c(k)
X
n∈S(o)
βod,c
o,n (k) · (1 −πo,n(k)) With regard to highway and railway arcs, it should be noted
that, for both types of arc, the transfer time is estimated as a
function of the total number of vehicles or trains present in the
connection. 1) ENTERING FLOWS Now let us analyze the receptive capacity of an arc accord-
ing to the load conditions it is experiencing in a given time
step. Then, let us define with wod,c
i,j (k) the amount of users of VOLUME 10, 2022 73591 73591 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks class c related to the od pair who want to enter arc (i, j) in
time step k only enter the rail network if they are enough to fully load
at least a freight train with capacity Cf . Since the transfer
of cargo units from road to rail is realized through a freight
intermodal arc, we consider that this arc behaves as a buffer
where cargo units wait until their number is sufficient to fill at
least one train and then leave the intermodal arc. The number
of cargo units possibly leaving a road-to-rail intermodal arc
(i, j) ∈AIf is then given by wod,c
i,j (k) = βod,c
i,j
(k)
X
n∈P(i)
ϵc
n,i · Sod,c
n,i (k)
+ ξc
i,j ·
Dod,c
K
+ lod,c(k)
(30) + ξc
i,j ·
Dod,c
K
+ lod,c(k)
(30) (30) (30) In (30), wod,c
i,j (k) includes the potential outflow from the
previous arcs Sod,c
n,i (k), better detailed in Section V-2, and the
demand associated with the pair od and the eventual queue
length at the node i if it coincides with the origin o. Sod,2
i,j
(k) =
nod,2
i,j
(k)
Cf
Cf
(35) (35) g
g
Therefore, the total amount of units that potentially enter
arc (i, j) at time step k is given by: Now let us discuss the potential outflow for all classes c for
the arcs (i, j) ∈AH ∪AR, for passengers in arcs (i, j) ∈AIp
and for freight in rail-to-road intermodal arcs (i, j) ∈AIf . 1) ENTERING FLOWS More in details, for each highway arc (i, j) ∈AH,
the transfer time ti,j(k) is computed according to the current
traffic conditions through the steady-state relation between
speed and number of vehicles given by (33) whereas the relative queue of units at the origin node o is
given by lod,c(k + 1) = lod,c(k) + Dod,c
K
−qod,c(k)
(34) (34) VI. RESULTS AND DISCUSSION The focus of this section is to show the potential benefits
that can be obtained by adopting the proposed multi-class
intermodal modeling scheme, simulating a perturbation on
the network and then analyzing how this event can affect the
rest of the network. Specifically, the considered perturbation
is the failure of a connection in the intermodal network. The experimental tests have been performed in two distinct
scenarios: • Scenario pre-disruption: network at the equilibrium
before the advent of the disruption; TABLE 5. Main parameters referred to railway arcs. TABLE 5. Main parameters referred to railway arcs. • Scenario post-disruption: network once a new equilib-
rium is reached some time after the initial perturbation. The results have been obtained by adopting a test network
derived from the well-known Nguyen-Dupuis network prop-
erly modified to consider the intermodal case (for further
information see [44]). This network is composed of 14 nodes
and 21 arcs, as depicted in Fig. 3. The critical event is simu-
lated considering the total loss of functionality of the railway
arc 12-8. simulation has been set equal to one hour, that corresponds to
K = 60 time steps. The transportation demand is expressed
with two origin-destination matrices, one referring to passen-
gers, as shown in Table 6, and one referring to freight, as indi-
cated in Table 7. The mobility demand of both passengers and
freight is assumed to be perfectly inelastic and therefore it is
the same in the two tested scenarios. The main parameters of the highway and railway arcs
are reported in Table 4 and Table 5, respectively. The other
parameters have been set as follows: the conversion factor
η, representing the average number of passengers per car,
is equal to 1.45, the congestion wave speed ωi,j is equal to
30 [km/h], ∀(i, j) ∈AH, the average time headway hi,j is
15 minutes and the minimum average space headway smin
i,j is
equal to 2 [km], ∀(i, j) ∈AR, while the constant parameter
αi,j has been set equal to 15, ∀(i, j) ∈AIp and γi,j equal to 30,
∀(i, j) ∈AIf . The capacity of a freight train Cf is 25 rail
wagons, while the capacity of a passenger train Cp is chosen
equal to 700 passengers. 2) EXITING FLOWS TABLE 4. Main parameters referred to highway arcs. TABLE 5
Main parameters referred to railway arcs Ood,c
i,j (k) = Sod,c
i,j
(k)
X
n∈S(j)
βod,c
j,n (k)
1 −πj,n(k)
(40) (40) VI. RESULTS AND DISCUSSION Note finally that, in both scenarios described below, the
splitting rates βod,c
i,j
(k) computed by the intermodal traffic
assignment are constant throughout the simulation period,
and, then, k is omitted for the sake of simplicity, i.e.,
βod,c
i,j
(k) = βod,c
i,j
. 2) EXITING FLOWS Vi,j(ntot
i,j (k))
= min
vH
i,j,
wi,j
ntot
i,j (k)1i,j
nmax
i,j
1i,j
−
ntot
i,j (k)
1i,j
(38) Even with respect to outflows from an arc, these are calcu-
lated based on the residual capacity of the arcs they wish to
enter. For this reason, we distinguish Ood,c
i,j (k), i.e., the units
actually exiting arc (i, j), from Sod,c
i,j
(k), i.e., the units that
would like to exit arc (i, j) at time step k. (38) Relation (38) has been derived from a triangular funda-
mental diagram, as the one proposed in [43], and expressed
in terms of number of vehicles. With regard to railway arcs,
the steady-state speed relation Vi,j(N tot
i,j (k)) for all (i, j) ∈AR Let us start by describing the relation that defines the
potential outflow from an intermodal freight arc (i, j) ∈AIf
preceded by a highway arc. We assume that cargo units can 73592 73592 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks FIGURE 3. Sketch of the test intermodal transport network. may be formulated as
Vi,j(N tot
i,j (k)) =
vR
i,j
if
N tot
i,j (k)
1i,j
≤
1
hi,jvR
i,j + L
1
hi,j
1i,j
N tot
i,j (k) −L
if
1
hi,jvR
i,j + L <
N tot
i,j (k)
1i,j
≤
1
smin
i,j
(39) may be formulated as may be formulated as
Vi,j(N tot
i,j (k)) =
vR
i,j
if
N tot
i,j (k)
1i,j
≤
1
hi,jvR
i,j + L
1
hi,j
1i,j
N tot
i,j (k) −L
if
1
hi,jvR
i,j + L <
N tot
i,j (k)
1i,j
≤
1
smin
i,j
(39) (39) It is worth noting that, for each highway or railway arc,
condition (1) with (38) and (39) implies that the transfer
time ti,j(k) is never lower than T, ensuring the validity of
the conservation equations. For further details concerning the
development of this relationship, please refer to Appendix. d p
p p
pp
Finally, given Sod,c
i,j
(k) and πi,j(k), we can compute the
outflow Ood,c
i,j (k) which represent the units of class c referred
to the pair od that actually exit the arc (i, j) as FIGURE 3. Sketch of the test intermodal transport network. TABLE 4. Main parameters referred to highway arcs. A. PERFORMANCE INDICATORS Let us begin with the Total Travel Time,
that is an indicator that computes the total time spent by each
unit, both of passengers and freight type, in a connection,
considering that an arc can belong to multiple paths at the
same time. This indicator refers to each arc (i, j) ∈A and is
calculated as follows: • od pair 1-2: β12,1
1,12 = 1, β12,1
5,6 = 1, β12,1
6,7 = 1,β12,1
7,11 = 1,
β12,1
8,2 = 1, β12,1
11,2 = 1, β12,1
12,8 = 1;
• od pair 1-3: β13,1
1,5
= 0.92, β13,1
1,12 = 0.92, β13,1
5,6
=
0.36, β13,1
5,9
= 0.64, β13,1
6,7
= 1, β13,1
7,11 = 1, β13,1
9,10 =
0.12,β13,1
9,14 = 1, β13,1
10,11 = 1, β13,1
11,3 = 1, β13,1
12,6 = 1,
β13,1
13,3 = 1, β13,1
14,3 = 1;
• od pair 4-2: β42,1
4,5
= 0.62, β42,1
4,9
= 0.38, β42,1
5,6
= 0.75,
β42,1
5,9
= 0.25, β42,1
6,7
= 0.67, β42,1
6,10 = 0.33, β42,1
7,11 = 1,
β42,1
9,10 = 1, β42,1
10,11 = 1, β42,1
11,2 = 1;
• od pair 4-3: β43,1
4,5
= 0.18, β43,1
4,9
= 0.82, β43,1
5,6
= 1,
β43,1
6,7
= 1, β43,1
7,11 = 1, β43,1
9,10 = 0.28, β43,1
9,14 = 0.72,
β43,1
10,11 = 1, β43,1
11,3 = 1, β43,1
13,3 = 1, β43,1
14,13 = 1. TTTi,j = T
K
X
k=1
2
X
c=1
X
o∈JO
X
d∈JD
nod,c
i,j (k)
(41) (41) • od pair 4-2: β42,1
4,5
= 0.62, β42,1
4,9
= 0.38, β42,1
5,6
= 0.75,
β42,1
5,9
= 0.25, β42,1
6,7
= 0.67, β42,1
6,10 = 0.33, β42,1
7,11 = 1,
β42,1
9,10 = 1, β42,1
10,11 = 1, β42,1
11,2 = 1;
• od pair 4-3: β43,1
4,5
= 0.18, β43,1
4,9
= 0.82, β43,1
5,6
= 1,
β43,1
6,7
= 1, β43,1
7,11 = 1, β43,1
9,10 = 0.28, β43,1
9,14 = 0.72,
β43,1
10,11 = 1, β43,1
11,3 = 1, β43,1
13,3 = 1, β43,1
14,13 = 1. B. SCENARIO PRE-DISRUPTION It should be noted that among these routes, only one is of
intermodal type, with a change from road to rail, one route
is entirely by railway, while the remaining ones involve only
the use of highway arcs. The methodology presented in Section IV has been used to
allocate the passengers demand on possible routes and the
resulting assignment is shown in Fig. 4. It is worth reminding
that the feasible paths are those that have at most one modal
shift. The paths used at the equilibrium, before the disruption,
are reported in Table 8: one path adopts only the rail mode,
five are highway routes, while the remaining eight require the
use of both modes of transport. A. PERFORMANCE INDICATORS The Mean Arc Occupancy is an indicator for assessing the
average occupancy of an arc, either road or rail, given the
total number of vehicles or trains occupying it on average,
therefore the Mean Arc Occupancy is given by MAOi,j =
1
K
XK
k=1 ntot
i,j (k)
if (i, j) ∈AH
1
K
XK
k=1 N tot
i,j (k)
if (i, j) ∈AR
(42) (42) ,
,
,
,
The freight flows are fixed and their distribution is shown
again in Fig. 4, while the paths are reported in Table 9. The corresponding non-zero splitting rates, again considered
constant throughout the simulation, are the following: Finally, the Mean Arc Saturation relates the Mean Arc
Occupancy to the capacity of an arc, i.e. Finally, the Mean Arc Saturation relates the Mean Arc
Occupancy to the capacity of an arc, i.e. • od pair 1-2: β12,2
1,12 = 1, β12,2
8,2 = 1, β12,2
12,8 = 1;
• od pair 1-3: β13,2
1,5 = 1, β13,2
5,9 = 1, β13,2
9,14 = 1, β13,2
13,3 = 1,
β13,2
14,13 = 1; MASi,j =
MAOi,j
nmax
i,j
· 100
if (i, j) ∈AH
MAOi,j
N max
i,j
· 100
if (i, j) ∈AR
(43) (43) • od pair 4-2: β42,2
4,5
= 0.75, β42,2
4,9
= 0.24 β42,2
5,6
= 0.17,
β42,2
5,9
= 0.82 β42,2
6,7
= 1, β42,2
7,11 = 1, β42,2
11,2 = 1, β42,2
9,10 =
1, β42,2
10,11 = 1; Note that the Mean Arc Occupancy and Mean Arc Satura-
tion indicators are calculated for highway and rail arcs only,
as for intermodal arcs we assume no capacity restrictions. ,
• od pair 4-3: β43,2
4,9
= 0.21, β43,2
4,5
= 0.78, β43,2
5,9
= 1,
β43,2
9,14 = 1, β43,2
13,3 = 1, β43,2
14,13 = 1. A. PERFORMANCE INDICATORS • od pair 1-2: β12,1
1,12 = 1, β12,1
5,6 = 1, β12,1
6,7 = 1,β12,1
7,11 = 1,
β12,1
8,2 = 1, β12,1
11,2 = 1, β12,1
12,8 = 1;
• od pair 1-3: β13,1
1,5
= 0.92, β13,1
1,12 = 0.92, β13,1
5,6
=
0.36, β13,1
5,9
= 0.64, β13,1
6,7
= 1, β13,1
7,11 = 1, β13,1
9,10 =
0.12,β13,1
9,14 = 1, β13,1
10,11 = 1, β13,1
11,3 = 1, β13,1
12,6 = 1,
β13,1
13,3 = 1, β13,1
14,3 = 1;
• od pair 4-2: β42,1
4,5
= 0.62, β42,1
4,9
= 0.38, β42,1
5,6
= 0.75,
β42,1
5,9
= 0.25, β42,1
6,7
= 0.67, β42,1
6,10 = 0.33, β42,1
7,11 = 1,
β42,1
9,10 = 1, β42,1
10,11 = 1, β42,1
11,2 = 1;
• od pair 4-3: β43,1
4,5
= 0.18, β43,1
4,9
= 0.82, β43,1
5,6
= 1,
β43,1
6,7
= 1, β43,1
7,11 = 1, β43,1
9,10 = 0.28, β43,1
9,14 = 0.72,
β43,1
10,11 = 1, β43,1
11,3 = 1, β43,1
13,3 = 1, β43,1
14,13 = 1. TABLE 6. Passengers origin-destination matrix. TABLE 7. Freight origin-destination matrix. TABLE 8. Paths used by passengers in the pre-disruption scenario. TABLE 8. Paths used by passengers in the pre-disruption scenario. TABLE 9
Paths used by freight in the pre disruption scenario TABLE 6. Passengers origin-destination matrix. TABLE 7. Freight origin-destination matrix. TABLE 6. Passengers origin-destination matrix. TABLE 6. Passengers origin-destination matrix. TABLE 7. Freight origin-destination matrix. TABLE 9. Paths used by freight in the pre-disruption scenario. have been developed using some of the dynamic variables
introduced in Section V to describe the behavior of the system
over time and space. Specifically, the proposed indicators
are the Total Travel Time, the Mean Arc Occupancy, and the
Mean Arc Saturation. Let us begin with the Total Travel Time,
that is an indicator that computes the total time spent by each
unit, both of passengers and freight type, in a connection,
considering that an arc can belong to multiple paths at the
same time. This indicator refers to each arc (i, j) ∈A and is
calculated as follows: have been developed using some of the dynamic variables
introduced in Section V to describe the behavior of the system
over time and space. Specifically, the proposed indicators
are the Total Travel Time, the Mean Arc Occupancy, and the
Mean Arc Saturation. A. PERFORMANCE INDICATORS As for the dynamic model, a sample time T equal to
one minute has been adopted, while the time horizon of the In order to evaluate the effects of the mobility demand on the
intermodal transport network, some performance indicators VOLUME 10, 2022 73593 73593 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks TABLE 8. Paths used by passengers in the pre-disruption scenario. TABLE 9. Paths used by freight in the pre-disruption scenario. • od pair 1-2: β12,1
1,12 = 1, β12,1
5,6 = 1, β12,1
6,7 = 1,β12,1
7,11 = 1,
β12,1
8,2 = 1, β12,1
11,2 = 1, β12,1
12,8 = 1;
• od pair 1-3: β13,1
1,5
= 0.92, β13,1
1,12 = 0.92, β13,1
5,6
=
0.36, β13,1
5,9
= 0.64, β13,1
6,7
= 1, β13,1
7,11 = 1, β13,1
9,10 =
0.12,β13,1
9,14 = 1, β13,1
10,11 = 1, β13,1
11,3 = 1, β13,1
12,6 = 1,
β13,1
13,3 = 1, β13,1
14,3 = 1;
• od pair 4-2: β42,1
4,5
= 0.62, β42,1
4,9
= 0.38, β42,1
5,6
= 0.75,
β42,1
5,9
= 0.25, β42,1
6,7
= 0.67, β42,1
6,10 = 0.33, β42,1
7,11 = 1,
β42,1
9,10 = 1, β42,1
10,11 = 1, β42,1
11,2 = 1;
• od pair 4-3: β43,1
4,5
= 0.18, β43,1
4,9
= 0.82, β43,1
5,6
= 1,
β43,1
6,7
= 1, β43,1
7,11 = 1, β43,1
9,10 = 0.28, β43,1
9,14 = 0.72,
β43,1
10 11 = 1, β43,1
11 3 = 1, β43,1
13 3 = 1, β43,1
14 13 = 1. TABLE 8. Paths used by passengers in the pre-disruption scenario. TABLE 9. Paths used by freight in the pre-disruption scenario. C. SCENARIO POST-DISRUPTION As mentioned earlier, the disruption is represented in this
example by removing the railway arc 12-8. Regarding passen-
gers, the new path configuration for each origin-destination The non-zero splitting rates are in this case: 73594 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks FIGURE 4. Pre-disruption equilibrium. TABLE 10. Paths used by passengers after the disruption. FIGURE 4. Pre-disruption equilibrium. TABLE 10. Paths used by passengers after the disruption. TABLE 11. Paths used by freight after the disruption. FIGURE 5. Post-disruption equilibrium. TABLE 11. Paths used by freight after the disruption. FIGURE 5. Post-disruption equilibrium. FIGURE 4. Pre-disruption equilibrium. FIGURE 4. Pre-disruption equilibrium. FIGURE 4. Pre-disruption equilibrium. TABLE 10. Paths used by passengers after the disruption. TABLE 10. Paths used by passengers after the disruption. FIGURE 5. Post-disruption equilibrium. pair is shown in Table 10. As can be seen, the railway path
[1-12-8-2] no longer appears since it includes the damaged
arc. As shown in Fig. 5, there is a shift of od pair 1-2 flows
and, as a consequence, a greater load on the central arcs of the
network, in particular on path [1-12-6-7-8-2]. On the other
hand, not surprisingly, the southern paths in the network for
pair 4-3 are the least perturbed as testified by the fact that
the flows do not undergo excessive redistribution, meaning
that for those users the most attractive routes have remained
the same. The splitting rates for passenger flows are the
following: As far as freight flows are concerned, similar considera-
tions can be made, since the railway path can no longer be
used and the freight flow of od pair 1-2 is reassigned to the
remaining path, as reported in Table 11 with the resulting
assignment shown in Fig. 5. C. SCENARIO POST-DISRUPTION The corresponding non-zero
splitting rates are as follows: • od pair 1-2: β12,2
1,12 = 1, β12,2
6,7
= 0.83, β12,2
6,10 = 0.16,
β12,2
6,7 = 1, β12,2
10,11 = 1, β12,2
11,2 = 1, β12,2
12,6 = 1;
• od pair 1-3: β13,2
1,5 = 1, β13,2
5,9 = 1, β13,2
9,14 = 1, β13,2
13,3 = 1,
β13,2
14,13 = 1;
• od pair 4-2: β42,2
4,5
= 0.65, β42,2
4,9
= 0.35, β42,2
5,9
= 1,
β42,2
6,7 = 1,β42,2
7,11 = 1 β42,2
9,10 = 1, β42,2
10,11 = 1, β42,2
11,2 = 1;
• od pair 4-3: β43,2
4,5
= 0.89, β43,2
4,9
= 0.11, β43,2
5,9
= 1, • od pair 1-2: β12,2
1,12 = 1, β12,2
6,7
= 0.83, β12,2
6,10 = 0.16,
β12,2
6,7 = 1, β12,2
10,11 = 1, β12,2
11,2 = 1, β12,2
12,6 = 1;
• od pair 1-3: β13,2
1,5 = 1, β13,2
5,9 = 1, β13,2
9,14 = 1, β13,2
13,3 = 1,
β13,2
14,13 = 1;
• od pair 4-2: β42,2
4 5
= 0 65 β42,2
4 9
= 0 35 β42,2
5 9
= 1 • od pair 1-2: β12,2
1,12 = 1, β12,2
6,7
= 0.83, β12,2
6,10 = 0.16,
β12,2
6,7 = 1, β12,2
10,11 = 1, β12,2
11,2 = 1, β12,2
12,6 = 1;
13 2
13 2
13 2
13 2 • od pair 1-3: β13,2
1,5 = 1, β13,2
5,9 = 1, β13,2
9,14 = 1, β13,2
13,3 = 1,
β13,2
14,13 = 1; g
• od pair 1-2: β12,1
1,5
= 0.41, β12,1
1,12 = 0.59, β12,1
5,6
= 0.85,
β12,1
5,9
= 0.15, β12,1
6,7
= 0.69, β12,1
6,10 = 0.31, β12,1
7,8
=
0.27, β12,1
7,11 = 0.73, β12,1
8,2
= 1, β12,1
9,10 = 1, β12,1
10,11 = 1,
β12,1
11,2 = 1, β12,1
12,6 = 1;
• od pair 1-3: β13,1
1,5
= 0.52, β13,1
1,12 = 0.48, β13,1
5,6
= 0.1,
β13,1
5,9
= 0.9, β13,1
6,7
= 0.65, β13,1
6,10 = 0.35, β13,1
7,11 = 1,
β13,1
9,14 = 1, β13,1
10,11 = 1, β13,1
11,3 = 1, β13,1
12,6 = 1, β13,1
13,3 = 1,
β13,1
14,3 = 1;
42 1
42 1
42 1 • od pair 1-2: β12,1
1,5
= 0.41, β12,1
1,12 = 0.59, β12,1
5,6
= 0.85,
β12,1
5,9
= 0.15, β12,1
6,7
= 0.69, β12,1
6,10 = 0.31, β12,1
7,8
=
0.27, β12,1
7,11 = 0.73, β12,1
8,2
= 1, β12,1
9,10 = 1, β12,1
10,11 = 1,
β12,1
11,2 = 1, β12,1
12,6 = 1;
• od pair 1-3: β13,1
1,5
= 0.52, β13,1
1,12 = 0.48, β13,1
5,6
= 0.1,
β13,1
5,9
= 0.9, β13,1
6,7
= 0.65, β13,1
6,10 = 0.35, β13,1
7,11 = 1,
β13,1
9,14 = 1, β13,1
10,11 = 1, β13,1
11,3 = 1, β13,1
12,6 = 1, β13,1
13,3 = 1,
β13,1
14 3 = 1; • od pair 1-2: β12,1
1,5
= 0.41, β12,1
1,12 = 0.59, β12,1
5,6
= 0.85,
β12,1
5,9
= 0.15, β12,1
6,7
= 0.69, β12,1
6,10 = 0.31, β12,1
7,8
=
0.27, β12,1
7,11 = 0.73, β12,1
8,2
= 1, β12,1
9,10 = 1, β12,1
10,11 = 1,
β12,1
11,2 = 1, β12,1
12,6 = 1;
13 1
13 1
13 1 • od pair 4-2: β42,2
4,5
= 0.65, β42,2
4,9
= 0.35, β42,2
5,9
= 1,
β42,2
6,7 = 1,β42,2
7,11 = 1 β42,2
9,10 = 1, β42,2
10,11 = 1, β42,2
11,2 = 1;
• od pair 4-3: β43,2
4,5
= 0.89, β43,2
4,9
= 0.11, β43,2
5,9
= 1,
β43,2
9,14 = 1, β43,2
13,3 = 1, β43,2
14,13 = 1. VOLUME 10, 2022 C. SCENARIO POST-DISRUPTION The deviation from
the unperturbed case is not reported for this indicator because,
being derived from the Mean Arc Occupancy, this indicator
has the same variations. As can be seen the disruption of 12-8 arc implies that
TABLE 13. The Mean Arc Occupancy for highway and railway arcs. FIGURE 7. Mean Arc Occupancy for some of the most affected highway
arcs. Time, by means of the dynamic model presented in this pap
allows therefore to identify the elements of the network th
will be more stressed after the perturbation. Analyzing Tables 13 and 14, it is possible to observe th
the disruptive event has effects on Mean Arc Occupancy an FIGURE 6. Total Travel Time absolute variations. FIGURE 6. Total Travel Time absolute variations. TABLE 13. The Mean Arc Occupancy for highway and railway arcs. FIGURE 6. Total Travel Time absolute variations. TABLE 12. The Total Travel Time for each arc. TABLE 12. The Total Travel Time for each arc. TABLE 12. The Total Travel Time for each arc. FIGURE 7. Mean Arc Occupancy for some of the most affected highway
arcs. the values of the Mean Arc Saturation for each roadway and
railway arc, in the pre-disruption scenario, i.e., MASE
i,j, and in
the post-disruption scenario, i.e., MASD
i,j. The deviation from
the unperturbed case is not reported for this indicator because,
being derived from the Mean Arc Occupancy, this indicator
has the same variations. Time, by means of the dynamic model presented in this paper,
allows therefore to identify the elements of the network that
will be more stressed after the perturbation. Analyzing Tables 13 and 14, it is possible to observe that
the disruptive event has effects on Mean Arc Occupancy and
Mean Arc Saturation even for arcs that are not connected
with the collapsed one. In addition, it can be seen that the
disturbance affects not only the railway arcs but also the high-
way arcs, as highlighted in Fig. 7. Referring to the highway
arcs, the most significant change in the average occupancy
evaluated in a time interval of one minute is experienced for
arc 6-10 which approximately doubles this indicator. As can be seen, the disruption of 12-8 arc implies that
freight and passenger flows, which previously used the
peripheral rail route, are re-assigned in more internal routes,
particularly intermodal routes. C. SCENARIO POST-DISRUPTION Table 12 shows, for each arc, TTT E
i,j, i.e., the Total Travel Table 12 shows, for each arc, TTT E
i,j, i.e., the Total Travel
Time computed in the pre-disruption scenario, TTT D
i,j, i.e.,
the Total Travel Time in the post-disruption scenario, and
1TTTi,j, i.e., the absolute deviations of the same metric. Analogously, Fig. 6 shows, for each arc, the Total Travel Time
percent variation by exhibiting the effects of disruption more
clearly. Table 13 shows instead the values of the Mean Arc
Occupancy for each roadway and railway arc, specifically
MAOE
i,j is the Mean Arc Occupancy computed in the pre-
disruption scenario, MAOD
i,j is the Mean Arc Occupancy in
the post-disruption scenario, and 1MAOi,j is the variation of
MAOD
i,j with respect to MAOE
i,j. Fig. 7 depicts the Mean Arc
Occupancy in pre- and post-disruptive scenarios for some of
the most affected highway arcs. Finally, Table 14 displays • od pair 1-3: β13,1
1,5
= 0.52, β13,1
1,12 = 0.48, β13,1
5,6
= 0.1,
β13,1
5,9
= 0.9, β13,1
6,7
= 0.65, β13,1
6,10 = 0.35, β13,1
7,11 = 1,
β13,1
9,14 = 1, β13,1
10,11 = 1, β13,1
11,3 = 1, β13,1
12,6 = 1, β13,1
13,3 = 1,
β13,1
14,3 = 1; ,
• od pair 4-2: β42,1
4,5
= 0.57, β42,1
4,9
= 0.43, β42,1
5,6
= 0.8,
β42,1
5,9
= 0.2, β42,1
6,7
= 0.78, β42,1
6,10 = 0.22, β42,1
7,8
= 0.54,
β42,1
7,11 = 0.46, β42,1
8,2
= 1, β42,1
9,10 = 1, β42,1
10,11 = 1,
β42,1
11,2 = 1; ,
• od pair 4-3: β43,1
4,5
= 0.19, β43,1
4,9
= 0.81, β43,1
5,9
= 1,
β43,1
6,10 = 1, β43,1
9,10 = 0.22, β43,1
9,14 = 0.78, β43,1
10,11 = 1,
β43,1
11,3 = 1, β43,1
13,3 = 1, β43,1
14,13 = 1. β43,1
11,3 = 1, β43,1
13,3 = 1, β43,1
14,13 = 1. 73595 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks FIGURE 6. Total Travel Time absolute variations. TABLE 12. The Total Travel Time for each arc. the values of the Mean Arc Saturation for each roadway and
railway arc, in the pre-disruption scenario, i.e., MASE
i,j, and in
the post-disruption scenario, i.e., MASD
i,j. VII. CONCLUSION In this paper, a two-stage modeling approach is proposed to
represent passenger and freight flows on a intermodal trans-
portation network, i.e., a network in which there are road-
ways, railways, and connections that allow the modal shifts. The modeling scheme consists of an assignment model and a
discrete-time dynamic model. The assignment model allows
to represent the choices of the users, both passengers and
freight, in terms of routes and transport modes. These route
choices provide the input to the dynamic model that allows
to represent the evolution in time and space of user flows,
allowing to evaluate some dynamic characteristics such as
speed and travel time on the network arcs. In order to provide
some insights about the proposed approach, this concluding
section has been structured into four parts, in order to further
discuss the results produced in this paper, to illustrate some
possible applications of the proposed modeling framework
for scenario evaluation, on the one hand, and for testing
regulatory policies and control actions, on the other hand,
and, finally, to argue about the possible weaknesses of this
methodology. Scenarios of particular relevance are those concerning the
occurrence of critical events. Indeed, large transport networks
are anyway susceptible to critical events that may have severe
implications on the whole activity system of a territory. These
disruptive events may be caused by natural phenomena (such
as floods, earthquakes, pandemics, etc.) or anthropogenic
causes (such as terrorist attacks, infrastructure failures, but
also planned maintenance works): whatever the cause, they
may affect the transport network as a variation of mobility
demand or as events that partially or totally deteriorate the
capacity of a transport network. In this regard, a topic that is
attracting particular attention in scientific research is the eval-
uation of the resilience of a transport network, i.e., its ability
to resist, adapt or change in order to maintain acceptable per-
formance in case of critical events. Although the concept of
resilience has been initially introduced to describe a property
of natural systems [45], recently it has been applied to trans-
port networks and in particular to road networks [46]–[48]
and railway networks [49]–[51]. However, as shown in the
case study presented in this paper, transport networks are
complex and highly interdependent systems and a critical
event affecting one mode of transport can have an impact on
other modes giving rise to a chain effect. VII. CONCLUSION What is lacking in
the literature, and what this paper has aimed to address, is the
possibility of using a tool that allows to quantify the effects
of these interdependencies and to evaluate the ability of a
transport network to maintain acceptable performance even
when it is affected by disruptions. C. SCENARIO POST-DISRUPTION analysis than the one which can be conducted using modeling
frameworks that contemplate a single mode of transport only. analysis than the one which can be conducted using modeling
frameworks that contemplate a single mode of transport only. 1) DISCUSSION OF THE PROPOSED TEST CASE The objective of the proposed case study has been to illustrate
the benefits of the proposed intermodal modeling scheme by
analyzing the behavior of a network subject to a disruptive
event, specifically the loss of functionality of a railway arc. This application revealed the ability of the intermodal model
to capture the ripple effects of such events that cannot be
gained if analyzed with models representing a single trans-
portation mode only. 3) POSSIBLE APPLICATIONS FOR DEVELOPMENT AND
TESTING OF REGULATORY POLICIES AND
CONTROL ACTIONS C. SCENARIO POST-DISRUPTION Consistently with an overall
load increase on the central arcs of the network, it is possible
to observe a shift, though small, of flows of the 4-3 pair in
favor of the outermost intermodal route [4-9-14-13-3]. Not
surprisingly, the arcs relatively most affected by the pertur-
bation are those in proximity to the disrupted arc, such as
arcs 12-6, 6-7 and 7-8. However, it can be seen that even the
central arc 6-10 experiences a 30% increase in Total Travel
Time. The computation of a metric such as the Total Travel From the analysis of these results, it can then be concluded
that the adoption of a modeling framework of this type per-
mits to grasp the interdependencies that exist between the
road and rail modes of transport, enabling a more accurate 73596 VOLUME 10, 2022 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks TABLE 14. The Mean Arc Saturation for highway and railway arcs. analysis than the one which can be conducted using modeling
frameworks that contemplate a single mode of transport only. TABLE 14. The Mean Arc Saturation for highway and railway arcs. collapsed arc. Specifically, some arcs, both of road and rail
type, have almost doubled in volume, making them obvi-
ously more critical to any further disruption. In addition,
the post-disruption scenario showed a significant increase in
the use of intermodal arcs, both for passengers and freight,
suggesting to a possible decision maker that the adoption of
more connections of this type can make the transport network
more robust to the occurrence of unexpected events. 2) POSSIBLE APPLICATIONS FOR SCENARIO EVALUATION
The proposed model can be adopted to evaluate the effects
of the decisions of the users on a transport network. These
effects can be quantified through the adoption of specific per-
formance indexes, such as those introduced above, or through
the development of other indicators. For example, through the
evaluation of the Mean Arc Saturation index, a preliminary
analysis can be conducted to identify, under specific mobility
demand conditions, which arcs are closest to saturation and
assess the effects of their failure. Furthermore, this model can
also be used to assess the sustainability of user choices by
integrating this modeling scheme with models that estimate
emissions or energy consumptions. 3) POSSIBLE APPLICATIONS FOR DEVELOPMENT AND
TESTING OF REGULATORY POLICIES AND
CONTROL ACTIONS The analysis showed that the collapse of a railway arc
can bring a substantial increase in travel time for some
highway arcs, which can reach a 30-40% increase, and an
even greater growth in the average occupancy of highway
and railway arcs that are not directly connected with the The modeling framework proposed in this paper may consti-
tute the basis for regulation and control approaches finalized VOLUME 10, 2022 73597 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks FIGURE 8. Triangular fundamental diagram for railway traffic. at defining routing and modal indications to be provided to
the users. First of all, this model can be used to provide
more detailed information to users about travel times or
route choices for improving sustainability. Moreover, specific
routing instructions can be defined for the users and, since
the modeling scheme is multi-class, such instructions can be
suitably defined for each class of users. The proposed two-stage modeling framework may be
adopted to test control policies that aim at fully exploiting
the mobility capacity of a large-scale intermodal transport
network by suggesting routes that may include one or more
transport modes. This can be done both in nominal conditions
of the network or when the transport system is affected by an
event which changes its structure or the mobility demand. FIGURE 8. Triangular fundamental diagram for railway traffic. g
y
Finally, this approach can be used to develop online jour-
ney re-planning strategies such as the one developed in [52]
for the re-routing of freight during disruptive events. FIGURE 9. Steady-state speed-headway functions for railway traffic. 4) POSSIBLE WEAKNESSES OF THE
PROPOSED METHODOLOGY The major weakness concerning the proposed methodology
is related to the collection and elaboration of data that must
describe both the transport supply, i.e., the infrastructural
and technical characteristics of the intermodal network, and
the mobility demand that must be expressed both in terms
of origin/destination matrix, to represent the willingness
of freight and passengers to move, and punctual measure-
ments on the network for the validation of the modeling
scheme. This phase is particularly challenging because, since
a large-scale intermodal network is considered, the involve-
ment of different actors over a large territory is required and,
then, it is necessary to integrate data from different, and pos-
sibly heterogeneous, data sources, as thoroughly discussed
in [53]. FIGURE 9. Steady-state speed-headway functions for railway traffic. fundamental diagram in the railway context can be defined
as follows REFERENCES [1] E. Cascetta, Transportation Systems Analysis: Models and Applications. Cham, Switzerland: Springer, 2009. [26] X. Pi, W. Ma, and Z. Qian, ‘‘A general formulation for multi-modal
dynamic traffic assignment considering multi-class vehicles, public transit
and parking,’’ Transp. Res. Proc., vol. 38, pp. 914–934, Jan. 2019. [2] S. P. Hoogendoorn and P. H. L. Bovy, ‘‘State-of-the-art of vehicular traffic
flow modeling,’’ Proc. Inst. Mech. Eng., I, J. Syst. Control Eng., vol. 215,
283–303, Jun. 2001. [27] A. Loder, L. Ambühl, M. Menendez, and K. W. Axhausen, ‘‘Empirics
of multi-modal traffic networks—Using the 3D macroscopic fundamental
diagram,’’ Transp. Res. C, Emerg. Technol., vol. 82, pp. 88–101, Sep. 2017. [3] A. Gille, M. Klemenz, and T. Siefer, ‘‘Applying multiscaling analysis to
detect capacity resources in railway networks,’’ WIT Trans. Built Environ.,
vol. 103, pp. 595–603, Sep. 2008. [28] W. Liu and N. Geroliminis, ‘‘Doubly dynamics for multi-modal networks
with park-and-ride and adaptive pricing,’’ Transp. Res. B, Methodol.,
vol. 102, pp. 162–179, Aug. 2017. [4] M. Ouyang, ‘‘Review on modeling and simulation of interdependent crit-
ical infrastructure systems,’’ Rel. Eng. Syst. Saf., vol. 121, pp. 43–60,
Jan. 2014. [29] A. Bucchiarone, M. De Sanctis, and N. Bencomo, ‘‘Agent-based frame-
work for self-organization of collective and autonomous shuttle fleets,’’
IEEE Trans. Intell. Transp. Syst., vol. 22, no. 6, pp. 3631–3643, Jun. 2021. [5] R. Pant, K. Barker, F. H. Grant, and T. L. Landers, ‘‘Interdependent
impacts of inoperability at multi-modal transportation container termi-
nals,’’ Transp. Res. E, Logistics Transp. Rev., vol. 47, no. 5, pp. 722–737,
Sep. 2011. [30] T. G. Crainic, G. Perboli, and M. Rosano, ‘‘Simulation of intermodal
freight transportation systems: A taxonomy,’’ Eur. J. Oper. Res., vol. 270,
no. 2, pp. 401–418, Oct. 2018. [6] P. Chen, C. Scown, H. S. Matthews, J. H. Garrett, and C. Hendrick-
son, ‘‘Managing critical infrastructure interdependence through economic
input-output methods,’’ J. Infrastruct. Syst., vol. 15, no. 3, pp. 200–210,
Sep. 2009. [31] L. Li, R. R. Negenborn, and B. D. Schutter, ‘‘Intermodal freight transport
planning—A receding horizon control approach,’’ Transp. Res. C, Emerg. Technol., vol. 60, pp. 77–95, Nov. 2015. p
[7] C. Pasquale, E. Siri, S. Sacone, and S. Siri, ‘‘A discrete-time model for
large-scale multi-modal transport networks,’’ IFAC-PapersOnLine, vol. 54,
no. 2, pp. 7–12, 2021. [32] T. H. Zunder and R. Jorna, ‘‘Performance evaluation of an online bench-
marking tool for European freight transport chains,’’ Benchmarking, Int. J., vol. 20, no. 2, pp. REFERENCES 233–250, Apr. 2013. [8] C. Pasquale, E. Siri, S. Sacone, and S. Siri, ‘‘A modeling framework for
passengers and freight in large-scale multi-modal transport networks,’’ in
Proc. 29th Medit. Conf. Control Autom. (MED), Jun. 2021, pp. 681–686. [33] S. Stoilova and S. Kunchev, ‘‘Method for optimal transportation using a
three-phase model,’’ Comp. Rendus de l’Académie Bulgare des Sci., Sci. Mathématiques et Naturelles, vol. 69, pp. 905–910, Jul. 2016. [9] C. Macharis and Y. M. Bontekoning, ‘‘Opportunities for OR in intermodal
freight transport research: A review,’’ Eur. J. Oper. Res., vol. 153, no. 2,
pp. 400–416, Mar. 2004. [34] S. Stoilova, ‘‘Evaluation efficiency of intermodal transport using multi-
criteria analysis,’’ Eng. Rural Develop., vol. 17, pp. 2030–2039, Jan. 2018. pp
[10] A. Caris, C. Macharis, and G. K. Janssens, ‘‘Decision support in inter-
modal transport: A new research agenda,’’ Comput. Ind., vol. 64, no. 2,
pp. 105–112, Feb. 2013. [35] M. Patriksson, The Traffic Assignment Problem: Models and Methods. New York, NY, USA: Dover, 2015. [36] J. G. Wardrop, ‘‘Road paper. Some theoretical aspects of road traffic
research,’’ Proc. Inst. Civil Eng., vol. 1, no. 3, pp. 325–362, May 1952. [11] M. Steadieseifi, N. P. Dellaert, W. Nuijten, T. Van Woensel, and R. Raoufi,
‘‘Multimodal freight transportation planning: A literature review,’’ Eur. J. Oper. Res., vol. 233, no. 1, pp. 1–15, Feb. 2014. [37] M. J. Beckmann, C. B. McGuire, and C. B. Winsten, Studies in the
Economics of Transportation. Santa Monica, CA, USA: Rand Corporation,
1995. [12] M. J. Basallo-Triana, C. J. Vidal-Holguín, and J. J. Bravo-Bastidas, ‘‘Plan-
ning and design of intermodal hub networks: A literature review,’’ Comput. Oper. Res., vol. 136, Dec. 2021, Art. no. 105469. [38] D.-Y. Lin and P. W. Leong, ‘‘An N-path user equilibrium for transportation
networks,’’ Appl. Math. Model., vol. 38, no. 2, pp. 667–682, Jan. 2014. [13] P. Arnold, D. Peeters, and I. Thomas, ‘‘Modelling a rail/road intermodal
transportation system,’’ Transp. Res. E, Logistics Transp. Rev., vol. 40,
no. 3, pp. 255–270, May 2004. [39] M. Borchers, P. Breeuwsma, W. Kern, J. Slootbeek, G. Still, and W. Tibben,
‘‘Traffic user equilibrium and proportionality,’’ Transp. Res. B, Methodol.,
vol. 79, pp. 149–160, Sep. 2015. [14] K. Sörensen, C. Vanovermeire, and S. Busschaert, ‘‘Efficient metaheuris-
tics to solve the intermodal terminal location problem,’’ Comput. Oper. Res., vol. 39, no. 9, pp. 2079–2090, Sep. 2012. [40] T. F. Rossi, S. McNeil, and C. REFERENCES Hendrickson, ‘‘Entropy model for consistent
impact-fee assessment,’’ J. Urban Planning Develop., vol. 115, no. 2,
pp. 51–63, Sep. 1989. [15] M. Oudani, ‘‘A simulated annealing algorithm for intermodal trans-
portation on incomplete networks,’’ Appl. Sci., vol. 11, no. 10, p. 4467,
May 2021. [41] C. Caballini, C. Pasquale, S. Sacone, and S. Siri, ‘‘An event-triggered
receding-horizon scheme for planning rail operations in maritime ter-
minals,’’ IEEE Trans. Intell. Transp. Syst., vol. 15, no. 1, pp. 365–375,
Feb. 2014. [16] E. Demir, M. Hrusovsky, W. Jammernegg, and T. Van Woensel, ‘‘Green
intermodal freight transportation: Bi-objective modelling and analysis,’’
Int. J. Prod. Res., vol. 57, pp. 6162–6180, Oct. 2019. [42] C. Caballini, S. Fioribello, S. Sacone, and S. Siri, ‘‘An MILP optimization
problem for sizing port rail networks and planning shunting operations
in container terminals,’’ IEEE Trans. Autom. Sci. Eng., vol. 13, no. 4,
pp. 1492–1503, Oct. 2016. [17] I. C. Bilegan, T. G. Crainic, and Y. Wang, ‘‘Scheduled service network
design with revenue management considerations and an intermodal barge
transportation illustration,’’ Eur. J. Oper. Res., vol. 300, no. 1, pp. 164–177,
Jul. 2022. [43] C. F. Daganzo, ‘‘The cell transmission model: A dynamic representation
of highway traffic consistent with the hydrodynamic theory,’’ Transp. Res. B, Methodol., vol. 28, no. 4, pp. 269–287, Aug. 1994. [18] T.-S. Chang, ‘‘Best routes selection in international intermodal networks,’’
Comput. Oper. Res., vol. 35, no. 9, pp. 2877–2891, Sep. 2008. [44] S. Nguyen and C. Dupuis, ‘‘An efficient method for computing traffic
equilibria in networks with asymmetric transportation costs,’’ Transp. Sci.,
vol. 18, no. 2, pp. 185–202, May 1984. [19] C. Shao, H. Wang, and M. Yu, ‘‘Multi-objective optimization of customer-
centered intermodal freight routing problem based on the combination of
DRSA and NSGA-III,’’ Sustainability, vol. 14, no. 5, p. 2985, Mar. 2022. [45] C. S. Holling, ‘‘Resilience and stability of ecological systems,’’ Annu. Rev. Ecol. Systematics, vol. 4, no. 1, pp. 1–23, Nov. 1973. [20] S. C. Dafermos, ‘‘Integrated equilibrium flow models for transportation
planning,’’ in Traffic Equilibrium Methods. Berlin, Germany: Springer,
1976, pp. 106–118. [46] P. Gauthier, A. Furno, and N.-E. El Faouzi, ‘‘Road network resilience: How
to identify critical links subject to day-to-day disruptions,’’ Transp. Res. Rec., J. Transp. Res. Board, vol. 2672, no. 1, pp. 54–65, Dec. 2018. [21] M. Florian, ‘‘A traffic equilibrium model of travel by car and public transit
modes,’’ Transp. Sci., vol. 11, no. 2, pp. ACKNOWLEDGMENT [23] Y. Sheffiand C. Daganzo, ‘‘Hypernetworks and supply-demand equi-
librium obtained with disaggregate demand models,’’ Transp. Res. Rec.,
vol. 673, pp. 113–121, Jan. 1978. In this article, the opinions expressed are those of the authors
and do not represent the official position of the European
Commission. [24] H. K. Lo, C. W. Yip, and K. H. Wan, ‘‘Modeling transfer and non-linear
fare structure in multi-modal network,’’ Transp. Res. B, Methodol., vol. 37,
no. 2, pp. 149–170, Feb. 2003. [25] X. Fu, W. H. K. Lam, and B. Y. Chen, ‘‘A reliability-based traffic assign-
ment model for multi-modal transport network under demand uncertainty,’’
J. Adv. Transp., vol. 48, no. 1, pp. 66–85, Jan. 2014. APPENDIX In the following, the main steps that have led to the definition
of the function (39) expressing the steady-state speed with
respect to the number of trains are briefly sketched. Let us
start by considering a generic graphical train timetable and let
us define with 1X the spatial interval, 1T the time interval
and with N the number of trains present in that frame, hence
the average space headway ¯s can be defined as ¯s = 1X
N while
the average time headway ¯h is given by ¯h = 1T
N . Let us also
define the relation between the average space headway and
the average time headway that is given by ¯s = ¯h·v+L, where
v is the train speed and L is the average length of trains. Q
1
¯s
=
1
¯s · vmax
if 1
¯s ≤
1
¯h · vmax + L
1
¯h
1 −L
¯s
if
1
¯h · vmax + L < 1
¯s ≤
1
smin
(44) (44) where Q
1
¯s
is the ‘‘flow of trains’’ in function of the aver-
age space headway. The corresponding steady-state speed-
headway relation V
1
¯s
is given by V
1
¯s
=
vmax
if 1
¯s ≤
1
¯h · vmax + L
1
¯h(¯s −L)
if
1
¯h · vmax + L < 1
¯s ≤
1
smin
(45) Inspired by the traffic fundamental diagram, we can
assume that, for a given average time headway, the maximum
flow of trains in an arc corresponds to an average space
headway equal to ¯s = ¯h·vmax+L, where vmax is the maximum
speed allowed. At the same time, it has to be remembered that
trains have to maintain a minimum distance smin that allows
them to stop safely; consequently, as the number of trains
on an arc increases, they have to reduce their speed in order
to guarantee this safety distance. Then, a triangular traffic (45) Thus, equation (39), has been derived on the basis of (45)
and by considering that the inverse of the average space
headway has an equivalent meaning of ‘‘density of trains’’
in an arc that is
N tot
i,j (k)
1i,j . 73598 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks REFERENCES 166–179, May 1977. [47] E. Siri, S. Siri, and S. Sacone, ‘‘A progressive traffic assignment procedure
on networks affected by disruptive events,’’ in Proc. Eur. Control Conf. (ECC), May 2020, pp. 130–135. [22] M. Florian and S. Nguyen, ‘‘A combined trip distribution modal split
and trip assignment model,’’ Transp. Res., vol. 12, no. 4, pp. 241–246,
Aug. 1978. 73599 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks SILVIA SIRI (Member, IEEE) received the M.Sc. degree in management engineering and the Ph.D. degree in information and communication tech-
nologies from the University of Genoa, Italy, in
2002 and 2006, respectively. She is currently an
Associate Professor of automatic control. She is a
coauthor of more than 120 papers in international
journals, chapters, and conference proceedings. Her research interests include control of freeway
traffic systems, planning of logistics systems and
intermodal terminals, and optimal management of electric mobility systems. From 2019 to 2021, she was a member of the Board of Governors of the
IEEE Intelligent Transportation Systems Society. Currently, she is a Senior
Editor of the IEEE TRANSACTIONS ON INTELLIGENT TRANSPORTATION SYSTEMS,
an Associate Editor of the IEEE TRANSACTIONS ON AUTOMATION SCIENCE AND
ENGINEERING, and an Editor of the IEEE INTELLIGENT TRANSPORTATION SYSTEMS
SOCIETY NEWSLETTER. [48] E. Siri, S. Siri, and S. Sacone, ‘‘Network performance evaluation under
disruptive events through a progressive traffic assignment model,’’ IFAC
World Congr., vol. 53, no. 2, pp. 15017–15022, 2020. [49] K. Adjetey-Bahun, B. Birregah, E. Châtelet, and J.-L. Planchet, ‘‘A model
to quantify the resilience of mass railway transportation systems,’’ Rel. Eng. Syst. Saf., vol. 153, pp. 1–14, Sep. 2016. [50] R. Dorbritz, ‘‘Assessing the resilience of transportation systems in case
of large-scale disastrous events,’’ in Proc. Int. Conf. Environ. Eng., vol. 8,
2011, p. 1070. [51] N. Bešinović, ‘‘Resilience in railway transport systems: A literature review
and research agenda,’’ Transp. Rev., vol. 40, no. 4, pp. 457–478, Jul. 2020. [52] M. Hrušovský, E. Demir, W. Jammernegg, and T. Van Woensel, ‘‘Real-time
disruption management approach for intermodal freight transportation,’’
J. Cleaner Prod., vol. 280, Jan. 2021, Art. no. 124826. [53] C. Ferrari, A. Bottasso, M. Conti, E. Musso, C. Pasquale, P. P. Puliafito,
S. Sacone, M. Santagata, S. Siri, and A. Tei. (2022). New Economic Reg-
ulation for Transport in Case of Emergency Events. Handbook. [Online]. REFERENCES Available: https://cieli.unige.it/SRSP CECILIA PASQUALE (Member, IEEE) received
the bachelor’s degree in civil engineering from the
Polytechnic University of Turin, Italy, in 2009,
and the master’s degree in transports and logistics
engineering and the Ph.D. degree in monitoring
of systems and environmental risk management
from the University of Genoa, Italy, in 2012 and
2016, respectively. Currently, she is a Postdoctoral
Research Fellow with the University of Genoa. Her
research interests include modeling, optimization,
and control methods applied to the field of transportation systems. Currently,
she serves as an Associate Editor for the IEEE OPEN JOURNAL OF INTELLIGENT
TRANSPORTATION SYSTEMS and IEEE TRANSACTIONS ON INTELLIGENT VEHICLES. SIMONA SACONE (Member, IEEE) received
the master’s degree in electronic engineering and
the Ph.D. degree in electronic engineering and
computer science, in 1992 and 1997, respec-
tively. Currently, she is an Associate Professor of
automatic control with the Department of Infor-
matics, Bioengineering, Robotics and Systems
Engineering, University of Genoa, Italy, where she
acts as the Coordinator of the Ph.D. course on
systems engineering. She has authored and coau-
thored more than 150 papers published on international journals, interna-
tional books, and international conference proceedings. Since 2019, she
has been a member of the Board of Governors of the IEEE Intelligent
Transportation Systems Society. She is the Chair of the Technical Committee
on Planning and Control of Transportation Networks of the IEEE Intelligent
Transportation Systems Society. Currently, she serves as an Senior Editor for
the IEEE TRANSACTIONS ON INTELLIGENT TRANSPORTATION SYSTEMS, an Associate
Editor for the IEEE Control Systems Magazine, and an Associate Editor for
the IEEE TRANSACTIONS ON CONTROL SYSTEMS TECHNOLOGY. ENRICO SIRI (Member, IEEE) received the
B.Sc. degree in industrial engineering and the
M.Sc. degree in management engineering from
the University of Genoa, in 2016 and 2018,
respectively, where he is currently pursuing the
Ph.D. degree with the Department of Informat-
ics, Bioengineering, Robotics and Systems Engi-
neering. His research interests include systems
engineering, traffic systems modeling, assignment
models, and optimization and control. 73600 VOLUME 10, 2022
|
https://openalex.org/W3174314089
|
https://journal.ipb.ac.id/index.php/jabm/article/download/32520/21751
|
Indonesian
| null |
FORMULASI STRATEGI UNIT BISNIS LAUNDRY SEPATU (STUDI KASUS DARMAWAN WASH SHOE BOGOR)
|
Jurnal Aplikasi Bisnis dan Manajemen
| 2,021
|
cc-by
| 5,339
|
1 Corresponding author:
Email: danangwicak72@gmaiI.com Jurnal Aplikasi Manajemen dan Bisnis, Vol. 7 No. 2, Mei 2021
Permalink/DOI: http://dx.doi.org/10.17358/jabm.7.2.356
Tersedia online http://journal.ipb.ac.id/index.php/jabm Jurnal Aplikasi Manajemen dan Bisnis, Vol. 7 No. 2, Mei 2021
Permalink/DOI: http://dx.doi.org/10.17358/jabm.7.2.356
Tersedia online http://journal.ipb.ac.id/index.php/jabm TERAKREDITASI PERINGKAT 2
Direktur Jenderal Penguatan Riset dan Pengembangan,
Kemenrisek DIKTI No. 30/E/KPT/2018 Kata kunci: EFE, formuIasi strategi, IFE, QSPM, SWOT Danang Wicaksono*)1, Lukman M. Baga**), dan Tanti Novianti*** Danang Wicaksono*)1, Lukman M. Baga**), dan Tanti Novianti***) *) Sekolah Bisnis, IPB University
Jl. Raya Pajajaran, Bogor 16151, Indonesia
**) Departemen Agribisnis, Fakultas Ekonomi dan Manajemen, IPB University
Jl. Agatis Kampus IPB Dramaga, Bogor 16680, Indonesia
**) Departemen Ilmu Ekonomi, Fakultas Ekonomi dan Manajemen, IPB University
Jl. Agatis Kampus IPB Dramaga, Bogor 16680, Indonesia *) Sekolah Bisnis, IPB University
Jl. Raya Pajajaran, Bogor 16151, Indonesia
**) Departemen Agribisnis, Fakultas Ekonomi dan Manajemen, IPB University
Jl. Agatis Kampus IPB Dramaga, Bogor 16680, Indonesia
**) Departemen Ilmu Ekonomi, Fakultas Ekonomi dan Manajemen, IPB University
Jl. Agatis Kampus IPB Dramaga, Bogor 16680, Indonesia Abstract: Shoes are not only used for daily needs now, but people rather consider that
shoes are an important fashion item for their appereance. Despite the growing culture of
Indonesian sneakerhead, Darmawan Wash Shoe had recently experiencing decreased sales
from 2017 until 2019. This research aimed to 1) identifying internal and external factors
which are strengths, weaknesses, opportunities and threats that influences the sales of
Darmawan Wash Shoe; 2) formulate alternative strategies that can be implemented to
increase sales based on the influences of each internal and external factors on Darmawan
Wash Shoe; 3) determine the priority from those alternative strategies to be implemented
to achieve sales targets in Darmawan Wash Shoe. The methods used in this study are: 1)
identification of Darmawan Wash Shoe internal factors using the Internal Factor Evaluation
(IFE) method and identification of Darmawan Wash Shoe external factors using the External
Factor Evaluation (EFE) method, positioning Darmawan Wash Shoe business using the
Internal External (IE) matrix; 2) aIternative strategy formulations using the SWOT matrix
method; 4) formulating priority strategies with the Quantitative Stratategy Planning Matrix
(QSPM) method. The results of internal and external analysis found that the position of
Darmawan Wash Shoe is in quadrant V, the strategy that must be done is market penetration
and development product in the QSPM analysis, the main priority strategy is to conduct
onIine discount promotions. Keywords: EFE, IFE, QSPM, Startegy Formulation, SWOT Keywords: EFE, IFE, QSPM, Startegy Formulation, SWOT Mengacu pada data yang
berasal dari Kementerian Koperasi dan UMKM
RepubIik Indonesia mengenai Perkembangan Usaha
Mikro, Kecil, Menengah (UMKM) dan Usaha Besar
(UB) Tahun 2012-2018, UMKM di Indonesia pada
tahun 2018 berjumlah sebanyak 64,2 juta unit serta
diharuskan mencukupi tenaga kerja sebesar 97 % (116,9
juta) dilihat pada nilai masukan pada Produk Domestik
Bruto (PDB) nasional sebesar 61,07 % (8,5 miliar
rupiah). Peningkatan jumlah UMKM daIam kurun
waktu 2012 hingga 2018 dengan tingkat perkembangan
sebesar 15,5 % (8,9 juta unit) terIihat cukup pesat. Sehingga dapat diklasifikasikan dari total unit usaha
yang ada menjadi 99,99% UMKM, 99,99% usaha
mikro dan 1,22% usaha kecil. Hal tersebut mengartikan
bahwa UMKM memiIiki peran yang cukup strategis
daIam kemajuan ekonomi di Indonesia. Pengusaha laundry sepatu memikat calon pelanggan
dengan cara inovasi dan pengembangan yang
berkelanjutan. Nuriyawan (2011) menyatakan bahwa
harga, fasilitas dan kuaIitas pelayanan adalah faktor
utama kepuasan konsumen Iaundry sepatu. Penelitian
yang dilakukan Asmawati (2018) mengklasifikasikan
beberapa faktor internal yang harus menjadi concern
pada usaha bisnis Iaundry sepatu, yaitu harga, kualitas
produk, pelayanan one day service, diversifikasi
produk, komunikasi internal unit bisnis, jumlah dan
kinerja tenaga kerja dan quality control. Faktor-faktor
strategis baik internal maupun eksternal yang digunakan
dalam menyusun alternatif strategi pada penelitian
terhadap DWS, diperoleh langsung dari kondisi real
yang dihadapi perushaan. Penelitian ini menggunakan
3 tahap perumusan strategi, yaitu input, matching dan
decision. Penyebab muncuInya jenis usaha baru yang salah
satunya adalah bisnis laundry sepatu disebabkan oleh
peningkatan UMKM dan industri sepatu di Indonesia. Pada tahun 2013, laundry sepatu di Indonesia ramai
diperbincangkan seiring dengan perkembangan industri
sepatu itu sendiri. Industri pencucian sepatu atau
Iaundry sepatu sudah merada pada semua kota besar
di Indonesia. Bisnis ini tidak terlepas dari rasa ingin
yang tinggi untuk membeli oIeh masyarakat terutama
pada model sepatu jenis kats dan sneakers. Rahaju
dan Sumarlan (2013) menyatakan bahwa penyebab
pengaruh motivasi konsumen mengggunakan jasa
Iaundry adalah faktor ekonomi, gaya hidup dan faktor
alam. Dapat dilihat pada Gambar 1, penjualan DWS
cenderung menurun dari tahun 2017 hingga 2019. Menurut data yang diperoleh, DWS memiliki penjualan
tertinggi pada triwulan II tahun 2017 sebanyak 1.969
pasang sepatu. SeteIah itu penjualan DWS berfluktuasi
tidak menentu, namun hingga triwulan IV tahun
2019 tidak pernah mencapai titik penjualan tertinggi
pada triwulan II tahun 2017. Sebaliknya, penjualan
terendah terjadi pada triwulan II 2019 sebanyak 806
pasang sepatu. Pada Tahun 2019 adalah titik terendah
DWS dicerminkan dengan total penjualan yang sangat
rendah. Keywords: EFE, IFE, QSPM, Startegy Formulation, SWOT Abstrak: Sepatu saat ini tidak hanya dipakai untuk kebutuhan sehari-hari, sebagian besar
masyarakat menganggap sepatu merupakan icon penting daIam kehidupan. Darmawan
Wash Shoe memiIiki penjualan cenderung menurun dari tahun 2017 hingga 2019. Penelitian
ini memiliki tujuan sebagai berikut, yaitu 1) mengidentifikasi faktor internal dan eksternal
yang menjadi kekuatan, kelemahan, peluang dan ancaman dalam memengaruhi penjuaIan
jasa Darmawan Wash Shoe; 2) merumuskan alternatif strategi yang dapat dilakukan
untuk meningkatkan penjuaIan berdasarkan pengaruh masing-masing faktor internal dan
eksternal pada Darmawan Wash Shoe; 3) memilih prioritas strategi yang dapat digunakan
Darmawan Wash Shoe. Penelitian ini menggunakan metode, yaitu 1) identifikasi faktor
internaI Darmawan Wash Shoe menggunakan metode InternaI Factor EvaIuation (IFE) dan
identifikasi faktor eksternaI Darmawan Wash Shoe menggunakan metode ExternaI Factor
EvaIuation (EFE), penentuan posisi bisnis Darmawan Wash Shoe dengan menggunakan
matriks InternaI ExternaI (IE); 2) formulasi alternatif strategi menggunakan metode matriks
SWOT; 3) perumusan strategi prioritas dengan metode Quantitative Stratategy PIanning
Matrix (QSPM). Hasil analisis internal dan eksternal didapatkan posisi Darmawan Wash
Shoe berada pada kuadran V yaitu pada posisi sedang, sehingga grand strategy yang tepat
adalah penetrasi pasar dan perkembangan produk. Hasil dari alternatif strategi dengan
QSPM, didapatkan melakukan promosi diskon secara daring sebagai prioritas strategi. 356 Copyright © 2020, ISSN: 2528-5149/EISSN: 2460-7819 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 PENDAHUIUAN Salah satu faktor penyebab industri laundry sepatu
berkembang adalah kota-kota besar. Jumlah penduduk
Kota Bogor yang mencapai 1.081.009 juta jiwa
dengan laju pertumbuhan 1,53 % pertahunnya (Badan
Pusat Stastistik, 2018) menjadi bidang strategis yang
menguntungkan bagi para pelaku usaha laundry
sepatu. Banyaknya industri sejenis yang tersebar di
daerah usaha DWS, membuat persaingan laundry
sepatu di Kota Bogor semakin ketat dan membawa
dampak kurang baik bagi kelangsungan bisnis DWS. Penentuan strategi yang sesuai bagi perusahaan berasal
dari pemahaman yang baik dilihat pada aspek kondisi
internal dan eksternal (Chan, 2011). Komaryatin (2007)
dan Kurniawati dan Sari (2009) menyatakan bahwa
untuk memprediksi persaingan yang semakin ketat,
setiap perusahaan harus mampu merumuskan strategi
secara visioner. Perkembangan Usaha Mikro Kecil dan Menengah
(UMKM) menjadi saIah satu sektor vital guna
memajukan perekonomian di era industri 4.0,
khususnya di Indonesia. Keywords: EFE, IFE, QSPM, Startegy Formulation, SWOT DWS dapat meningkatkan volume penjualan
dan bertahan pada persaingan industri laundry sepatu
apabila memanfaatkan kelebihan dan meminimalisasi
kekurangan yang dimiliki. Seiring
dengan
perkembangan
industri
sepatu,
khususnya laundry sepatu, Darmawan Wash Shoe atau
DWS berdiri. DWS adalah salah satu UMKM di bidang
laundry sepatu yang berdiri sejak tahun 2016. DWS
memiIiki segmentasi pasar baru dan luas berdasarkan
dengan perkembangan industri sepatu di Indonesia,
dan perubahan gaya hidup konsumen di Indonesia
seperti sneakerhead atau kolektor sepatu sneakers yang
menjadikan ragam jenis sepatu sebagai tanda status
sosial. Philip (2019), menyatakan perubahan gaya hidup
yang konsumtif adalah variabel yang berpengaruh
positif signifikan terhadap keputusan pembeIian sepatu
oIeh konsumen. 357 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 07-5434 E-ISSN: 2407-7321
d by Ministry of RTHE Number 32a/E/KPT/2017
Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021
Gambar 1. Penjualan Darmawan Wash Shoe Gambar 1. Penjualan Darmawan Wash Shoe Gambar 1. Penjualan Darmawan Wash Shoe Uraian permasalahan menunjukkan perlunya langkah
strategis yang tepat melalui formulasi strategi. Oleh
karena itu, perlu dilakukan analisis lingkungan internal
dan eksternal pada DWS untuk mendapatkan alternatif
dan strategi prioritas yang dapat diterapkan dalam
persaingan untuk meningkatkan pendapatan penjualan
dan bertahan di industri laundry sepatu. Tujuan
penelitian ini adalah mengidentifikasi faktor internal
dan eksternal yang memengaruhi penjualan jasa DWS;
Merumuskan alternatif strategi DWS; Menentukan
strategi penjualan yang menjadi prioritas pada DWS. Penelitian ini mencakup ruang lingkup terbatas meliputi
melakukan pengamatan kondisi internal dan eksternal
DWS, memformulasi alternatif strategi penjualan dan
memprioritaskan strategi aIternatif berdasarkan data
penjualan DWS pada tahun 2017-2019. Uraian permasalahan menunjukkan perlunya langkah
strategis yang tepat melalui formulasi strategi. Oleh
karena itu, perlu dilakukan analisis lingkungan internal
dan eksternal pada DWS untuk mendapatkan alternatif
dan strategi prioritas yang dapat diterapkan dalam
persaingan untuk meningkatkan pendapatan penjualan
dan bertahan di industri laundry sepatu. Tujuan
penelitian ini adalah mengidentifikasi faktor internal
dan eksternal yang memengaruhi penjualan jasa DWS;
Merumuskan alternatif strategi DWS; Menentukan
strategi penjualan yang menjadi prioritas pada DWS. Penelitian ini mencakup ruang lingkup terbatas meliputi
melakukan pengamatan kondisi internal dan eksternal
DWS, memformulasi alternatif strategi penjualan dan
memprioritaskan strategi aIternatif berdasarkan data
penjualan DWS pada tahun 2017-2019. Penelitian ini menggunakan jenis dan sumber berupa
data primer dan sekunder. Data primer, diperoleh
langsung dari responden hasil wawancara. Keywords: EFE, IFE, QSPM, Startegy Formulation, SWOT Responden
dalam penelitian ini berjumlah 2 orang internal
perusahan yaitu pemiIik unit bisnis dan manajer
dan 120 konsumen DWS. Rumusan masalah dan
perumusan pengembangan bisnis dijawab dengan
melihat data primer penelitian yang dikaitkan dengan
tujuan spesifik penelitian (Sarwono, 2006). Data
sekunder, diperoleh dalam bentuk dokumen dan dapat
berupa berbagai sumber rujukan atau literatur berupa
dokumen-dokumen yang berhubungan dengan topik
penelitian ini. Membaca, melihat dan mendengarkan
oleh peneliti adalah data sekunder yang sudah tersedia
(Sarwono, 2006). Penentuan responden menggunakan teknik purposive
sampling (penentuan responden secara sengaja),
yaitu pihak yang menjawab berbagai pertanyaan
dari
kuisioner
untuk
kepentingan
penelitian. Responden dipilih berdasarkan kompetensi keahian
dalam pemberian penilaian strategi pengembangan
bisnis pada DWS dikarenakan mengetahui kondisi
lapangan perusahaan. Teknik purposive sampIing
mempertimbangkan responden yang dipilih memiIiki
pengetahuan, keahlian dan pengalaman dalam bidang
yang di teliti (Sugiyono, 2013). METODE PENELITIAN Data penelitian berasal dari salah satu UMKM yang
bergerak di bidang jasa laundry sepatu yaitu DWS
yang berada di JaIan KoI. Ahmad Syam, Bogor. Penelitian ini dilakukan pada bulan Maret-Juni 2020. Data penelitian dikumpulkan dalam beberapa tahap,
yaitu tahap pertama pengumpulan data melalui
wawancara dengan stakeholders yang terkait dengan
dunia usaha. Pada tahap pertama, tujuannya adalah
untuk menyaring persepsi dan informasi dari sumber
DWS untuk mengetahui faktor internal yaitu kelebihan
dan kekurangan, dan faktor eksternal yaitu peIuang
dan ancaman DWS. Tahap selanjutnya adalah dengan
memberikan kuesioner kepada 120 konsumen yang
diIakukan untuk mengidentifikasi alasan konsumen
memilih Iayanan DWS. AnaIisis IFE (Internal Factor Evaluation) digunakan
untuk mengidentifikasi faktor-faktor strategis berupa
kekuatan dan kelemahan internal. Sedangkan, EFE
(External
Factor
Evaluation)
digunakan
untuk
menganalisis faktor strategis berupa peIuang dan
ancaman perusahaan. Masalah ini menjadi sangat
penting karena kinerja perusahaan akan dipengaruhi
secara langsung maupun tidak langsung oleh faktor 358 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 7-7321
E Number 32a/E/KPT/2017
Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 internal dan eksternal. Selain itu, hasil pembobotan
IFE dan EFE akan dimasukkan ke daIam kuadran atau
matriks berbentuk segiempat IE. AnaIisis IE merupakan
kombinasi dari faktor internal dan eksternal. Parameter
yang digunakan meliputi faktor internal dan eksternal
yang dihadapi perusahaan. Menurut Raymond et aI. (2012) matriks IE adalah alat perumusan strategi
yang menguraikan dan mengevaluasi kekuatan dan
kelemahan utama area fungsional bisnis, dan juga
berfungsi sebagai dasar untuk mengidentifikasi dan
mengevaluasi hubungan antara area tersebut. menentukan daya tarik relatif (reIative attractiveness)
dari strategi alternatif yang dipilih, untuk menentukan
strategi mana yang dianggap paIing tepat untuk
diimplementasikan (David dan David, 2011). AnaIisis
QSPM digunakan untuk menentukan strategi yang
terbaik dalam pemutusan keputusan yang akan
dijalankan oleh perusahaan dalam jangka pendek
maupun panjang (Isnandar et al. 2016). DWS adalah UMKM yang bergerak di bidang jasa
laundry sepatu di Kota Bogor. Penurunan penjualan
dan tidak stabilnya penjualan terjadi pada Darmawan
Wash Shoe. Menganalisis faktor eksternal dan internal
Darmawan Wash Shoe untuk mendapatkan faktor
strategis penting untuk merumuskan alternatif strategi
DWS. Faktor-faktor tersebut kemudian diidentifikasi
dan dipiIih mana yang menjadi kekuatan, kelemahan,
peIuang dan ancaman dengan mengunakan analisis
SWOT dan IE. Identifikasi Faktor Internal HASIL HASIL Analisis internal merupakan analisis yang digunakan
untuk mengidentifikasi kekuatan dan kelemahan suatu
perusahaan atas kualitas sumber daya dan kemampuan
yang dimiliki (Capps dan GIissmeyer, 2012). Kekuatan
merupakan faktor internal yang memberi perusahaan
keunggulan komparatif di pasar (Kotler dan Keller,
2016). Pada penelitian Asmawati (2018), harga yang
terjangkau adalah salah satu kekuatan yang harus
dimiliki unit usaha laundry. Penelitian yang diIakukan
Umar et aI. (2016), kuaIitas pelayanan yang baik
merupakan salah satu kekuatan yang harus dimiliki unit
usaha laundry. Kekuatan yang diidentifikasi pada DWS
yaitu harga yang ditawarkan terjangkau konsumen,
kuaIitas pelayanan yang baik, terdapatnya Iayanan
fast cIeaning, banyaknya varian parfum, komunikasi
yang baik terhadap konsumen, terdapat garansi/cuci
uIang. Kelemahan yang diidentifikasi yaitu belum
menggunakan teknologi pengering sepatu, manajemen
keuangan yang belum baik, SOP lapangan yang suIit
direalisasikan, komunikasi yang kurang baik pada
internal unit usaha, administrasi yang belum baik dan
modal promosi yang terbatas. Skor rata-rata keseluruhan
faktor internal adalah 2,30 yang menunjukkan bahwa
DWS memiliki kondisi internal sedang. Hasil matriks
IFE selengkapnya pada TabeI 1. Gambaran Umum Unit Bisnsi Darmawan Wash
Shoe Darmawan Wash Shoe merupakan salah satu unit usaha
yang berdiri pada tahun 2016 bergerak daIam bidang
jasa industri yaitu washing shoes oIeh Vicky Darmawan. DWS melihat pangsa pasar baru dan terbilang Luas
dengan melihat perkembangan industri sepatu dan
perubahan gaya hidup konsumen di Indonesia seperti
sneakerhead atau kolektor sepatu sneaker yang
menjadikan koleksi sepatu sebagai tanda status sosial. DWS mendirikan outlet di Jalan Kol. Ahmad Syam,
Bogor dimana lokasi tersebut dapat dikatakan sebagai
Iokasi yang strategis untuk melakukan kegiatan bisnis. Motivasi Vicky sebagai pemilik bisnis dalam mendirikan
bisnis laundry sepatu yaitu karena dirinya adalah salah
satu sneakerhead atau koIektor sepatu sneaker. Vicky
mendapat peluang bisnis yang baru, setiap jenis sepatu
terbuat dari berbagai jenis dan bahan yang berbeda. Perbedaan jenis setiap bahan sepatu tentu memiIiki
perawatan yang berbeda. DWS menyediakan layanan
laundry sepatu, perusahaan sudah menentukan setiap
jenis bahan sepatu dan jenis perawatan yang sesuai. DWS tidak hanya menerima laundry sepatu, tetapi juga
menerima berbagai perawatan sepatu Iainnya. TabeI 1. METODE PENELITIAN Kemudian diIakukan formulasi dalam
penentuan alternatif strategi dari berbagai faktor yang
menjadi kekuatan, kelemahan, peluang dan ancaman. Analisis QSPM digunakan untuk merumuskan strategi
apa yang akan menjadi prioritas dalam pencapaian target
penjualan pada DWS. Adapun kerangka pemikiran
penelitian dapat dilihat pada Gambar 2. Matriks SWOT digunakan sebagai alat untuk
mengembangkan strategi alternatif. Matriks SWOT
secara jeIas menggambarkan beberapa peIuang
dan ancaman eksternal yang dihadapi perusahaan
berdasarkan kekuatan dan kelemahannya. Langkah
seIanjutnya adalah mengembangkan opsi strategis
meIaIui perbandingan berpasangan. Matriks SWOT
dapat membuat perbandingan berpasangan antara
kekuatan dan peluang (SO), kekuatan dan ancaman
(ST), peluang dan kelemahan (WO), serta kelemahan
dan ancaman (WT). SeteIah didapatkan alternatif
strategi analisis SWOT, maka penentuan prioritas
strategi akan menggunakan Quantitative Strategic
Planning Matrix (QSPM). QSPM digunakan untuk Gambar 2. Kerangka pemikiran penelitian Gambar 2. Kerangka pemikiran penelitian 359 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Identifikasi Faktor Eksternal yang dikatakan menjadi peluang DWS adalah Iokasi
yang strategis dengan skor 0,61. Sedangkan faktor
yang menjadi ancaman utama DWS adalah munculnya
pesaing di area unit bisnis dengan skor 0,23. total skor
rata-rata dari faktor eksternal adalah 2,92 seIengkapnya
pada Tabel 2. AnaIisis
faktor
eksternal
perusahaan
meliputi
lingkungan industri dari berbagai faktor yang akan
menyebabkan perusahaan menghadapi peIuang dan
ancaman. Penelitian yang dilakukan Rofik (2017)
mengidentifikasi beberapa faktor peluang yang
diidentifikasi, yaitu pelanggan tetap, perkembangan
penduduk sekitar usaha, bahan baku yang murah dan
relatif melimpah, harga layanan yang relatif murah
dibanding kompetitor, lokasi strategis dan modal usaha
yang relatif tidak membutuhkan modal besar. Ancaman
yang diidentifikasi pada penelitian Eko (2013) adalah
Banyaknya pesaing di perusahaan sejenis, kenaikan
harga bahan bakar yang relatif tinggi, juga kenaikan
harga dasar listrik, serta perusahaan sejenis atau pesaing
dengan dana besar dan cuaca yang tidak menentu. Identifikasi Faktor Internal HasiI matriks IFE
Bobot
Peringkat
Skor
Ranking
Kekuatan
Harga yang ditetapkan terjangkau
0,06
3
0,18
6
KuaIitas peIayanan yang baik
0,09
4
0,36
2
MemiIiki peIayanan Fast cIeaning/One day service
0,08
3
0,24
4
Banyak piIihan varian parfum
0,07
3
0,21
5
MemiIiki hubungan dan komuniasi yang baik
0,10
4
0,40
1
Konsumen mendapatkan garansi/cuci uIang
0,08
4
0,32
3
KeIemahan
BeIum menggunakan teknoIogi pengering sepatu
0,05
2
0,10
4
SOP Iapangan suIit direaIisasikan
0,09
1
0,09
5
Komunikasi pada internaI bisnis kurang
0,09
1
0,09
3
Administrasi beIum baik
0,08
2
0,16
6
Manajemen keuangan yang beIum baik
0,08
1
0,08
2
ModaI promosi terbatas
0,07
1
0,07
1
TotaI Skor Faktor Internal
2,30 TabeI 1. HasiI matriks IFE TabeI 1. HasiI matriks IFE 360 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Matriks Internal-Eksternal (IE) Dwiastuti (2008), menyatakan bahwa kuadran IE
berdasar pada nilai total matriks IFE berbobot sumbu
X dan nilai total matriks EFE berbobot sumbu Y. Analisis matriks IE adalah strategi yang meringkas
dan mengevaluasi keunggulan dan kelemahan utama
pada kuadran bisnis, serta menjadi landasan untuk
mengklasifikasikan dan mengidentifikasi hubungan
area tersebut (Raymond et aI. 2012). Hasil penggunaan
matriks IFE untuk menganalisis faktor internal
mendapatkan skor 2,30, dan hasil penggunaan matriks
EFE untuk analisis eksternal mendapatkan skor 2,92. Dengan demikian terlihat bahwa posisi DWS saat ini
berada pada kuadran V yang digambarkan pada Gambar
3. Dilihat dari matriks IE, maka posisi perusahaan
berada pada kuadran V dengan posisi dalam kategori
sedang yang dapat diIihat pada Gambar 3. HaI ini
menandakan grand strategy yang paling tepat diIakukan
oIeh DWS adalah hold and maintain strategy. Menurut
David (2016) strategi yang dapat dikembangkan adalah
market penetration dan product development. Peluang
yang
diidentifikasi
pada
DWS
yaitu
perkembangan jumIah penduduk, perubahan gaya hidup
masyarakat, segmentasi relatif luas, kemajuan teknoIogi
internet/sosial media, lokasi strategis dan melakukan
diversifikasi produk. Ancaman yang diidentifikasi pada
DWS yaitu munculnya pesaing baru sejenis, harga sewa
Iahan yang selalu naik, musim hujan. Penelitian yang
diIakukan Siregar (2019) mengidentifikasi beberapa
faktor peIuang yang diidentifikasi, yaitu pertumbuhan
jumlah penduduk yang mengimplikasinya pertumbuhan
pangsa pasar ikut meningkat dan fasilitas laundry yang
memadai. Berdasarkan hasil analisis tersebut faktor TabeI 2. HasiI matriks EFE
Bobot
Peringkat
Skor
Ranking
PeIuang
Perkembangan jumIah penduduk
0,06
3
0,20
6
Perubahan gaya hidup masyarakat
0,11
4
0,47
2
Segmentasi pasar reIatif Iuas
0,10
4
0,43
3
Kemajuan teknoIogi internet/sociaI media
0,12
3
0,38
4
Iokasi strategis
0,15
4
0,61
1
MeIakukan diversifikasi produk
0,09
3
0,29
5
Ancaman
MuncuInya pesaing di area unit bisnis
0,11
2
0,23
1
Biaya sewa Iahan yang seIaIu naik
0,12
1
0,12
3
Musim hujan
0,07
2
0,15
2
TotaI Skor Faktor EnternaI
2,92 TabeI 2. HasiI matriks EFE Strategi S-O Perumusan tersebut didasarkan pada ide korporat
untuk memanfaatkan peluang dengan menggunakan
semua kelebihannya. Strategi S-O digunakan untuk
melihat kekuatan internal perusahaan sebagaimana
dimanfaatkan daIam melihat peluang eksternal. Dalam
matrik SWOT dirumuskan strategi SO, yaitu promosi
diskon online diIakukan untuk meningkatkan jumIah
pelanggan. Strategi promosi diskon secara daring
untuk meningkatkan jumlah kosumen sejaIan dengan
penelitian yang dilakukan Varatisha (2017). HaI ini
dengan menggunakan kekuatan DWS seperti S1: harga
yang ditetapkan terjangkau konsumen, untuk merebut
dan memanfaatkan peluang yaitu O1: perkembangan
jumIah penduduk, O2: gaya hidup masyarakat,
O4: kemajuan teknologi internet dan sosial media. Selanjutnya, melakukan reminder wash shoe secara
berkala dengan memanfaatkan sosial media, hal ini
dengan menggunakan kekuatan DWS seperti S5:
Menjaga komunikasi yang baik dengan konsumen
untuk merebut dan memanfaatkan peluang, yaitu O4:
kemajuan teknoIogi internet dan sosial media. PeIuang 361 -ISSN: 2407-5434 E-ISSN: 2407-7321
Accredited by Ministry of RTHE Number 32a/E/KPT/2017
Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Gambar 3. Matriks IE Darmawan Wash Shoe Gambar 3. Matriks IE Darmawan Wash Shoe Strategi W-O Strategi W-O diterapkan dengan tujuan meminimaIisir
kelemahan DWS dan meIihat keberuntungan atau
peluang yang ada, Strategi W-O. Pada matriks SWOT
terdapat strategi mengadakan agenda gathering atau
berwisata ke tempat-tempat yang sedang trend secara
berkala dengan memanfaatkan kelemahan W2: SOP
lapangan yang sulit direaIisasikan, W3: kurangnya
hubungan dan komunikasi yang baik pada internal
bisnis, W4: administrasi yang belum baik, W5:
manajemen keuangan yang belum baik, untuk meraih
peluang O2: gaya hidup masyarakat yang konsumtif. Strategi ini ditujukan kepada bagian internal bisnis. Strategi SWOT DWS seperti S2: kualitas pelayanan yang baik, S3:
terdapatnya pelayanan Fast CIeaning/One Day Service
yang diminati konsumen, S4: banyaknya pilihan varian
parfum, S5: memiliki hubungan dan komunikasi yang
baik dengan konsumen, S6: terdapat garansi/cuci
uIang, untuk mengatasi ancaman, yaitu T2: harga sewa
Iahan yang selalu naik. Selanjutnya, meningkatkan
daya saing dengan peningkatan kualitas pelayanan,
haI ini dengan menggunakan kekuatan DWS seperti
S1: harga yang ditetapkan terjangkau konsumen, S2:
kualitas pelayanan yang baik, S4: banyaknya pilihan
varian parfum, S5: memiliki hubungan dan komunikasi
yang baik dengan konsumen, S6: terdapat garansi cuci
uIang, untuk mengatasi ancaman, yaitu T1: munculnya
pesaing baru di area unit bisnis. Strategi tersebut
konsisten serta tepat, yaitu memiliki daya saing dan
kualitas pelayanan yang terus meningkat, DWS akan
memiliki brand awareness yang baik bagi konsumen
dan dapat menjadi laundry sepatu yang unggul dari
competitor (Dewi, 2018). Strategi S-T Perumusan disusun berdasarkan keunggulan korporat
dalam mencegah terjadinya ancaman. Strategi S-T
melihat
keunggulan
internal
perusahaan
untuk
mengatasi ancaman eksternal. DaIam matriks SWOT,
strategi pemberian hak istimewa dirumuskan kepada
pemiIik lahan berupa layanan gratis yang terdapat
pada DWS, haI ini dengan menggunakan kekuatan 362 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Selanjutnya, memanfaatkan investor untuk penambahan
modal promosi daIam upaya meningkatkan daya saing
dan pangsa pasar dengan memanfaatkan kelemahan,
yaitu W6: modal promosi yang terbatas, untuk meraih
peluang O1: perkembangan jumIah penduduk, O2: gaya
hidup masyarakat, O3: segmentasi pasar relatif Luas. DWS dapat mempertimbangkan untuk melakukan
penambahan modal baik mandiri ataupun dari pihak
investor, dimana diharapkan dengan modal yang cukup
maka promosi akan diIakukan lebih maksimal sehingga
penetrasi pasar akan berjaIan dengan baik. Selanjutnya, memanfaatkan investor untuk penambahan
modal promosi daIam upaya meningkatkan daya saing
dan pangsa pasar dengan memanfaatkan kelemahan,
yaitu W6: modal promosi yang terbatas, untuk meraih
peluang O1: perkembangan jumIah penduduk, O2: gaya
hidup masyarakat, O3: segmentasi pasar relatif Luas. DWS dapat mempertimbangkan untuk melakukan
penambahan modal baik mandiri ataupun dari pihak
investor, dimana diharapkan dengan modal yang cukup
maka promosi akan diIakukan lebih maksimal sehingga
penetrasi pasar akan berjaIan dengan baik. (TAS) tertinggi hingga terendah, semua alternatif
strategi yang dianilisis dengan menggunakan QSPM
pada Darmawan Wash Shoe selengkapnya pada Tabel
6. Tabel 6. Alternatif strategi hasil analisis QSPM
NO
Alternatif strategi
TAS
1
Melakukan promosi diskon secara daring
6,15
2
Mengadakan kerja sama dengan
beberapa unit bisnis seperti coffee shop
dan barbershop
6,07
3
Melakukan reminder wash shoe secara
berkala
5,80
4
Meningkatkan daya saing dengan
peningkatan kualitas layanan
5,64
5
Memberikan privilege kepada pemilik
lahan berupa layanan gratis
5,56
6
Mengadakan agenda gathering dan
training dengan ke tempat-tempat yang
sedang trend
5,11
7
Menggunakan alat berteknologi
pengering sepatu
4,86
8
Memanfaatkan investor untuk
penambahan modal dan biaya promosi
4,78 Tabel 6. ImpIikasi ManajeriaI Prioritas strategis utama yang direkomendasikan
untuk DWS adalah meIakukan promosi diskon
secara daring untuk meningkatkan meningkatkan
daya saing dan jumIah kosumen. Perusahaan harus
dapat mempertahankan dan meningkatkan jumIah
konsumen baru. DWS dapat membuat potongan harga
berupa voucher dan didistribusikan kepada setiap
konsumen dengan memanfaatkan media sosiaI onIine
seperti Instagram, Youtube, Facbook, Iine dan Iain
sebagainya. Prioritas strategi kedua yang disarankan adalah
mengadakan kerja sama dan kemitraan dengan beberapa
unit bisnis seperti coffee shop dan barbershop daIam
upaya melakukan promosi dan meningkatkan daya
saing. Perkembangan zaman dan perkembangan gaya
hidup masyarakat sangat berarti bagi upaya perusahaan
untuk menjalin kerja sama dengan departemen bisnis
jenis lain, sehingga perusahaan dapat memasuki pasar
yang lebih luas. DWS dapat mengerjakan sesuatu haI
yang unik seperti membuat suatu event seperti expo
dan menjadikan mitra sebagai drop zone sepatu dengan
bekerja sama atau bermitra dengan jenis unit usaha Strategi S-T Alternatif strategi hasil analisis QSPM
NO
Alternatif strategi
TAS
1
Melakukan promosi diskon secara daring
6,15
2
Mengadakan kerja sama dengan
beberapa unit bisnis seperti coffee shop
dan barbershop
6,07
3
Melakukan reminder wash shoe secara
berkala
5,80
4
Meningkatkan daya saing dengan
peningkatan kualitas layanan
5,64
5
Memberikan privilege kepada pemilik
lahan berupa layanan gratis
5,56
6
Mengadakan agenda gathering dan
training dengan ke tempat-tempat yang
sedang trend
5,11
7
Menggunakan alat berteknologi
pengering sepatu
4,86
8
Memanfaatkan investor untuk
penambahan modal dan biaya promosi
4,78 Strategi W-T Strategi ini bersifat defensif dengan meminimaIisir
kelemahan dan mengkaji ulang ancaman terhadap DWS. Strategi W-T bertujuan meminimaIisir kelemahan
internal dengan mengkaji ulang ancaman eksternal. DaIam matriks SWOT, strategi W-T dirumuskan, yaitu
menggunakan aIat berteknoIogi sebagai pengering
sepatu berdasarkan jenis dan bahan sepatu dalam
upaya mengontrol perubahan cuaca yang memengaruhi
kuaIitas
pelayanan
pencucian
sepatu
dengan
meminimalkan kelemahan W1: belum menggunakan
teknologi mesin pengering, untuk menghindari
ancaman T3: musim hujan. Strategi ini merupakan
strategi difensif yang sangat baik untuk menghindari
ancaman penurunan penjualan akibat musim hujan. SeIanjutnya, mengadakan kerja sama dan kemitraan
dengan beberapa unit bisnis dengan meminimalkan
kelemahan W6: modal promosi yang terbatas, untuk
menghindari ancaman T1: munculnya pesaing baru di
area unit bisnis. Penelitian yang dilakukan Hutahaean
(2017) menghasilkan strategi yang serupa, yaitu
mengadakan kerja sama dengan bisnis lain. Strategi ini
merupakan bentuk difensif atau bertahan yang sangat
baik jika dilakukan oIeh DWS. daIam kerja sama akan
memperoIeh keuntungan. DAFTAR PUSTAKA Asmawati H. 2018. Strategi pengembangan usaha
dengan metode anaIisa SWOT pada usaha
Istiqomah Samarinda. JournaI of administration
business 6(1): 65-76. [BPS] Badan Pusat Statistik. 2018. Kota Bogor
daIam angka 2017. https://bogorkota.bps.go.id/
statictabIe/2018/10/01/183/jumIah-penduduk-
dan-Iaju-pertumbuhan-penduduk-menurut-
kecamatan-di-kota-bogor-2010-2016-dan-2017. htmI [2 Mar 2020]. Saran Strategi prioritas ketiga yang disarankan adalah
meIakukan reminder wash shoe secara berkala dengan
memanfaatkan media sosial online. Strategi ini bertujuan
menjadi pengingat kepada setiap konsumen dan bahkan
seluruh penduduk Kota Bogor yang memiliki sepatu
untuk membersihkan sepatu yang sudah kotor. Strategi
tersebut adalah hal lumrah yang diIakukan oIeh
perusahaan besar, dengan tujuan menjaga performa dan
kualitas produk. Hasil penelitian ini dibatasi hingga perumusan prioritas
strategi pada bisnis laundry sepatu DWS, sehingga
penelitian seIanjutnya disarankan untuk meIakukan
analisis
terhadap
implementasi
strategi
yang
sudah dihasilkan dan melakukan evaluasi terhadap
pelaksanaan strategi. Penelitian selanjutnya disarankan
untuk mencari pembanding terhadap shoes laundry lain
sebagai penentu faktor strategis. Penelitian selanjutnya
disarankan untuk melakukan identifikasi ulang terhadap
persaingan industri selama pandemic Covid-19. Strategi prioritas keempat yang disarankan adalah
meningkatkan daya saing dengan peningkatan kuaIitas
layanan. Memiliki daya saing dan kualitas pelayanan
yang unggul, membuat konsumen cenderung akan Iebih
memilih suatu merek. Strategi prioritas selanjutnya
adalah menggunakan alat berteknologi pengering
sepatu dalam upaya mengontrol musim hujan yang
memengaruhi volume penjualan dan kualitas pelayanan
laundry sepatu. Perusahaan harus segera membuat
perencanaan pembelian alat teknologi pengering
sepatu, dengan mengimplementasikan strategi kelima,
diharapkan perusahaan dapat menghindari ancaman
penurunan penjualan akibat musim hujan. KESIMPULAN DAN SARAN Capps CJ, GIissmeyer MD. 2012. Extending the
competitive profiIe matrix using internal factor
evaIuation and externaI factor evaIuation matrix
concepts. JournaI of AppIied Business Research. 28(5):1059-1062. AnaIisis Matriks QSPM MeIihat analisis lingkungan internal dan eksternal
dengan matriks IFE dan EFE serta dipadankan dengan
matriks IE dan SWOT. Selanjutnya, adalah tahap
pengambiIan putusan prioritas strategis dengan QSPM. AnaIisis SWOT dan Quantitative Strategic Planning
Matrix (QSPM) adalah analisis tingkat tinggi yang
biasa digunakan untuk menentukan strategi manajemen
(Setyorini, 2017). Berdasarkan hasil analisis QSPM,
strategi diperoIeh nilai Total Attractiveness Score 363 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 berteknologi pengering sepatu. Strategi prioritas yang
dapat diusulkan adalah meIakukan promosi diskon
secara daring untuk meningkatkan jumIah kosumen
dengan skor TAS 6,15. lainnya. Dua strategi di atas diutamakan karena strategi
melakukan promosi dan bekerja sama dengan unit
usaha lain akan dengan cepat meningkatkan daya saing
atau peningkatan pangsa pasar sehingga perusahaan
dapat menyiapkan strategi lain. KesimpuIan Hasil analisis internal dengan menggunakan matriks
IFE adalah 2,30, dan hasil analisis eksternal dengan
menggunakan matriks EFE adalah 2,92. Posisi
perusahaan pada matriks IE berada pada kondisi
perusahaan yang sedang. Strategi yang dapat dirumuskan
adalah market penetration dan product development. Analisis SWOT DWS menghasilkan 8 alternatif
strategi ,yaitu melakukan promosi diskon secara daring,
mengadakan kerja sama dan kemitraan dengan beberapa
unit, meningkatkan daya saing dengan peningkatan
kualitas layanan, melakukan reminder wash shoe
secara, memberikan privilege kepada pemiIik lahan
berupa layanan gratis, mengadakan agenda training
dalam bentuk gathering, memanfaatkan investor untuk
penambahan modal dan biaya promosi daIam upaya
meningkatkan segmentasi pasar, menggunakan alat Chan X. 2011. A SWOT study of the development
strategy of haier group as one of the most
successfuI chinese enterprises. InternationaI
JurnaI of Business and SociaI Science 2(11):
147–153. David FR. 2016. Manajemen Strategik Suatu Pendekatan
KeungguIan
Bersaing. Puspasari,
Novita,
penerjemah; Dedy A, editor. Jakarta: SaIemba
Empat. Terjemahan dari: Strategic Management. A Competitive Advantage Approach, Concepts
amd Case. David ME, David FR. 2011. The quantitative strategic
pIanning matrix (QSPM) appIied to a retaiI
computer store. The CoastaI Business JournaI. 8(1):42-52. 364 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM),
Vol. 7 No. 2, Mei 2021 branded oIeh generasi z di Surabaya. JurnaI
Manajemen Bisnis 7(2): 4-5. Dewi NR. 2018. Formulasi strategi pengembangan
bisnis salon xyz. Jurnal ApIikasi Manajemen
dan Bisnis 4(3):372. Rahaju EE, Sumarlan. 2013. Identifikasi variabeI
yang memotivasi konsumen menggunakan jasa
laundry. JurnaI business review 2(2):55-56. Dwiastuti I. 2008. Analisis manajemen strategi industri
aIternatif (Studi Kasus Biofuel). Jurnal Ekonomi
dan Pembangunan 16(1):21-33. Raymond MIB, Ine M, Iwang G. 2012. AnaIisis
pengembangan usaha pemindangan ikan di
Kecamatan bekasi Barat. JurnaI Perikanan dan
KeIautan 3(1): 17–24. Eko JAS. 2013. Strategi keunggulan bersaing pada
Diva Iaundry daIam menghadapi persaingan
antar usaha jasa di Mojokerto. Jurnal IImiah
Universitas Brawijaya 2(1):5-7. Rofik A. 2017. The Marketing strategy of Sshoe
washing services in Surabaya (case study of
farcIean shoes washing in Surabaya). JurnaI
Manajemen Kinerja 3(2):7-9. Hutahaean R. 2017. Perumusan strategi bisnis PT
XYZ. JurnaI ApIikasi Manajemen dan Bisnis
3(3):454. Sarwono J. 2006. Metode Penelitian Kuantitatif dan
KuaIitatif . Yogyakarta: Graha IImu. Isnandar FR, Firdaus M, Maulana A. 2016. Strategi
peningkatan aset PT BPR Syariah Harta Insan
Karimah (HIK) CiIedug. JurnaI ApIikasi Bisnis
dan Manajemen 2(1): 12–22. Setyorini R, Rey RO. 2017. KesimpuIan AnaIisis model bisnis pada
eighteen nineteen laundry dengan pendekatan
business modeI canvas. Jurnal Sekretariat
Administrasi Bisnis 1(1):70-81. Kementerian Koperasi dan UMKM 2020. Perkembangan
Data Usaha Mikro, KeciI, Menengah (UMKM)
dan Usaha Besar (UB) Tahun 2012-2-18. http://
www.depkop.go.id. [2 Mar 2020]. Siregar IV. 2019. AnaIisis prospek dan strategi
pengembangan usaha jasa laundry berbasis
syariah di kota Medan. Jurnal Ilmu Manajemen
dan Bisnis Islam 5(1):115-117. KotIer P, KeIIer KI. 2016. Marketing Management,
15th. New Jersey: Pearson Pretice HaII, Inc. Sugiyono. 2013. Metode Penelitian Pendidikan
Pendekatan Kuantitatif, Kualitatif, dan R&D. Bandung: AIfabeta Kurniawati T, Sari KBDK. 2009. Analisis dan piIihan
Strategi: membangun eksistensi perusahaan di
masa kritis. JournaI Ekonomi Bisnis 14(3): 179–
190. Umar A, Sasongko AH, Aguzman G, Sugiharto. 2016. Analisa SWOT pada bisnis rumahan studi kasus
pada bisnis laundry kiloan. Jurnal Bisnis dan
Manajemen 2(2): 91-94. Nuriyawan. 2011. Faktor-faktor yang memengaruhi
kepuasan konsumen daIam memakai jasa cuci
kiIoan pada D’Iaundry dan dry cIean di Ktintang
Baru SeIatan Surabaya [tesis]. Surabaya:
Universitas Pembangunan NasionaI. Varatisha AB. 2017. Sosial media sebagai pasar bagi
masyarakat modern sebuah kritik terhadap
budaya popuIer. JournaI UIN AIauddin 18(1):
116-130. PhiIip. 2019. Pengaruh gaya hidup terhadap keputusan
pembeIian dengan periIaku konsumtif sebagai
variabeI intervening pada pembeIian sneakers 365
|
https://openalex.org/W3005209155
|
https://thericejournal.springeropen.com/track/pdf/10.1186/s12284-019-0354-2
|
English
| null |
How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis
|
Rice
| 2,020
|
cc-by
| 14,017
|
Abstract The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing
technology has led to great progress in monocot research; however, several factors need to be considered for the
efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA
are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted
mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need
to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key
factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes,
many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the
evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing
technology has led to great progress in monocot research; however, several factors need to be considered for the
efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA
are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted
mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need
to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key
factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes,
many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the
evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA
ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of
sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can
reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce
new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Abstract Four
topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to
SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to
encompass a full spectrum of information for genome editing in monocot crops. Keywords: Cas12a, Genome editing, Plant transformation, Promoter, Protoplast REVIEW Open Access © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. * Correspondence: cslin99@gate.sinica.edu.tw
†Jin-Jun Yue, Chwan-Yang Hong, Pengcheng Wei and Yu-Chang Tsai
contributed equally to this work.
5Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
Full list of author information is available at the end of the article Yue et al. Rice (2020) 13:9
https://doi.org/10.1186/s12284-019-0354-2 Yue et al. Rice (2020) 13:9
https://doi.org/10.1186/s12284-019-0354-2 Yue et al. Rice (2020) 13:9
https://doi.org/10.1186/s12284-019-0354-2 How to start your monocot CRISPR/Cas
project: plasmid design, efficiency
detection, and offspring analysis Jin-Jun Yue1†, Chwan-Yang Hong2†, Pengcheng Wei3†, Yu-Chang Tsai4† and Choun-Sea Lin5* Design and Construction of Plasmids for Genome
Editing in Monocots double-strand breaks (DSBs), thereby generating a short
deletion or insertion. Genome-edited plants can be gen-
erated either by stable or transient transformation. For
stable
transformations,
the
Agrobacterium-mediated
transformation method is typically used to deliver trans-
fer DNA (T-DNA) into the plant cell, where it is then
inserted into the plant genome (Mikami et al. 2015a;
Nandy et al. 2019). For transient transformations, par-
ticle bombardment and polyethylene glycol (PEG)-medi-
ated
methods
are
used
to
deliver
plasmids
or
ribonucleoproteins (RNPs) into the plant cells (Woo
et al. 2015; Svitashev et al. 2016; Zhang et al. 2016; Lin
et al. 2018). Plasmids used for the stable genome editing
of plants require a selection cassette, known as a sgRNA
cassette, and a clustered regularly interspaced short pal-
indromic repeat (CRISPR)/CRISPR-associated protein 9 The CRISPR/Cas9 system has been successfully used for
genome editing in a variety of monocots, including rice
(Oryza sativa), wheat (Triticum sp.), barley (Hordeum
vulgare), and maize (Zea mays) (Feng et al. 2018; Gas-
paris et al. 2018; Hu et al. 2018; Kis et al. 2019; Okamoto
et al. 2017; Wang et al. 2017; Zhang et al. 2016). To per-
form CRISPR/Cas9-mediated genome editing, the Cas9
endonuclease is guided by a single guide RNA (sgRNA)
to recognize the complementary sequence and create * Correspondence: cslin99@gate.sinica.edu.tw
†Jin-Jun Yue, Chwan-Yang Hong, Pengcheng Wei and Yu-Chang Tsai
contributed equally to this work. 5Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
Full list of author information is available at the end of the article Page 2 of 13 Yue et al. Rice Yue et al. Rice (2020) 13:9 (2020) 13:9 Fig. 1 Schematic of the T-DNA region in a binary vector for genome editing in monocots. PPol III: Polymerase III promoter. PPol II: Polymerase II
promoter. PUbi1: maize Ubiquitin 1 promoter and the first exon. Hpt: Hygromycin phosphotransferase. (Modified from Howells et al. 2018) (Cas9) cassette in the T-DNA region, while the selection
cassette is not necessary for transient transformations
(Fig. 1). addition to promoters, the specificity of the sgRNA for
its target DNA sequence is another factor that affects
the efficiency of genome editing. Several resources
available on the web can be used to design highly spe-
cific sgRNAs for use with the CRISPR/Cas9 system
(Table 1; modified from Zhang lab, https://zlab.bio/
guide-design-resources). Nevertheless, even if these
20-nt sequences perfectly match the target gene, some
sgRNAs do not work well. Design and Construction of Plasmids for Genome
Editing in Monocots The online tools listed in
Table 1 recommend targets with a low risk of an off-
target match, but not all predicted target sequences
may result in an efficient mutation. In addition, tar-
geted DNA sequences with GC contents higher than
50% have higher genome-editing efficiencies (88.5–
89.6%) than those with GC contents lower than 50%
(77.2% efficiency) (Ma et al. 2015). Successive Ts in
20-nt target sequence is not good when sgRNA ex-
pression is driven by the U3 or U6 promoters (Wu
et al. 2014). 1. Selection cassette: In monocots, several genes have
served as useful selection markers for the efficient
selection of transgenic plants, such as neomycin-
phosphotransferase (NPTII), bar, mutated
acetolactate synthase (ALS), plant phosphomannose
isomerase, and hygromycin phosphotransferase (Hpt)
(Miki and McHugh 2004; Hu et al. 2016). Among
these genes, Hpt is the most widely used selection
marker which confers tolerance to the herbicide
hygromycin because several crops have a natural
tolerance to kanamycin. Plant phosphomannose
isomerase can also be used as a selectable marker
for rice transformation (Hu et al. 2016). The
expression of Hpt is usually driven by a strong
constitutive promoter, such as maize Ubiquitin 1
(ZmUbi1), rice ACTIN 1, or Cauliflower mosaic
virus (CaMV) 35S, for the ubiquitous expression of
the antibiotic-tolerance gene (Mikami et al. 2015a). 1. Selection cassette: In monocots, several genes have
served as useful selection markers for the efficient
selection of transgenic plants, such as neomycin-
phosphotransferase (NPTII), bar, mutated
acetolactate synthase (ALS), plant phosphomannose
isomerase, and hygromycin phosphotransferase (Hpt)
(Miki and McHugh 2004; Hu et al. 2016). Among
these genes, Hpt is the most widely used selection
marker which confers tolerance to the herbicide
hygromycin because several crops have a natural
tolerance to kanamycin. Plant phosphomannose
isomerase can also be used as a selectable marker
for rice transformation (Hu et al. 2016). The
expression of Hpt is usually driven by a strong
constitutive promoter, such as maize Ubiquitin 1
(ZmUbi1), rice ACTIN 1, or Cauliflower mosaic
virus (CaMV) 35S, for the ubiquitous expression of
the antibiotic-tolerance gene (Mikami et al. 2015a). )
. Cas9 cassette: Aspects of the Cas9 cassette that
affect the mutation rate during genome editing
include the expression level and codon usage of
Cas9 (Ma et al. 2015; Xie et al. 2015). Design and Construction of Plasmids for Genome
Editing in Monocots Several
strategies have been conducted to improve the
expression of Cas9, including the use of a strong
constitutive promoter, the addition of a
translational enhancer, and the addition of nuclear
localization signals. Constitutive promoters have
been used to direct the expression of Cas9,
including the ZmUbi1 and 35S. The Cas9 nucleases
from different bacteria may have variations in the
protospacer adjacent motif (PAM) sequence that
they require for cleavage; therefore, many Cas9
homologs with different PAM requirements have
been isolated from different bacteria. These Cas
proteins are introduced in ‘Alternatives to SpCas9’
section. Streptococcus pyogenes Cas9 (SpCas9) is the
most common Cas used in CRISPR/Cas9-mediated
genome editing. It has been codon-optimized for
maize (Xing et al. 2014) and rice (Miao et al. 2013)
to improve its expression levels in these monocots. 3. 2. sgRNA cassette: sgRNA is a programmable 20-
nucleotide (nt) sequence that recognizes the target
DNA sequence and an invariant scaffold sequence
(Ran et al. 2013), then directs the Cas nuclease to
cleave the target sequence. Two factors are important
for the function of sgRNA; promoter activity and the
specificity of the sgRNA. A mixed dual promoter sys-
tem is generally used in CRISPR/Cas9 system. In this
system, Cas9 is directed by the RNA Polymerase II
(Pol II) promoter while sgRNA expression is regulated
by a Pol III promoter such as U6 or U3. To increase
the transcription of the sgRNA, several monocot-
specific U3 or U6 promoters have been cloned and
used to direct the expression of the sgRNA, such as
those from rice (Ma et al. 2015), maize (Qi et al. 2018), and wheat (Xing et al. 2014). In rice, the sgRNA
driven by the OsU6 promoter produces more tran-
scripts than when driven by the OsU3 promoter
(Mikami et al. 2015a); however, several promoters, in-
cluding OsU3, OsU6a, OsU6b, and OsU6c, have been
used to direct the expression of sgRNA, and all of
them could effectively direct genome editing with mu-
tation rates of 81.4–90.0% (Ma et al. 2015; Shan et al. 2013; Xie and Yang 2013; Zhou et al. 2014). In Strategies for Multiplex Genome Editing in
Monocots One advantage of the CRISPR/Cas9 system over other
crop-breeding strategies is its flexibility for multiplex Yue et al. Rice (2020) 13:9 Page 3 of 13 Yue et al. Rice (2020) 13:9 Table 1 Web-based tools for sgRNA design
Name
Website
Reference
Benchling
https://www.benchling.com/crispr/
Benchling, CA
Broad Institute GPP
https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design
Doench et al. 2016
CHOPCHOP
http://chopchop.cbu.uib.no/
Labun et al. 2019
CRISPOR
http://crispor.tefor.net/
Concordet and Haeussler 2018
CRISPR-P
http://cbi.hzau.edu.cn/CRISPR2/. Liu et al. 2017
DeskGen
https://www.deskgen.com/landing/#/
Desktop Genetics, MA
E-CRISP
http://www.e-crisp.org/E-CRISP/designcrispr.html
Heigwer et al. 2014
Horizon Discovery
https://dharmacon.horizondiscovery.com/gene-editing/crispr-cas9/crispr-design-tool/
Horizon Discovery, UK
IDT
https://sg.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM
Integrated DNA Technologies, IA
Off-Spotter
https://cm.jefferson.edu/Off-Spotter/
Pliatsika and Rigoutsos 2015
Synthego
https://www.synthego.com/products/bioinformatics/crispr-design-tool
Synthego, CA
ZiFiT
http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx
Sander et al. 2010 Table 1 Web-based tools for sgRNA design
Name
Website
Reference
Benchling
https://www.benchling.com/crispr/
Benchling, CA
Broad Institute GPP
https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design
Doench et al. 2016
CHOPCHOP
http://chopchop.cbu.uib.no/
Labun et al. 2019
CRISPOR
http://crispor.tefor.net/
Concordet and Haeussler 2018
CRISPR-P
http://cbi.hzau.edu.cn/CRISPR2/. Liu et al. 2017
DeskGen
https://www.deskgen.com/landing/#/
Desktop Genetics, MA
E-CRISP
http://www.e-crisp.org/E-CRISP/designcrispr.html
Heigwer et al. 2014
Horizon Discovery
https://dharmacon.horizondiscovery.com/gene-editing/crispr-cas9/crispr-design-tool/
Horizon Discovery, UK
IDT
https://sg.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM
Integrated DNA Technologies, IA
Off-Spotter
https://cm.jefferson.edu/Off-Spotter/
Pliatsika and Rigoutsos 2015
Synthego
https://www.synthego.com/products/bioinformatics/crispr-design-tool
Synthego, CA
ZiFiT
http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx
Sander et al. 2010 Table 1 Web-based tools for sgRNA design genome editing (Wang et al. 2017; Wang et al. 2018). The editing of multiple functional genes allows for the
rapid improvement of multiple agronomic traits at one
time, while editing the cis-acting elements of a promoter
affects transcriptional regulation. The deletion of larger
fragments between two sgRNA-targeted sites on the same
chromosome following the generation of multiple DSBs
has been reported in many species. The CRISPR/Cas9 sys-
tem has been used to delete DNA fragments ranging from
dozens of bases to greater than 1 Mb (Mali et al. 2013;
Shan et al. 2013). In addition, targeted deletions of 10 bp
to over 200 kb between two target sites have been re-
ported in rice (Mikami et al. 2016; Zhou et al. 2014). The
elimination of the Tos17 retrotransposon using CRISPR/ genome editing (Wang et al. 2017; Wang et al. 2018). The editing of multiple functional genes allows for the
rapid improvement of multiple agronomic traits at one
time, while editing the cis-acting elements of a promoter
affects transcriptional regulation. The deletion of larger
fragments between two sgRNA-targeted sites on the same
chromosome following the generation of multiple DSBs
has been reported in many species. Strategies for Multiplex Genome Editing in
Monocots The CRISPR/Cas9 sys-
tem has been used to delete DNA fragments ranging from
dozens of bases to greater than 1 Mb (Mali et al. 2013;
Shan et al. 2013). In addition, targeted deletions of 10 bp
to over 200 kb between two target sites have been re-
ported in rice (Mikami et al. 2016; Zhou et al. 2014). The
elimination of the Tos17 retrotransposon using CRISPR/ Cas9 was reported in rice, providing a rapid breeding
route for making reverting the agronomically important
genes that have been inactivated by the insertion of trans-
posable elements (Saika et al. 2019). Multiplex genome editing can be achieved by the sim-
ultaneous delivery and expression of multiple sgRNAs;
however, since most CRISPR/Cas9 components are
transferred
into
plants
via
Agrobacterium-mediated
transformations, an efficient plasmid construction strat-
egy is required. Traditionally, multiple sgRNA expression
cassettes (including a Pol III promoter, a sgRNA, and a
terminator) can be stacked into one T-DNA (Fig. 2a);
however, this may increase the cloning difficulties due to
the limited restriction sites available, and the fact that Fig. 2 Diagram of a plasmid construct used for multiplex genome editing. a Stacking of multiple sgRNA expression cassettes in one T-DNA. P Pol
III: Polymerase III promoter. b Cloning of four tRNA-gRNAs into the CRISPR/Cas9 binary vector using a single Golden Gate ligation. (Modified from
Kurata et al. 2018) Fig. 2 Diagram of a plasmid construct used for multiplex genome editing. a Stacking of multiple sgRNA expression cassettes in one T-DNA. P Pol
III: Polymerase III promoter. b Cloning of four tRNA-gRNAs into the CRISPR/Cas9 binary vector using a single Golden Gate ligation. (Modified from
Kurata et al. 2018) Yue et al. Rice (2020) 13:9 Page 4 of 13 Page 4 of 13 large T-DNAs may decrease the transformation effi-
ciency. Alternative strategies have therefore been devel-
oped to facilitate multiplexed genome editing in plants. These alternative strategies are based on the expression
of multiple sgRNAs as a single transcript, after which
multiple functional sgRNAs are generated following the
processing of the transcripts by exogenous ribozymes
(Gao and Zhao 2014), Csy-type ribonuclease 4 (Csy4)
(Cermak et al. 2017), or the plant endogenous transfer
RNA (tRNA)-processing system (Xie et al. 2015). The
endogenous tRNA-processing system exists in almost all
organisms and has been successfully used to perform
multiplex genome editing in rice (Xie et al. 2015). Alternatives to SpCas9 p
The CRISPR/Cas systems are divided into two classes. The Class 1 systems possess multiple Cas protein sub-
units, whereas the Class 2 systems utilize a single, multi-
functional protein effector (Shmakov et al. 2015). Class 2
CRISPR systems are further divided into types II, V, and
VI. Along with the commonly used type II effector
SpCas9, other orthologs with RNA-guided site-specific
nuclease (SSN) activity have been engineered for use as
tools in the genome editing of eukaryotic cells. These
orthologs include proteins from Staphylococcus aureus
(SaCas9), Streptococcus thermophilus (St1Cas9), Franci-
sella
novicida
(FnCas9),
Neisseria
meningitidis
(NmCas9),
Brevibacillus
laterosporus
(BlCas9),
and
Campylobacter jejuni (CjCas9) (Cong et al. 2013; Hou
et al. 2013; Sampson et al. 2013; Ran et al. 2015; Karvelis
et al. 2015; Kim et al. 2017a; Chatterjee et al. 2018;
Edraki et al. 2019). These Cas9s can be a welcome com-
plement to the editing ability of SpCas9 in plants. Here,
we describe plant genome-editing tools developed from
these orthologs and discuss their advantages for research
in botany. Reducing off-Target Mutations and Lethality in Monocots
Several
reports
have
indicated
that
constitutively
expressed Cas9 produces an excess of sgRNA-Cas9,
which may increase the incidence of genome-wide off-
target mutations (Hsu et al. 2013; Hu et al. 2018; Patta-
nayak et al. 2013; Svitashev et al. 2016). By contrast, con-
ditionally or transiently expressing Cas9 significantly
reduces the frequency of off-target mutations (Srivastava
et al. 2017; Zhang et al. 2014). Increasing evidence has
indicated that conditionally expressing Cas9 at the plant
regeneration phase can markedly improve genome-
editing efficiency, and this kind of conditional targeting
could avoid the lethal phenotype caused by the cleavage
of genes essential for development (Srivastava et al. 2017; Zhang et al. 2014). The promoter of the gene en-
coding heat-shock protein 17.5E (Hsp17.5E) from soy-
bean
(Glycine
max)
has
been
used
to
direct
the
expression of Cas9 for genome editing in rice. The mu-
tation frequency was 16% and 50–63% among the trans-
genic
lines
before
and
after
a
heat
treatment,
respectively (Nandy et al. 2019). In maize, Cas9 driven
by
the
meiosis-specific
Disrupted
Meiotic
cDNA
1
(ZmDMC1) promoter was able to generate up to 66%
homozygous or bi-allelic mutants, and no off-target mu-
tations were detected using whole-genome sequencing
(Feng et al. 2018). The vector delivery of preassembled Strategies for Multiplex Genome Editing in
Monocots In the
tRNA-processing system, the Pol III promoter is used to
direct the expression of a single synthetic gene contain-
ing multiple tRNA-sgRNAs or a polycistronic tRNA-
sgRNA (PTG) gene. The PTG gene can be generated
using the Golden Gate assembly method (Lowder et al. 2015) (Fig. 2b). After transcribing the PTG, the en-
dogenous RNases specific to tRNA would recognize the
tRNA components and cleave the individual sgRNAs
from the PTG transcript. The resulting sgRNAs would
then guide Cas9 to multiple target sites for genome edit-
ing. Many toolboxes are currently available in the public
database Addgene (Cermak et al. 2017; Castel et al. 2019; Hahn et al. 2019), from where these vectors can be
purchased by academic researchers. Cas9-sgRNA RNPs instead of DNA has been reported to
significantly reduce the frequency of off-site cleavage in
both protoplast and zygote systems (Toda et al. 2019). In maize embryo cells, the delivery of DNA vectors con-
taining Cas9 and sgRNA showed a high frequency of
off-site mutations (50%) when compared with the Cas9-
sgRNA RNP complex (0%) (Svitashev et al. 2016). In
most plant species, the isolation, cultivation, and regen-
eration of protoplasts remains a challenge; however, re-
cent work demonstrated the direct delivery of a Cas9-
sgRNA
RNP
into
rice
zygotes
using
the
in
vitro
fertilization of isolated gametes, resulting in a targeted
mutation rate of 14–64%. This protoplast-free zygote
system makes the RNP-mediated genome-editing system
much easier to perform, which could be a potential av-
enue for crop improvement in many monocot species
(Toda et al. 2019). Type II Cas9 Systems Type II CRISPR systems are abundant in prokaryotic or-
ganisms and are the principal resource used to develop
gene-editing tools. In addition to the first-reported
SpCas9,
several
studies
have
described
the
Cas9
ortholog-mediated genetic engineering of plants. SaCas9
is most frequently used in place of SpCas9 and provides
comparable editing efficiency for eukaryotic genomes. Compared with SpCas9, SaCas9 may reduce the delivery
barrier of the CRISPR system because of its smaller size
(Friedland et al. 2015; Ran et al. 2015). SaCas9 and its
engineered
variant
SaKKH
(E782K/N968K/R1015H)
(Kleinstiver et al. 2015), which relaxes the canonical Page 5 of 13 Page 5 of 13 Yue et al. Rice (2020) 13:9 Yue et al. Rice (2020) 13:9 Yue et al. Rice NNGRRT PAM of SaCas9 to NNNRRT, have been used
to achieve the efficient targeted mutagenesis of Arabi-
dopsis thaliana, tobacco (Nicotiana tabacum), rice, and
citrus (Citrus sp.) (Kaya et al. 2016; Steinert et al. 2015;
Jia et al. 2017; Qin et al. 2019). temperature used in crop culture systems may facilitate
a higher editing efficiency when using Cas12a proteins. a higher editing efficiency when using Cas12a proteins. Cas12a proteins not only induce DSBs, but also
process their own pre-crRNA for maturation. This
RNase activity provides a simple pattern by which single
or multiplex crRNA(s) can be expressed from the same
transcription unit of Cas12a, facilitating the highly effi-
cient genome editing of plants (Wang et al. 2017; Ding
et al. 2018; Wang et al. 2018; Xu et al. 2019). In the gen-
ome editing mediated by the CRISPR/Cas9 system, RNA
could be used as a repair template for homology-
directed repair (Butt et al. 2017). The self-processing ac-
tivity of Cas12a means that homologous recombination-
mediated gene replacements can be generated using the
FnCas12a system (Li et al. 2019). In mammalian cells,
CBEs were also developed using Cas12a proteins to fa-
cilitate precise C-to-T conversions in A/T-rich regions
(Li et al. 2018); however, similar systems have not yet
been reported in plants. The engineered Alicyclobacillus
acidiphilus Cas12b (AaCas12b) has been used to edit
mammalian genomes under a wide range of tempera-
tures (31–59 °C; Teng et al. 2018), but it is still unclear
whether the high-temperature-preferring Cas12b sys-
tems function in plants. Type II Cas9 Systems SaCas9 and SaKKH have also been used to develop
plant base editors (BEs), including the cytosine BE (CBE)
responsible for a C·G to T·A conversion and the adenine
BE (ABE) responsible for the reverse substitution of A·T
to G·C. Similar to the BEs derived from SpCas9, the
SaCas9 BEs have been successfully used to induce spe-
cific base conversions in the rice genome (Hua et al. 2018; Hua et al. 2019; Qin et al. 2019). Notably, the edit-
ing windows of the SaCas9 BEs are much broader than
those of the SpCas9 BEs, possibly due to differences in
the formation of the R-loop complex (Kim et al. 2017d). Compared the conventional CRISPR/Cas9-targeted mu-
tagenesis system, BEs developed from SpCas9 tend to be
less efficient at editing certain targets and less flexible
for specific nucleotides. Because of their recognition of
different PAMs and the enlargement of the editing win-
dow, the SaCas9 BEs have provided alternative tools for
precise genome editing in plants. Another Cas9 ortholog, St1Cas9, has been used to in-
duce mutations in the Arabidopsis gene ALCOHOL DE-
HYDROGENASE 1 (ADH1) (Steinert et al. 2015), while
FnCas9 was used to confer molecular immunity against
RNA viruses in tobacco and Arabidopsis (Zhang et al. 2018). Type VI Cas Systems Type VI Cas Systems
The type VI CRISPR proteins, such as Cas13a, can target
and cleave the target RNA under the guidance of
crRNA. The heterologous expression of Leptotrichia
wadei Cas13a (LwaCas13a) causes the knockdown of
target RNAs in mammalian and plant cells (Abudayyeh
et al. 2017). Similarly, in both dicot and monocot plants,
the transient or stable expression of a Cas13a-crRNA
complex results in the targeting and degradation of the
RNA transcripts of endogenous genes and foreign re-
porters (Aman et al. 2018; Zhang et al. 2019a, 2019b). To precisely edit nucleotides in RNA, the ADAR2 ad-
enosine deaminase or an evolved ADAR2 cytidine deam-
inase
was
fused
to
a
catalytically
inactive
Cas13a
(dCas13a), generating C-to-U and A-to-I RNA editors in
mammalian cells (Cox et al. 2017; Abudayyeh et al. 2019). RNA editing, especially organellar RNA editing,
plays an irreplaceable role in plant growth and develop-
ment; therefore, similar Cas13a tools are highly antici-
pated to facilitate related research in plants. Type V Cas Systems Type V Cas Systems
The CRISPR RNA (crRNA)-guided SSN activity of the
type V Cas system, which includes Cas12a and Cas12b,
also generates DSBs. Several Cas12a (formerly Cpf1)
orthologs, such as F. novicida Cas12a (FnCas12a), Acida-
minococcus sp. Cas12a (AsCas12a), and Lachnospiraceae
sp. Cas12a (LbCas12a), have been engineered as a class
of genome-editing tools distinct from the Cas9 system
(Zetsche et al. 2015). The Cas12a proteins induce stag-
gered DSBs at sites distal to a 5′ T-rich PAM, generating
relatively longer deletions via the non-homologous end
joining repair pathway in various plant species, including
rice (Endo et al. 2016a; Begemann et al. 2017; Hu et al. 2017; Kim et al. 2017c; Tang et al. 2017; Xu et al. 2017). Similar to the SpCas9 systems, transgene-free Cas12a-
mediated mutants can be generated by simply segregat-
ing the T-DNA fragment in the transition from the T0
to T1 generations (Xu et al. 2017). In addition, mixing
RNP with Cas12a and crRNA has allowed transgene-free
genome editing in soybean and tobacco (Kim et al. 2017b). Interestingly, the Cas12a proteins have different
sensitivities to temperature in plants (Malzahn et al. 2019); therefore, the optimization of the incubation Single Protoplast Analysis When using protoplasts to validate CRISPR efficiency,
more than 100,000 protoplasts are typically used in each
transfection experiment. DNA is extracted from the
pooled protoplasts to enable the amplification of the tar-
get region using PCR. The pooled protoplasts also con-
tain unedited DNA, making mutations difficult to detect
if the mutagenesis efficiency is low (Lin et al. 2018). Mu-
tagenesis efficiency can also be assessed using next-
generation sequencing, from which the density ratio of
the target fragments or the editing percentage can be de-
termined. This method is relatively accurate, but it is ex-
pensive and time consuming. Recently, a convenient and
reliable protocol for evaluating CRISPR mutagenesis effi-
ciency from a single cell was established (Lin et al. 2018). In this approach, single cells can be isolated from
various species and subjected to two rounds of PCR
amplification and enzyme digestion without DNA purifi-
cation to identify successful mutants. The mutated se-
quences and the mutation efficiency could thus be
analyzed directly, allowing even low-efficiency mutation
events to be detected in maize. This single-cell analysis
technique could be used to improve the precision and
application
range
of
CRISPR
gene
editing
using
protoplasts. y
)
In comparison with the number of articles on stable
rice CRISPR transformations, very few have been pub-
lished using protoplasts for validation. One of the rea-
sons for this is the efficiency of protoplast transfection
and isolation. We have evaluated the different methods
of protoplast transfection in rice, including PEG, electro-
poration, and liposome delivery, which revealed that the
PEG method has highest transfection efficiency (Lin and
Hsu, personal comm.). The main bottleneck restricting
the application of rice protoplasts in the evaluation of
genome-editing reagents is thus considered to be proto-
plast isolation. We previously improved the protocol for
isolating
Arabidopsis
protoplasts
(Tape
Arabidopsis
Sandwich; Wu et al. 2009) to facilitate their use for vari-
ous purposes, and we are currently trying to develop a
convenient rice protoplast isolation method, modifying
two of the steps reported in previous rice mesophyll
protoplast isolation protocols (Chen et al. 2006; Zhang
et al. 2011). First, based on our observations, seedlings cut cross-
sectionally retain more cells within the leaf sheath after
digestion, meaning the mesophyll cells can be digested
but not released. Protoplasts and CRISPR
Protoplast Isolation and Validation Many different vectors are available for the expression of
Cas proteins using different promoters. Researchers
should choose a suitable vector based on their needs,
and importantly should evaluate the target sites and con-
structs using a transient expression system before per-
forming the stable transformation to reduce the time Yue et al. Rice (2020) 13:9 Yue et al. Rice (2020) 13:9 Page 6 of 13 Page 6 of 13 and labor required. Protoplasts are often used for plant
science investigations (Marx 2016), and the convenience
and speed of their transfection means they are an at-
tractive model in which to assess the mutagenesis effi-
ciency of a CRISPR/Cas system, including the validation
of Cas protein codon optimizations or modifications,
sgRNA target sites, the promoters used for sgRNA and
Cas9 proteins, and different vector designs (Andersson
et al. 2017; Butt et al. 2017; Cermak et al. 2017; Endo
et al. 2019; Hsu et al. 2019; Hsu et al. in preparation; Li
et al. 2013; Li et al. 2018; Liang et al. 2014; Lowder et al. 2015; Shan et al. 2013; Sun et al. 2015; Zong et al. 2017,
2018). Rice, tobacco, and soybean protoplasts have been
used to analyze Cas9 and Cas12a (Kim et al. 2017; Tang
et al. 2017), while Cas13a was examined in rice proto-
plasts (Abudayyeh et al. 2017). Protoplast transfection
can also be used to evaluate the efficiency of the use of
RNPs (Andersson et al. 2018; Hsu et al. in preparation;
Kim et al. 2017; Malnoy et al. 2016; Woo et al. 2015). (Yakult, Japan) solution typically used for rice (Zhang
et al. 2011). This protocol can also be applied to other
Poaceae species, including wheat, bamboo (Bambusa
oldhamii), millet (Setaria italica), and maize (Lin et al. 2018). We believe this convenient method will be of
benefit not only in rice, but also for Poaceae crop re-
search in general. We have also established protoplast
isolation protocols for use with the Solanaceae and Cru-
ciferae (Hsu et al. submitted). The protoplasts isolated
using these methods can be used to more rapidly evalu-
ate the genome-editing efficiency in crops (Lin et al. 2018). CRISPR-Edited Protoplast Regeneration transfection (Chen et al. 2018); however, the gene-
editing/mutation efficiencies of these methods are
low (less than 10%). The protoplast transformation
strategy had a higher efficiency; for example, over
50% of tobacco protoplasts were mutagenized using
this technique, which was similar to the numbers
transformed during the efficiency evaluation with-
out antibiotic selection. In addition to the validation of transformation efficiency,
mutated protoplasts have the advantage of being able to
regenerate into entire mutant plants. Protoplasts isolated
from meristematic tissues or totipotent cells were first
used for plant regeneration in the early 1970s (Takebe
et al. 1971), and just a few years later, researchers used
protoplasts as materials for plant transformation (Mar-
ton et al. 1979). The progeny of N. tabacum regenerated
from transformed protoplasts displayed the mutant
phenotype, indicating that the transgenic tumor markers
(octopine and nopaline) were inherited through meiosis
(Wullems et al. 1981a; Wullems et al. 1981b). Monocot
protoplast regeneration (Abdullah et al. 1986; Fujimura
et al. 1985; Rhodes et al. 1988a) and transformation
(Rhodes et al. 1988b; Shimamoto et al. 1989; Toriyama
et al. 1988) protocols have also been established. out antibiotic selection. Protoplast transformation can deliver more than
one reagent. The current Agrobacterium vectors
used for stable transformations are multiplex
sgRNAs. The genes encoding Cas proteins are large,
and no multiplex Cas protein vectors are currently
available. Three different Cas protein vectors
(SaCas9, FnCas12a, and nCas9-Target-AID) can be
delivered into a single protoplast to edit three dif-
ferent target sites without interference (Hsu et al. 2019). The introduction of these three Cas protein
vectors requires three subsequent Agrobacterium-
mediated transformations, the crossing of individual
mutants, or the co-transformation of Agrobacterium
harboring independent Cas9s and different selection
markers.. In addition to sgRNA and Cas proteins,
donor DNA fragments are also required for knock-
in genome editing, and must be delivered using
Agrobacterium (Endo et al. 2016b; Miki et al. 2018;,
Wolter and Puchta 2019). Using these protocols,
only a few copies of donor DNA can be used in
genome editing, which may be one of the reasons
for the low efficiency of knock-in genome editing. Although multiple T-DNAs can be delivered into
plant nuclei to increase the editing efficiency, this
also increases the difficulty of removing the T-
DNAs to produce marker-free plants. In proto-
plasts, however, the donor DNA can be delivered in
microgram quantities, which may help increase the
knock-in efficiency. CRISPR-Edited Protoplast Regeneration Not only can the sgRNAs, Cas
proteins, and donor DNAs be more easily delivered
into protoplasts, but the application time and
amounts of genome-editing reagents can be con-
trolled to improve the editing efficiency. 2. p
Cas proteins and sgRNAs are sufficient for CRISPR/
Cas genome editing and are no longer required once the
genes have been edited. Transient expression or the dir-
ect delivery of sgRNAs and Cas proteins into the cells is
sufficient for editing; therefore, the DNA encoding the
Cas proteins and sgRNAs does not need to be integrated
into the genome for their continued expression, making
the plants regenerated from these edited cells transgene-
free. In 2015, Prof. Jin-Soo Kim’s group published a
milestone article using protoplasts (Woo et al. 2015), in
which RNP was used as the genome-editing reagent to
edit lettuce (Lactuca sativa) protoplasts, which were
subsequently regenerated into transgene-free plants. There are several advantages to generating transgene-
free edited crops using protoplasts: 1. Protoplast transformation can be applied to edit
hybrid and long-juvenile-phase crops, which are
typically propagated using vegetative methods. Traditionally, the use of Agrobacterium tumefa-
ciens-mediated or other stable transformation tech-
niques means the transgene (selection markers,
sgRNA, and Cas9) must be integrated into the gen-
ome. In inbred crops, such as rice, this transgene
can be removed through crossing; however, this
causes the segregation of the desired traits in the
offspring. This separation issue also occurs in sys-
tems with a low editing efficiency, in which the
transformants must be crossed in order to obtain
the homozygous genotype. By contrast, homozy-
gous edited crops can be achieved from heterozy-
gous edited protoplasts using a second transfection
(Hsu et al. 2019). Several methods are available for
achieving this goal, including the delivery of RNP
into the callus using a biolistic approach (Liang
et al. 2017, 2019) or the transient expression of
Cas9 and sgRNA using an Agrobacterium-mediated 1. Protoplast transformation can be applied to edit
hybrid and long-juvenile-phase crops, which are
typically propagated using vegetative methods. Traditionally, the use of Agrobacterium tumefa-
ciens-mediated or other stable transformation tech-
niques means the transgene (selection markers,
sgRNA, and Cas9) must be integrated into the gen-
ome. In inbred crops, such as rice, this transgene
can be removed through crossing; however, this
causes the segregation of the desired traits in the
offspring. Single Protoplast Analysis The veins in rice run parallel to each
other; therefore, we changed the cut direction from a
random or cross-sectional cut to a longitudinal cut par-
allel to the veins, which allows the enzyme solution to
more easily access the cells and provides more surface
area from which the protoplasts are released. To in-
crease the efficiency of this process, multiple blades were
fixed by a holder, creating a tool that can increase the
speed of cutting (Lin et al. 2018). Second, we also
assessed the enzyme components required for the diges-
tion solution when using longitudinal cuts, revealing that
the less expensive Cellulase R10 (Yakult, Japan) could be
used in place of the more expensive Cellulase RS Although these convenient methods could be used for
mesophyll isolation to provide the materials for the
evaluation of CRISPR editing efficiency and accuracy, it
is important to consider the correlation between CRISPR
efficiencies using mesophyll protoplasts and stable trans-
formation, particularly in rice, for which a callus is typic-
ally used as the material for stable transformation (Kaya
et al. 2016). Certain target sites were found to have a
high CRISPR efficiency in stable transformation experi-
ments, but their use did not result in mutations in the
mesophyll protoplasts (Toki, Endo, and Lin, personal
comm.). We are therefore working on developing a rice
protoplast
isolation
protocol using callus
materials,
which will enable the assessment of the gene-editing re-
lationship between these protoplasts and the stably
transformed calli. Page 7 of 13 Yue et al. Rice (2020) 13:9 CRISPR-Edited Protoplast Regeneration This separation issue also occurs in sys-
tems with a low editing efficiency, in which the
transformants must be crossed in order to obtain
the homozygous genotype. By contrast, homozy-
gous edited crops can be achieved from heterozy-
gous edited protoplasts using a second transfection
(Hsu et al. 2019). Several methods are available for
achieving this goal, including the delivery of RNP
into the callus using a biolistic approach (Liang
et al. 2017, 2019) or the transient expression of
Cas9 and sgRNA using an Agrobacterium-mediated p
g
y
3. Using RNPs, the issues of promoter and codon
modification in different species can be solved. The
original Cas proteins were obtained from
microorganisms; therefore, their codons must be
modified for vector construction in plant species,
and their expression must be driven by plant
promoters. These issues have been investigated in
Arabidopsis, resulting in the identification of a T-
DNA architecture causing homozygous mutations
in the first generation after transformation (Castel
et al. 2019); however, the transcription and transla-
tion of these genes can still be problematic in differ-
ent target crops. The resulting amount of Cas 3. Using RNPs, the issues of promoter and codon
modification in different species can be solved. The
original Cas proteins were obtained from
microorganisms; therefore, their codons must be
modified for vector construction in plant species,
and their expression must be driven by plant
promoters. These issues have been investigated in
Arabidopsis, resulting in the identification of a T-
DNA architecture causing homozygous mutations
in the first generation after transformation (Castel
et al. 2019); however, the transcription and transla-
tion of these genes can still be problematic in differ-
ent target crops. The resulting amount of Cas Yue et al. Rice (2020) 13:9 Yue et al. Rice (2020) 13:9 Yue et al. Rice (2020) 13:9 Page 8 of 13 Page 8 of 13 Yue et al. Rice Poaceae species (Rhodes et al. 1988a, 1988b). Recent discoveries have elucidated the mechanisms
by which plants can regenerate, which could be
applied to improve protoplast regeneration in the
future (Lowe et al. 2016). In rice, it is possible to
solve the lack of an efficient protoplast regeneration
protocol using an alternative method; Cas9-sgRNA
RNPs could be directly delivered into rice zygotes,
which can then be cultured into mature plants
(Toda et al. 2019). CRISPR-Edited Protoplast Regeneration A total of 14–64% of the
transgene-free plants obtained using this method
were found to contain the target mutations. proteins in the cell can cause a low editing effi-
ciency; however, in protoplasts, RNPs can be used
to solve this problem and provide a higher delivery
efficiency for transfection. 4. With the exception of Arabidopsis, most
Agrobacterium-mediated transformation protocols
were performed using a tissue culture platform. In
dicots, the regenerated plants were derived using
organogenesis, meaning they were derived from
multiple cells. Many edited transformants are
therefore chimeric (Kaya et al2016). To validate
their edited sequences, the transformant DNAs
were sequenced. Diploids contain two alleles in
each cell; therefore, if the transformants contained
more than two alleles they were determined to be
chimeric (Kaya et al. 2016). If the edited alleles are
not present in the reproductive organs, the alleles
cannot be passed on to the progeny. In contrast,
protoplasts are single cells that are edited before the
first cell division is completed. The regenerates are
then derived from a single edited protoplast,
meaning all cells have same genomic background,
which enables the edited alleles to be transmitted to
the next generation. In our previous studies, non-
chimeric regenerates were derived from protoplasts
edited using the Cas proteins Cas9, Cas12a, and
Target-AID, and the genotypes were inherited in a
Mendelian manner (Hsu et al. 2019). This
phenomenon was also reported in lettuce (Woo
et al. 2015). g
Unexpected mutations can occur during protoplast
regeneration. When 15 protoplast potato
regenerants were sequenced, they were found to
include a variety of mutations, including insertions/
deletions, chromosome rearrangements, and
aneuploidy (Fossi et al. 2019). Indeed, somaclonal
mutations occur in all tissue culture strategies,
including micropropagation and somatic
embryogenesis. In our experience using bamboo,
somaclonal mutations occurred after a long-term
subculture (Lin and Chang 1998; Lin et al. 2006,
2007; Liu et al. 2007). Using a shorter period for the
protoplast regeneration or reducing the amount of
supplemental plant growth regulators provided may
reduce this mutation rate (Lin and Hsu, personal
comm.). CRISPR/Cas can also introduce off-target
mutations, with the mutation rate dependent on the
CRISPR/Cas system; for example, in rice, CBE but
not ABE induces genome-wide off-target mutations
(Jin et al. 2019). Fortunately, off-target mutations
do not have a major impact on crop breeding like
they do in medical applications (Tang et al. CRISPR-Edited Protoplast Regeneration 2019);
rather, they simply result in more than one edited
transformant for each transformation. In addition,
mutation breeding is a strategy used in traditional
breeding. We can select the transformants with
good traits and use these edited lines as parental
lines during crop production. In our opinion, al-
though mutations can occur during regeneration,
the protoplast regeneration techniques currently
available are useful tools for transgene-free genome
editing. 2. RNPs (Andersson et al. 2018; Woo et al. 2015) and
plasmids (Andersson et al. 2017; Zong et al. 2018) have
been delivered into lettuce and potato (Solanum tubero-
sum) protoplasts, which were subsequently regenerated
into transgene-free genome-edited plants. In our lab, we
established N. tabacum (Hsu et al. 2019; Lin et al. 2018),
rapid cycle brassica (Brassica oleracea), wild tomato (So-
lanum peruvianum), and N. benthamiana (Lin et al. in
preparation) protoplast regenerations, and used these
systems to establish RNP and plasmid DNA gene-editing
platforms (Hsu et al. in preparation). Some issues are yet
to be resolved in protoplast regeneration during genome
editing, however: Screening for Marker-Free CRISPR/Cas9-Induced
Mutants 1. The regeneration protocol is difficult to establish. Only a few protocols for genome editing and
protoplast regeneration have been developed, all of
which were achieved in dicots (Andersson et al. 2017, 2018; Hsu et al. 2019; Jin et al. 2019; Lin et al. 2018; Tuncel et al. 2019; Woo et al. 2015). Many
protoplast regeneration protocols are available in
other species, including rice (Shimamoto et al. 1989; Toriyama et al. 1988) and other important Genome editing is widely used produce new genetic var-
iants in plants. Several approaches for creating genome-
edited
crops
have
been
developed,
including
the
CRISPR/Cas system, which can be used with various tis-
sue types including protoplasts (as described in the pre-
vious section), callus, leaf discs, and germline cells. Agrobacterium-mediated,
PEG-mediated,
particle Yue et al. Rice (2020) 13:9 Page 9 of 13 bombardment,
and
virus
infection
transformation
methods are commonly used for the delivery of the
CRISPR/Cas system into plants, resulting in stable or
transient expression patterns. mitochondria and chloroplasts (Oung et al. 2015). These
elevated H2O2 levels can be visualized directly using 3,
3’diaminobenzidine (DAB) staining (Wu et al. 2019). This approach can be easily applied to most monocot
species. The transient expression of the CRISPR/Cas system
can deliver DNA-based sgRNA and Cas9 RNP sequences
or the proteins themselves (Chen et al. 2018; Zhang
et al. 2016). In a DNA-based CRISPR/Cas9 system, re-
combinant DNA can be transferred to the plant using
Agrobacterium- or PEG-mediated transformations or
particle bombardment, eliminating the need for herbi-
cide or antibiotic selection steps. This method allows the
expression of the CRISPR/Cas9 plasmid without requir-
ing its integration into the plant genome. This approach
can reduce regeneration time via tissue culture while
producing mutation frequencies similar to the stable ex-
pression of the CRISPR/Cas9 vector at the target site. More than 86% of wheat T0 mutants generated using
this technique were transgene free (Zhang et al. 2016). In the Transgene-free CRISPR/Cas9 system, RNPs are
assembled in vitro and directly delivered into the proto-
plasts using a PEG fusion approach. The RNP complex
directly targets the recognized sequences and induces
DSBs (Park and Choe 2019; Woo et al. 2015). The bind-
ing of the RNP complex to the target DNA is tight, and
the half-life for dissociation is slow (more than 6 hours
in vitro) (Didovyk et al. 2016). Identification of CRISPR/Cas9-Induced Mutations Identification of CRISPR/Cas9-Induced Mutations
Following the delivery of the CRISPR/Cas system into
monocot calli or other tissues, a T0 generation of plants
harboring edited genes is regenerated. Three categories
of
site-directed
nuclease
systems
(SDN1–3)
are
employed in genome-editing techniques (Podevin et al. 2013). SDN1 relies on the most common endogenous
processes of non-homologous end-joining to repair
DSBs in the plant DNA. This process is error prone, and
may result in random mutations at the break site (Bor-
tesi
and
Fischer
2015). SDN2
involves
homology-
directed repair using one or a few nucleotides as a tem-
plate (EFSA Panel on Genetically Modified Organisms
2012). SDN3 uses the same repair mechanism as SDN2
but with a longer nucleotide template. Unlike SDN1, the
repair processes used by SDN2 and SDN3 is not ran-
dom, and does not cause substitutions, insertions, or de-
letions at the repair sites. Researchers have developed several methods to in-
crease the efficiency of screening large numbers of mu-
tants (Table 2). These methods can detect on-target or
off-target variants and include the restriction enzyme
(RE) cleaved amplified polymorphic sequences (CAPS)
assay (Shan et al. 2014), RE site created assays (Hodgens
et al. 2017), T7 endonuclease I assays (T7E1) (Vouillot
et al. 2015), polyacrylamide gel electrophoresis (PAGE)-
based genotyping assay (Zhu et al. 2014), high-resolution
melting analysis (HRM) (Thomas et al. 2014), PCR- and
labeling-based method (Biswas et al. 2019), and anneal-
ing at critical temperature PCR (ACT-PCR) (Hua et al. 2017). The majority of genome-edited plants involve the
stable integration of the CRISPR/Cas9 system into the
plant
genome. CRISPR/Cas9
DNA
is
delivered
by
methods similar to those employed for transient expres-
sion, followed by a herbicide or antibiotic selection of
successful transformants containing a marker gene. Antibiotic and herbicide resistance markers have been
widely used in plant biotechnology (Wilmink and Dons
1993); however, genome-integrated CRISPR/Cas9 carries
a risk of increased off-target effects and requires re-
searchers to follow the current regulations for typical
genetically modified crops. To bypass these strict bio-
safety regulations, CRISPR/Cas binary vectors containing
selection markers or foreign DNA can be segregated in
the progeny by self-pollinating or crossing the transfor-
mants (Gao et al. 2016). The PCR amplification of vector
sequences can be used to verify the presence of foreign
DNA in the genome. A rapid method using antibiotics
to identify marker-free genome-edited plants was also
reported recently (Wu et al. 2019). Screening for Marker-Free CRISPR/Cas9-Induced
Mutants After dissociation, the
RNP complex is degraded quickly in the cell. Identification of CRISPR/Cas9-Induced Mutations 2014)
HRM
Homozygous DNA has a unique melting temperature (Tm),
while mutated heterozygous DNA has a lower Tm
Fast and efficient for detecting
SNPs and indels in mutants
Requires specific instrumentation
and sensitivity is affected by
amplicon size
(Thomas
et al. 2014)
ACT-PCR
A critical annealing temperature in PCR suppresses the
mismatched annealing of the primer to the template, inhibiting
the production of amplicons
Simple, fast, economical, and can
detect homozygous mutants
Requires designing specific
primers and is time consuming
and/or labor intensive
(Hua et al. 2017)
PCR- and labeling-based assay
Simple, effective, and sensitive
Not able to reveal the exact
nucleotide change in the mutant
(Biswas
et al. 2019)
Whole-genome sequencing
Identifies on-target and off-target
mutations
Costly and time consuming
(Tang
et al. 2018) Table 2 Advantages and disadvantages of different methods for CRISPR/Cas9-mediated mutant screening. (Adapted from Bao et al. 2019) Table 2 Advantages and disadvantages of different methods for CRISPR/Cas9-mediated mutant screening. (Adapted from Bao et al. 2019) PCR- and labeling-based assay Whole-genome sequencing method allows for the detection of indels with a high
sensitivity (down to ±1 bp). to distinguish between homozygous mutants and the
wild type. Other PCR-based analyses include PAGE, HRM, ACT-
PCR, and PCR−/labeling-based assays. To distinguish
genome-edited
mutations from
wild-type
target se-
quences, target region amplicons are migrated on a na-
tive polyacrylamide gel. Homoduplex DNA migrates
faster than heteroduplex DNA. To distinguish homozy-
gous mutants from the wild type, unknown amplicons
can be pre-mixed with wild-type amplicons before the
assay. The migration pattern of the pre-mixed homozy-
gous mutant and wild type will be similar to that of the
heterozygous mutant. HRM is a fluorescence-based
technique for determining the differences in the melting
temperatures of heteroduplex and homoduplex DNA
fragments. This technique can detect differences as small
as 0.1 °C; however, its sensitivity is influenced by the
amplicon length and variation of the mutated sequence. ACT-PCR assay can also distinguish homozygous mu-
tant sequences at the target site using optimal annealing
temperature and specific primers; however, the assay re-
quires the design of specific primers and is time con-
suming
and
labor
intensive. A
simple
PCR-
and
amplicon labeling-based method was recently used to
identify the CRISPR/Cas9-generated mutants in rice
(Biswas et al. 2019). This approach requires two pairs of
primers or a FAM-labeled allele-specific primer. Identification of CRISPR/Cas9-Induced Mutations Leaf sections from
the T1 progeny of genome-edited rice were incubated
with hygromycin B, an antibiotic commonly used for the
positive selection of transgenic plants. In the presence of
hygromycin B, the genome-edited rice plants that did
not retain CRISPR/Cas vectors in their genome could
produce reactive oxygen species (e.g., H2O2) in their The CAPS, indCAPS, and T7E1 cleavage assays for
identifying gene-edited mutants are based on an enzym-
atic approach. Wild-type and mutant sequences are
amplified using PCR then subjected to enzyme digestion. A typical CAPS assay can be used if a RE cutting site is
present at the CRISPR target site, which is disrupted
once the sequences are mutated; however, RE sites are
not always present within target regions. A similar
dCAPS assay has been developed for genome regions
not possessing different RE sites between the wild type
and the mutant. This dCAPS assay introduces or dis-
rupts a RE site near the mutation site by amplifying sev-
eral mismatched nucleotides, which greatly increases the
flexibility for selecting the sgRNA target sites. T7 endo-
nuclease 1 can be useful for digesting the mismatched
heteroduplexes formed between the wild-type and mu-
tated strands; however, this method lacks the sensitivity Page 10 of 13 Yue et al. Rice (2020) 13:9 Yue et al. Rice (2020) 13:9 Yue et al. Rice Table 2 Advantages and disadvantages of different methods for CRISPR/Cas9-mediated mutant screening. (Adapted from Bao et al. 2019)
Method
Advantages
Disadvantages
Reference
CAPS
A RE site within the DNA target site is destroyed by a genome-
editing mutation
Simple, fast, economical, and can
detect homozygous and
heterozygous mutants
Limited to the original target
sequences
(Shan
et al. 2014)
indCAPS
A RE site is created using mismatch primers next to a DNA
target site
More flexibility for different types
of indel
Requires designing specific
primers to distinguish known indel
alleles
(Hodgens
et al. 2017)
T7E1 cleavage assay
T7 endonuclease 1 digests mismatched heteroduplexes formed
between wild-type strands and mutated strands
Simple, fast, economical, and can
detect heterozygous mutants
Cannot detect homozygous
mutants
(Vouillot
et al. 2015)
PAGE
Homoduplex DNA migrates faster than heteroduplex DNA in
native PAGE
Simple, fast, economical, and can
detect homozygous and
heterozygous mutants
Time consuming and low
throughput
(Zhu et al. Acknowledgments Acknowledgments
We thank Miranda Loney and Fu-Hui Wu for editing. Authors’ contributions Authors contributions
JJY, CYH, PW, YCT, and CSL contributed to the writing. CSL organized and
prepared this manuscript. All authors read and approved the final
manuscript. Identification of CRISPR/Cas9-Induced Mutations The
sensitivity, precision, and reliability of the FAM-labeled Although all these PCR-based analyses enable the ef-
fective, accurate, and economical screening of CRISPR/
Cas9-generated mutants, the identification of sequence
changes resulting from SDN1-generated mutations re-
quires the Sanger sequencing of amplicons generated
from the target region. In addition, whole-genome se-
quencing is a powerful tool for identifying not only on-
target and off-target mutations, but also transgene-free
plants produced by genome editing. The only drawback
of whole-genome
sequencing
is its cost
and
time
requirement. Availability of data and materials
Not applicable. Funding g
This research was supported by Academia Sinica, the Innovative Translational
Agricultural Research Administrative Office (AS-KPQ-107-ITAR-10), and the
Ministry of Science and Technology (105–2313-B-001 -007 -MY3 and 108–
2313-B-001-11 to CSL; 105-2628-B-036-MY3 and 108-2313-B-002-055-MY3 to
CYH; 108-2313-B-002 -051 and107-2313-B-002 -023 to YCT), Taiwan. References Genome Biol 19:1 Bao A, Burritt DJ, Chen H, Zhou X, Cao D, Tran LP (2019) The CRISPR/Cas9 system
and its applications in crop genome editing. Crit Rev Biotechnol 39:321–336 Gao Y, Zhao Y (2014) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro
and in vivo for CRISPR-mediated genome editing. J Integr Plant Biol 56:343–349 Begemann MB, Gray BN, January E, Gordon GC, He Y, Liu H, Wu X, Brutnell TP,
Mockler TC, Oufattole M (2017) Precise insertion and guided editing of
higher plant genomes using Cpf1 CRISPR nucleases. Sci Rep 7:11606 Gasparis S, Kala M, Przyborowski M, Lyznik LA, Orczyk W, Nadolska-Orczyk A
(2018) A simple and efficient CRISPR/Cas9 platform for induction of single
and multiple, heritable mutations in barley (Hordeum vulgare L.). Plant
Methods 14:111 Biswas S, Li R, Yuan Z, Zhang D, Zhao X, Shi J (2019) Development of methods
for effective identification of CRISPR/Cas9-induced indels in rice. Plant Cell
Rep 38:503–510 Hahn F, Korolev A, Loures LS, Nekrasov V (2019) A modular cloning toolkit for
genome editing in plants. bioRxiv. https://doi.org/10.1101/738021 Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and
beyond. Biotechnol Adv 33:41–52 Hodgens C, Nimchuk ZL, Kieber JJ (2017) indCAPS: a tool for designing screening
primers for CRISPR/Cas9 mutagenesis events. PLoS one 12:e0188406 Butt H, Eid A, Ali Z, Atia MAM, Mokhtar MM, Hassan N, Lee CM, Bao G, Mahfouz
MM (2017) Efficient CRISPR/Cas9-mediated genome editing using a chimeric
single-guide RNA molecule. Front Plant Sci 8:1441 Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, Thomson JA
(2013) Efficient genome engineering in human pluripotent stem cells using
Cas9 from Neisseria meningitidis. Proc Natl Acad Sci U S A 110:15644–15649 Castel B, Tomlinson L, Locci F, Yang Y, Jones JDG (2019) Optimization of T-DNA
architecture for Cas9-mediated mutagenesis in Arabidopsis. PLoS One 14:
e0204778 Howells RM, Craze M, Bowden S, Wallington EJ (2018) Efficient generation of stable,
heritable gene edits in wheat using CRISPR/Cas9. BMC Plant Biol 18:215 Hsu CT, Cheng YJ, Yuan YH, Hung WF, Cheng QW, Wu FH, Lee LY, Gelvin
SB, Lin CS (2019) Application of Cas12a and nCas9-activation-induced
cytidine deaminase for genome editing and as a non-sexual strategy to
generate homozygous/multiplex edited plants in the allotetraploid
genome of tobacco. Plant Mol Biol. Received: 23 September 2019 Accepted: 2 December 2019 Feng C, Su H, Bai H, Wang R, Liu Y, Guo X, Liu C, Zhang J, Yuan J, Birchler JA, Han
F (2018) High-efficiency genome editing using a dmc1 promoter-controlled
CRISPR/Cas9 system in maize. Plant Biotechnol J 16:1848–1857 References https://doi.org/10.1007/s11103-019-
00907-w Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY, Konecna E, Belanto JJ,
Starker CG, Mathre JW, Greenstein RL, Voytas DF (2017) A multipurpose
toolkit to enable advanced genome engineering in plants. Plant Cell 29:
1196–1217 Chatterjee P, Jakimo N, Jacobson JM (2018) Minimal PAM specificity of a highly
similar SpCas9 ortholog. Sci Adv 4:eaau0766 Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu
X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting
specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832
Hu L, Li H, Qin R, Xu R, Li J, Li L, Wei P, Yang J (2016) Plant phosphomannose
isomerase as a selectable marker for rice transformation. Sci Rep 6:25921
Hu X, Meng X, Liu Q, Li J, Wang K (2018) Increasing the efficiency of CRISPR-
Cas9-VQR precise genome editing in rice. Plant Biotechnol J 16:292–297
Hu X, Wang C, Liu Q, Fu Y, Wang K (2017) Targeted mutagenesis in rice using
CRISPR-Cpf1 system. J Genet Genomics 44:71–73
Hua K, Tao X, Yuan F, Wang D, Zhu JK (2018) Precise a· T to G· C base editing in
the rice genome. Mol Plant 11:627–630
Hua K, Tao X, Zhu JK (2019) Expanding the base editing scope in rice by using
Cas9 variants. Plant Biotechnol J 17:499–504
Hua Y, Wang C, Huang J, Wang K (2017) A simple and efficient method for
CRISPR/Cas9-induced mutant screening. J Genet Genomics 44:207–213 Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu
X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting
specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832 Chen, S., Tao, L., Zeng, L., Vega‐Sanchez, M.E., Umemura, K. and Wang, G.L. (2006) A
highly efficient transient protoplast system for analyzing defense gene expression
and protein‐protein interactions in rice. Mol. Plant Pathol. 7, 417– 427. Hu L, Li H, Qin R, Xu R, Li J, Li L, Wei P, Yang J (2016) Plant phosphomannose
isomerase as a selectable marker for rice transformation. Sci Rep 6:25921 Chen L, Li W, Katin-Grazzini L, Ding J, Gu X, Li Y, Gu T, Wang R, Lin X, Deng Z,
McAvoy RJ, Gmitter FG Jr, Deng Z, Zhao Y, Li Y (2018) A method for the
production and expedient screening of CRISPR/Cas9-mediated non-
transgenic mutant plants. References Fossi M, Amundson K, Kuppu S, Britt A, Comai L (2019) Regeneration of Solanum
tuberosum plants from protoplasts induces widespread genome instability. Plant Physiol 180:78–86 Abdullah R, Cocking EC, Thompson AJ (1986) Efficient plant regeneration
from rice protoplasts through somatic embryogenesis. Nat Biotechnol 4:
1087–1090 Friedland AE, Baral R, Singhal P, Loveluck K, Shen S, Sanchez M, Marco E, Gotta
GM, Maeder ML, Kennedy EM (2015) Characterization of Staphylococcus
aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and
paired nickase applications. Genome Biol 16:257 Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ,
Verdine V, Cox DBT, Kellner MJ, Regev A (2017) RNA targeting with CRISPR-
Cas13. Nature 550:280 Abudayyeh OO, Gootenberg JS, Franklin B, Koob J, Kellner MJ, Ladha A, Joung J,
Kirchgatterer P, Cox DBT, Zhang F (2019) A cytosine deaminase for
programmable single-base RNA editing. Science 365:382–386 Abudayyeh OO, Gootenberg JS, Franklin B, Koob J, Kellner MJ, Ladha A, Joung J,
Kirchgatterer P, Cox DBT, Zhang F (2019) A cytosine deaminase for
programmable single-base RNA editing. Science 365:382–386
Aman R, Ali Z, Butt H, Mahas A, Aljedaani F, Khan MZ, Ding S, Mahfouz M (2018)
RNA virus interference via CRISPR/Cas13a system in plants. Genome Biol 19:1
Bao A, Burritt DJ, Chen H, Zhou X, Cao D, Tran LP (2019) The CRISPR/Cas9 system
and its applications in crop genome editing. Crit Rev Biotechnol 39:321–336 Fujimura T, Sakurai M, Akagi H, Negishi T, Hirose A (1985) Regeneration of Rice
plants from protoplasts. Plant Tiss Cult Letters 2:74–75 j
g
g
g
plants from protoplasts. Plant Tiss Cult Letters 2:74–75
Gao X, Chen J, Dai X, Zhang D, Zhao Y (2016) An effective strategy for reliably
isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/
Cas9-mediated genome editing. Plant Physiol 171:1794–1800
Gao Y, Zhao Y (2014) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro
and in vivo for CRISPR-mediated genome editing. J Integr Plant Biol 56:343–349 Gao X, Chen J, Dai X, Zhang D, Zhao Y (2016) An effective strategy for reliably
isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/
Cas9-mediated genome editing. Plant Physiol 171:1794–1800 Aman R, Ali Z, Butt H, Mahas A, Aljedaani F, Khan MZ, Ding S, Mahfouz M (2018)
RNA virus interference via CRISPR/Cas13a system in plants. Availability of data and materials
Not applicable. Page 11 of 13 Yue et al. Rice (2020) 13:9 Yue et al. Rice (2020) 13:9 Yue et al. Rice Yue et al. Rice Yue et al. Rice Ethics approval and consent to participate
Not applicable. Ding D, Chen K, Chen Y, Li H, Xie K (2018) Engineering introns to express RNA
guides for Cas9- and Cpf1-mediated multiplex genome editing. Mol Plant 11:
542–552 Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I,
Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE. (2016)
Optimized sgRNA design to maximize activity and minimize off-target effects
of CRISPR-Cas9. Nat Biotechnol. 34:184-191. Competing interests Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y, Gallant J, Xue
W, Rivera-Pérez JA, Sontheimer EJ (2019) A compact, high-accuracy Cas9
with a dinucleotide PAM for in vivo genome editing. Mol Cell 73:714–726.e4 Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y, Gallant J, Xue
W, Rivera-Pérez JA, Sontheimer EJ (2019) A compact, high-accuracy Cas9
with a dinucleotide PAM for in vivo genome editing. Mol Cell 73:714–726.e4
EFSA Panel on Genetically Modified Organisms (2012) Scientific opinion addressing
the safety assessment of plants developed using zinc finger nuclease 3 and
other site-directed nucleases with similar function. EFSA J 10:2943 p
g
The authors declare that they have no competing interests. Author details
1 EFSA Panel on Genetically Modified Organisms (2012) Scientific opinion addressing
the safety assessment of plants developed using zinc finger nuclease 3 and
other site-directed nucleases with similar function. EFSA J 10:2943 1Research Institute of Subtropical Forestry, Chinese Academy of Forestry,
Hangzhou, China. 2Department of Agricultural Chemistry, College of
Bioresources and Agriculture, National Taiwan University, Taipei, Taiwan. 3Key
Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research
Institute, Anhui Academy of Agricultural Sciences, Hefei, China. 4Department
of Agronomy, National Taiwan University, Taipei, Taiwan. 5Agricultural
Biotechnology Research Center, Academia Sinica, Taipei, Taiwan. 1Research Institute of Subtropical Forestry, Chinese Academy of Forestry,
Hangzhou, China. 2Department of Agricultural Chemistry, College of
Bioresources and Agriculture, National Taiwan University, Taipei, Taiwan. 3Key
Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Endo A, Masafumi M, Kaya H, Toki S (2016a) Efficient targeted mutagenesis of rice
and tobacco genomes using Cpf1 from Francisella novicida. Sci Rep 6:38169 Endo M, Mikami M, Endo A, Kaya H, Itoh T, Nishimasu H, Nureki O, Toki S (2019)
Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM. Nat Plants 5:14–17 Endo M, Mikami M, Toki S (2016b) Biallelic gene targeting in Rice. Plant Physiol
170:667–677 Received: 23 September 2019 Accepted: 2 December 2019 References Mol Cell 60:385–397 Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y,
Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG
(2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex
genome editing in monocot and dicot plants. Mol Plant 8:1274–1284
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013)
RNA-guided human genome engineering via Cas9. Science 339:823–826
Malzahn AA, Tang X, Lee K, Ren Q, Sretenovic S, Zhang Y, Chen H, Kang M, Bao
Y, Zheng X, Deng K, Zhang T, Salcedo V, Wang K, Zhang Y, Qi Y (2019)
Application of CRISPR-Cas12a temperature sensitivity for improved genome Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y,
Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG
(2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex
genome editing in monocot and dicot plants. Mol Plant 8:1274–1284
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013)
RNA-guided human genome engineering via Cas9. Science 339:823–826 Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y,
Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG
(2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex
genome editing in monocot and dicot plants. Mol Plant 8:1274–1284 Srivastava V, Underwood JL, Zhao S (2017) Dual-targeting by CRISPR/Cas9 for
precise excision of transgene from rice genome. Plant Cell Tissue Organ Cult
129:153–160 Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013)
RNA-guided human genome engineering via Cas9. Science 339:823–826 Steinert J, Schiml S, Fauser F, Puchta H (2015) Highly efficient heritable plant
genome engineering using Cas9 orthologues from Streptococcus
thermophilus and Staphylococcus aureus. Plant J 84:1295–1305 Malzahn AA, Tang X, Lee K, Ren Q, Sretenovic S, Zhang Y, Chen H, Kang M, Bao
Y, Zheng X, Deng K, Zhang T, Salcedo V, Wang K, Zhang Y, Qi Y (2019)
Application of CRISPR-Cas12a temperature sensitivity for improved genome
editing in rice, maize, and Arabidopsis. References The Crop Journal 6:314–320 Li X, Wang Y, Liu Y, Yang B, Wang X, Wei J, Lu Z, Zhang Y, Wu J, Huang X (2018)
Base editing with a Cpf1–cytidine deaminase fusion. Nat Biotechnol 36:324–327 Li X, Wang Y, Liu Y, Yang B, Wang X, Wei J, Lu Z, Zhang Y, Wu J, Huang X (2018)
Base editing with a Cpf1–cytidine deaminase fusion. Nat Biotechnol 36:324–327
Liang Z, Chen K, Gao C (2019) Biolistic delivery of CRISPR/Cas9 with
ribonucleoprotein complex in wheat. Methods Mol Biol 1917:327–335 Qin R, Li J, Li H, Zhang Y, Liu X, Miao Y, Zhang X, Wei P (2019) Developing a
highly efficient and wildly adaptive CRISPR-SaCas9 toolset for plant genome
editing. Plant Biotechnol J 17:706–708 Liang Z, Chen K, Gao C (2019) Biolistic delivery of CRISPR/Cas9 with
ribonucleoprotein complex in wheat. Methods Mol Biol 1917:327–335 Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, Liu J, Zhang H, Liu C, Ran Y, Gao
C (2017) Efficient DNA-free genome editing of bread wheat using CRISPR/
Cas9 ribonucleoprotein complexes. Nat Commun 8:14261 Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O,
Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F (2015) In vivo genome
editing using Staphylococcus aureus Cas9. Nature 520:186 Lin CS, Chang WC (1998) Micropropagation of Bambusa edulis through nodal
explants of field-grown culms and flowering of regenerated plantlets. Plant
Cell Rep 17:617–620 Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome
engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308 Rhodes CA, Lowe KS, Ruby KL (1988a) Plant regeneration from protoplasts
isolated from embryogenic maize cell cultures. Nat Biotechnol 6:56–60 Lin CS, Lai YH, Sun CW, Liu NT, Tsay HS, Chang WC, Chen JJW (2006)
Identification of ESTs differentially expressed in green and albino mutant
bamboo (Bambusa edulis) by suppressive subtractive hybridization (SSH) and
microarray analysis. Plant Cell Tissue Organ Cult 86:169–175 Rhodes CA, Pierce DA, Mettler IJ, Mascarenhas D, Detmer JJ (1988b) Genetically
transformed maize plants from protoplasts. Science 240:204–207 Lin CS, Liang CJ, Hsaio HW, Lin MJ, Chang WC (2007) In vitro flowering of green
and albino Dendrocalamus latiflorus. New For 34:177–186 Saika H, Mori A, Endo M, Toki S (2019) Targeted deletion of rice retrotransposon
Tos17 via CRISPR/Cas9. References Nat Methods 13:551–554
Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu LJ (2013) Gao C (2019) Cytosine, but not adenine, base editors induce genome-wide
off-target mutations in rice. Science 364:292–295 Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu LJ (2013)
Targeted mutagenesis in rice using CRISPRCas system. Cell Res 23:1233–1236 Karvelis T, Gasiunas G, Young J, Bigelyte G, Silanskas A, Cigan M, Siksnys V (2015)
Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence
elements. Genome Biol 16:253 Mikami M, Toki S, Endo M (2015) Comparison of CRISPR/Cas9 expression
constructs for efficient targeted mutagenesis in rice. Plant Mol Biol 88:
561–572 Kaya H, Mikami M, Endo A, Endo M, Toki S (2016) Highly specific targeted
mutagenesis in plants using Staphylococcus aureus Cas9. Sci Rep 6:26871 Mikami M, Toki S, Endo M (2016) Precision targeted mutagenesis via Cas9 paired
nickases in rice. Plant Cell Physiol 57:1058–1068 Kim E, Koo T, Park SW, Kim D, Kim K, Cho HY, Song DW, Lee KJ, Jung MH, Kim S,
Kim JH, Kim JH, Kim JS (2017a) In vivo genome editing with a small Cas9
orthologue derived from Campylobacter jejuni. Nat Commun 8:14500 Miki B, McHugh S (2004) Selectable marker genes in transgenic plants:
applications, alternatives and biosafety. J Biotechnol 107:193–232 Kim H, Kim ST, Ryu J, Kang BC, Kim JS, Kim SG (2017b) CRISPR/Cpf1-mediated
DNA-free plant genome editing. Nat Commun 8:14406 Miki D, Zhang W, Zeng W, Feng Z, Zhu JK (2018) CRISPR/Cas9-mediated gene
targeting in Arabidopsis using sequential transformation. Nat Commun 9:1967 Miki D, Zhang W, Zeng W, Feng Z, Zhu JK (2018) CRISPR/Cas9-mediated gene
targeting in Arabidopsis using sequential transformation. Nat Commun 9:1967 Kim HK, Song M, Lee J, Menon AV, Kim H (2017c) In vivo highthroughput
profiling of CRISPRCpf1 activity. Nat Methods 14:153 Nandy S, Pathak B, Zhao S, Srivastava V (2019) Heat-shock-inducible CRISPR/Cas9
system generates heritable mutations in rice. Plant Direct 3:e00145 Nandy S, Pathak B, Zhao S, Srivastava V (2019) Heat-shock-inducible CRISPR/Cas9
system generates heritable mutations in rice. Plant Direct 3:e00145
Okamoto T, Ohnishi Y, Toda E (2017) Development of polyspermic zygote and
possible contribution of polyspermy to polyploid formation in angiosperms. References J Plant Res 130:485–490 Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR (2017d) Increasing the
genome-targeting scope and precision of base editing with engineered
Cas9-cytidine deaminase fusions. Nat Biotechnol 35:371–376 Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR (2017d) Increasing the
genome-targeting scope and precision of base editing with engineered
Cas9-cytidine deaminase fusions. Nat Biotechnol 35:371–376 Okamoto T, Ohnishi Y, Toda E (2017) Development of polyspermic zygote and
possible contribution of polyspermy to polyploid formation in angiosperms. J Plant Res 130:485–490 Kis A, Hamar E, Tholt G, Ban R, Havelda Z (2019) Creating highly efficient
resistance against wheat dwarf virus in barley by employing CRISPR/Cas9
system. Plant Biotechnol J 17:1004–1006 Kis A, Hamar E, Tholt G, Ban R, Havelda Z (2019) Creating highly efficient
resistance against wheat dwarf virus in barley by employing CRISPR/Cas9
system. Plant Biotechnol J 17:1004–1006 Oung HM, Lin KC, Wu TM, Chandrika NNP, Hong CY (2015) Hygromycin B-
induced cell death is partly mediated by reactive oxygen species in rice
(Oryza sativa L.). Plant Mol Biol 89:577–588 Park J, Choe S (2019) DNA-free genome editing with preassembled CRISPR/Cas9
ribonucleoproteins in plants. Transgenic Res 28(Suppl 2):61–64 Kleinstiver BP, Prew MS, Tsai SQ, Nguyen NT, Topkar VV, Zheng Z, Joung JK
(2015) Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9
by modifying PAM recognition. Nat Biotechnol 33:1293–1298 Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR (2013) High-
throughput profiling of off-target DNA cleavage reveals RNA-programmed
Cas9 nuclease specificity. Nat Biotechnol 31:839–843 Kurata M, Wolf NK, Lahr WS, Weg MT, Kluesner MG, Lee S, Hui K, Shiraiwa M,
Webber BR, Moriarity BS (2018) Highly multiplexed genome engineering
using CRISPR/Cas9 gRNA arrays. PLoS One 13:e0198714 Podevin N, Davies HV, Hartung F, Nogue F, Casacuberta JM (2013) Site-directed
nucleases: a paradigm shift in predictable, knowledge-based plant breeding. Trends Biotechnol 3:375–383 Li S, Li J, He Y, Xu M, Zhang J, Du W, Zhao Y, Xia L (2019) Precise gene
replacement in rice by RNA transcript-templated homologous
recombination. Nat Biotechnol 37:445 Qi X, Dong L, Liu C, Mao L, Liu F, Zhang X, Cheng B, Xie C (2018) Systematic
identification of endogenous RNA polymerase III promoters for efficient RNA
guide-based genome editing technologies in maize. References Plant Cell Rep 38:455–458 Sampson TR, Saroj SD, Llewellyn AC, Tzeng YL, Weiss DS (2013) A CRISPR/Cas
system mediates bacterial innate immune evasion and virulence. Nature 497:
254–257 Liu NT, Jane WN, Tsay HS, Wu H, Chang WC, Lin CS (2007) Chloroplast genome
aberration in micropropagation-derived albino Bambusa edulis mutants, ab1
and ab2. Plant Cell Tissue Organ Cult 88:147–156 Shan Q, Wang Y, Chen K, Liang Z, Li J, Zhang Y, Zhang K, Liu J, Voytas DF, Zheng
X, Zhang Y, Gao C (2013) Rapid and efficient gene modification in rice and
Brachypodium using TALENs. Mol Plant 6:1365–1368
Shan Q, Wang Y, Li J, Gao C (2014) Genome editing in rice and wheat using the
CRISPR/Cas system. Nat Protoc 9:2395–2410
Shimamoto K, Terada R, Izawa T, Fujimoto H (1989) Fertile transgenic rice plants Shan Q, Wang Y, Chen K, Liang Z, Li J, Zhang Y, Zhang K, Liu J, Voytas DF, Zheng
X, Zhang Y, Gao C (2013) Rapid and efficient gene modification in rice and
Brachypodium using TALENs. Mol Plant 6:1365–1368 Lowder LG, Zhang D, Baltes NJ, Paul JW 3rd, Tang X, Zheng X, Voytas DF, Hsieh
TF, Zhang Y, Qi Y (2015) A CRISPR/Cas9 toolbox for multiplexed plant
genome editing and transcriptional regulation. Plant Physiol 169:971–985 Shan Q, Wang Y, Li J, Gao C (2014) Genome editing in rice and wheat using the
CRISPR/Cas system. Nat Protoc 9:2395–2410 Lowe K, Wu E, Wang N, Hoerster G, Hastings C, Cho MJ, Scelonge C,
Lenderts B, Chamberlin M, Cushatt J, Wang L, Ryan L, Khan T, Chow-Yiu
J, Hua W, Yu M, Banh J, Bao Z, Brink K, Igo E, Rudrappa B, Shamseer PM,
Bruce W, Newman L, Shen B, Zheng P, Bidney D, Falco C, Register J,
Zhao ZY, Xu D, Jones T, Gordon-Kamm W (2016) Morphogenic
regulators Baby boom and Wuschel improve monocot transformation. Plant Cell 28:1998–2015 Shimamoto K, Terada R, Izawa T, Fujimoto H (1989) Fertile transgenic rice plants
regenerated from transformed protoplasts. Nature 338:274–276 Shmakov S, Abudayyeh OO, Makarova KS, Wolf YI, Gootenberg JS, Semenova E,
Minakhin L, Joung J, Konermann S, Severinov K, Zhang F, Koonin EV (2015)
Discovery and functional characterization of diverse class 2 CRISPR-Cas
systems. References Hortic Res 5:13 Hu X, Meng X, Liu Q, Li J, Wang K (2018) Increasing the efficiency of CRISPR-
Cas9-VQR precise genome editing in rice. Plant Biotechnol J 16:292–297 Hu X, Wang C, Liu Q, Fu Y, Wang K (2017) Targeted mutagenesis in rice using
CRISPR-Cpf1 system. J Genet Genomics 44:71–73 Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W,
Marraffini LA (2013) Multiplex genome engineering using CRISPR/Cas
systems. Science 339:819–823 Hua K, Tao X, Yuan F, Wang D, Zhu JK (2018) Precise a· T to G· C base editing in
the rice genome. Mol Plant 11:627–630 Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F
(2017) RNA editing with CRISPR-Cas13. Science 358:1019–1027 Hua K, Tao X, Zhu JK (2019) Expanding the base editing scope in rice by using
Cas9 variants. Plant Biotechnol J 17:499–504 Didovyk A, Borek B, Tsimring L, Hasty J (2016) Transcriptional regulation with
CRISPR-Cas9: principles, advances, and applications. Curr Opin Biotechnol 40:
177–184 Hua Y, Wang C, Huang J, Wang K (2017) A simple and efficient method for
CRISPR/Cas9-induced mutant screening. J Genet Genomics 44:207–213 Hua Y, Wang C, Huang J, Wang K (2017) A simple and efficient method for
CRISPR/Cas9-induced mutant screening. J Genet Genomics 44:207–213 Page 12 of 13 Page 12 of 13 Page 12 of 13 Yue et al. Rice (2020) 13:9 Yue et al. Rice (2020) 13:9 Jia H, Xu J, Orbović V, Zhang Y, Wang N (2017) Editing citrus genome via SaCas9/
sgRNA system. Front Plant Sci 8:2135 Marton L, Wullems GJ, Molendijk L, Schilperoort RA (1979) In vitro transformation
of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens. Nature 277:129–131 Jin S, Zong Y, Gao Q, Zhu Z, Wang Y, Qin P, Liang C, Wang D, Qiu JL, Zhang F,
Gao C (2019) Cytosine, but not adenine, base editors induce genome-wide
off-target mutations in rice. Science 364:292–295 Jin S, Zong Y, Gao Q, Zhu Z, Wang Y, Qin P, Liang C, Wang D, Qiu JL, Zhang F, Marx V (2016) Plants: a tool box of cell-based assays. Nat Methods 13:551–554
Mi
J G
D Zh
J H
Q Qi
G Zh
X W
J G
H Q
LJ (2013) Marx V (2016) Plants: a tool box of cell-based assays. References BMC Biol 17:9 Svitashev S, Schwartz C, Lenderts B, Young JK, Mark Cigan A (2016) Genome
editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat
Commun 7:13274 Page 13 of 13 Yue et al. Rice (2020) 13:9 Takebe I, Labib G, Melchers G (1971) Regeneration of whole plants from isolated
mesophyll protoplasts of tobacco. Naturwissenschaften 58:318–320 Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, Qiu JL, Gao C (2016) Efficient
and transgene-free genome editing in wheat through transient expression of
CRISPR/Cas9 DNA or RNA. Nat Commun 7:12617 Tang J, Chen L, Liu YG (2019) Off-target effects and the solution. Nat Plants 5:341–342 Zhang Y, Malzahn AA, Sretenovic S, Qi Y (2019a) The emerging and uncultivated
potential of CRISPR technology in plant science. Nat Plants 5:778–794 Tang X, Liu G, Zhou J, Ren Q, You Q, Tian L, Xin X, Zhong Z, Liu B, Zheng X,
Zhang D, Malzahn A, Gong Z, Qi Y, Zhang T, Zhang Y (2018) A large-scale
whole-genome sequencing analysis reveals highly specific genome editing
by both Cas9 and Cpf1 (Cas12a) nucleases in rice. Genome Biol 19:84 Zhou H, Liu B, Weeks DP, Spalding MH, Yang B (2014) Large chromosomal
deletions and heritable small genetic changes induced by CRISPR/Cas9 in
rice. Nucleic Acids Res 42:10903–10914 Tang X, Lowder LG, Zhang T, Malzahn AA, Zheng X, Voytas DF, Zhong Z, Chen Y,
Ren Q, Li Q, Kirkland ER, Zhang Y, Qi Y (2017) A CRISPR-Cpf1 system for
efficient genome editing and transcriptional repression in plants. Nat Plants
3:17013 Zhu X, Xu Y, Yu S, Lu L, Ding M, Cheng J, Song G, Gao X, Yao L, Fan D, Meng S,
Zhang X, Hu S, Tian Y (2014) An efficient genotyping method for genome-
modified animals and human cells generated with CRISPR/Cas9 system. Sci
Rep 4:6420 Teng F, Cui T, Feng G, Guo L, Xu K, Gao Q, Li T, Li J, Zhou Q, Li W (2018)
Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell
Discov 4:63 Wu X, Scott DA, Kriz AJ, Chiu AC, Hsu PD, Dadon DB, Cheng AW, Trevino AE,
Konermann S, Chen S, et al. (2014) Genome-wide binding of the CRISPR
endonuclease Cas9 in mammalian cells. Nat Biotechnol 32, 670–676. Thomas HR, Percival SM, Yoder BK, Parant JM (2014) High-throughput genome
editing and phenotyping facilitated by high resolution melting curve
analysis. Plant Biotechnol J. https://doi.org/10.1111/pbi.13137 Vouillot L, Thelie A, Pollet N (2015) Comparison of T7E1 and surveyor mismatch
cleavage assays to detect mutations triggered by engineered nucleases. G3
(Bethesda) 5:407–415 Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J
(2013) Multiplex and homologous recombination-mediated genome editing
in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature Biotechnol 31:688-691. Wang M, Mao Y, Lu Y, Tao X, Zhu JK (2017) Multiplex gene editing in rice using
the CRISPR-Cpf1 system. Mol Plant 10:1011–1013 Liang Z, Zhang K, Chen K, Gao C (2014) Targeted mutagenesis in Zea mays using
TALENs and the CRISPR/Cas system. J Genet Gen 41:63-68 Wang M, Mao Y, Lu Y, Wang Z, Tao X, Zhu JK (2018) Multiplex gene editing in
rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems. J Integr Plant Biol
60:626–631 Sun X, Hu Z, Chen R, Jiang Q, Song G, Zhang H, Xi Y (2015) Targeted
mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep 5:10342 Wilmink A, Dons JJM (1993) Selective agents and marker genes for use in
transformation of monocotyledonous plants. Plant Mol Biol Rep 11:165–185 Zong Y, Wang Y, Li C, Zhang R, Chen K, Ran Y, Qiu JL, Wang D, Gao C (2017)
Precise base editing in rice, wheat and maize with a Cas9-cytidine
deaminase fusion. Nature Biotechnol 35:438-440. Wolter F, Puchta H (2019) In planta gene targeting can be enhanced by the use
of CRISPR/Cas12a. Plant J. https://doi.org/10.1111/tpj.14488 g
g
g
of CRISPR/Cas12a. Plant J. https://doi.org/10.1111/tpj.14488 Zong Y, Song Q, Li C, Jin S, Zhang D, Wang Y, Qiu JL, Gao C. 2018 Efficient C-to-T
base editing in plants using a fusion of nCas9 and human APOBEC3A. Nature
Biotechnol 36, 950–953. Woo JW, Kim J, Kwon SI, Corvalan C, Cho SW, Kim H, Kim SG, Kim ST, Choe S,
Kim JS (2015) DNA-free genome editing in plants with preassembled CRISPR-
Cas9 ribonucleoproteins. Nat Biotechnol 33:1162–1164 Kim H, Kim ST, Ryu J, Kang BC, Kim JS, Kim SG (2017) CRISPR/Cpf1-mediated
DNA-free plant genome editing. Nature Commun 8:14406. Wu TM, Huang JZ, Oung HM, Hsu YT, Tsai YC, Hong CY (2019) H2O2-based
method for rapid detection of transgene-free rice plants from segregating
CRISPR/Cas9 genome-edited progenies. Int J Mol Sci 20:3885 Malnoy M, Viola R, Jung MH, Koo OJ, Kim S, Kim JS, Velasco R, Nagamangala
Kanchiswamy C (2016) DNA-free genetically edited grapevine and apple
protoplast using CRISPR/Cas9 ribonucleoproteins. Plant Biotechnol J. https://doi.org/10.1111/pbi.13137 Frontiers Plant Sci 7:1904. Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing
capability with the endogenous tRNA-processing system. Proc Natl Acad Sci
U S A 112:3570–3575 Wu FH, Shen SC, Lee LY, Lee SH, Chan MT, Lin CS (2009) Tape- Arabidopsis Sandwich -
a simpler Arabidopsis protoplast isolation method. Plant Methods 5:16 Xie K, Yang Y (2013) RNA-guided genome editing in plants using a CRISPR-Cas
system. Mol Plant 6:1975–1983 Zhang Y, Su J, Duan S, Ao Y, Dai J, Liu J, Wang P, Li Y, Liu B, Feng D, Wang J,
Wang H (2011) A highly efficient rice green tissue protoplast system for
transient gene expression and studying light/chloroplast‐related processes. Plant Methods 7: 30 Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ (2014)
A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol
14:327 Wullems GJ, Molendijk L, Ooms G, Schilperoort RA (1981a) Differential expression of
crown gall tumor markers in transformants obtained after in vitro Agrobacterium
tumefaciens-induced transformation of cell wall regenerating protoplasts
derived from Nicotiana tabacum. Proc Natl Acad Sci USA 78:4344-4348 Xu R, Qin R, Li H, Li D, Li L, Wei P, Yang J (2017) Generation of targeted mutant
rice using a CRISPR-Cpf1 system. Plant Biotechnol J 15:713–717 Xu R, Qin R, Li H, Li J, Yang J, Wei P (2019) Enhanced genome editing in rice using
single transcript unit CRISPR-LbCpf1 systems. Plant Biotechnol J 17:553–555 Wullems GJ, Molendijk L, Ooms G, Schilperoort RA (1981b) Retention of tumor
markers in F1 progeny plants from in vitro induced octopine and nopaline
tumor tissues. Cell 24:719-727 Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS,
Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang
F (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas
system. Cell 163:759–771 References PLoS One 9:e114632 Lin CS, Hsu CT, Yang LH, Lee LY, Fu JY, Cheng QW, Wu FH, Hsiao HC, Zhang Y,
Zhang R, Chang WJ, Yu CT, Wang W, Liao LJ, Gelvin SB, Shih MC (2018)
Application of protoplast technology to CRISPR/Cas9 mutagenesis: from
single-cell mutation detection to mutant plant regeneration. Plant Biotechnol
J 16:1295-1310 Toda E, Koiso N, Takebayashi A, Ichikawa M, Kiba T, Osakabe K, Osakabe Y, Sakakibara
H, Kato N, Okamoto T (2019) An efficient DNA- and selectable-marker-free
genome-editing system using zygotes in rice. Nat Plants 5:363–368 Andersson M, Turesson H, Nicolia A, Falt AS, Samuelsson M, Hofvander P (2017)
Efficient targeted multiallelic mutagenesis in tetraploid potato ( Solanum
tuberosum) by transient CRISPR-Cas9 expression in protoplasts. Plant Cell Rep
36:117-128. Toriyama K, Arimoto Y, Uchimiya H, Hinata K (1988) Transgenic rice plants after
direct gene transfer into protoplasts. Nat Biotechnol 6:1072–1074 Tuncel A, Corbin KR, Ahn-Jarvis J, Harris S, Hawkins E, Smedley MA, Harwood W,
Warren FJ, Patron NJ, Smith AM (2019) Cas9-mediated mutagenesis of potato
starch-branching enzymes generates a range of tuber starch phenotypes. Plant Biotechnol J. https://doi.org/10.1111/pbi.13137 Andersson M, Turesson H, Olsson N, Falt AS, Ohlsson P, Gonzalez MN,
Samuelsson M, Hofvander P (2018) Genome editing in potato via CRISPR-
Cas9 ribonucleoprotein delivery. Physiol Plant 64:378-384 Publisher’s Note Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu
N, Zhu JK (2014) The CRISPR/Cas9 system produces specific and
homozygous targeted gene editing in rice in one generation. Plant
Biotechnol J 12:797–807 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. Zhang T, Zhao Y, Ye J, Cao X, Xu C, Chen B, An H, Jiao Y, Zhang F, Yang X, Zhou G
(2019b) Establishing CRISPR/Cas13a immune system conferring RNA virus
resistance in both dicot and monocot plants. Plant Biotechnol J 17:1185–1187 Zhang T, Zheng Q, Yi X, An H, Ma S, Zhou G (2018) Establishing RNA virus
resistance in plants by harnessing CRISPR immune system. Plant Biotechnol J
16:1415–1423
|
https://openalex.org/W4383052364
|
https://zenodo.org/records/8033098/files/stomatology-2023-2-19.pdf
|
Russian
| null |
COMPLEX METHODS OF TREATMENT OF CHILDREN WITH ODONTOGENIC INFLAMMATION
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,023
|
cc-by
| 3,769
|
¬
i
W
|
Yfc`S^dea_Sea^aV[[[
]cS`[agSi[S^o`nh[dd^XWaUS`[\
ea_`a_Xc
ɮɮɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ
ISSN 2181-0966
Doi Journal 10.26739/2181-0966 ISSN 2181-0966
Doi Journal 10.26739/2181-0966 TОШКЕНТ-2023 TОШКЕНТ-2023 ɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\
ɮɮɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ
№2 (2023) DOI http://dx.doi.org/10.26739/ 2181-0966-2023-2 ɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\
ɮɮɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ
№2 (2023) DOI http://dx.doi.org/10.26739/ 2181-0966-2023-2 ɮɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\№2
Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\
№2 (2023) DOI http://dx.doi.org/10.26739/ 2181-0966-2023-2 agSi[S^o`nh[dd^XWaUS`[\№2 | 2023
Si[S^o`nh[dd^XWaUS`[\
6739/ 2181-0966-2023-2
\]a^^XV[[
Заместитель главного редактора:
Юлдашев Абдуазим Абдувалиевич
октор медицинских наук, доцент Ташкентского
Государственного Стоматологического
института, Узбекистан
рзахмедов
Контакт редакций журналов. www.tadqiqot.uz
ООО Tadqiqot город Ташкент,
улица Амира Темура пр.1, дом-2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz
Тел: (+998-94) 404-0000
Аветиков Давид Саломонович
доктор медицинских наук, профессор, Украина
Амхадова Малкан Абдурашидовна
доктор медицинских наук, профессор, Россия
Копбаева Майра Тайтолеуовна
доктор медицинских наук, профессор, Казахстан
Грудянов Александр Иванович
доктор медицинских наук, профессор, Россия
Лосев Фёдор Фёдорович
доктор медицинских наук, профессор, Россия
Шаковец Наталья Вячеславовна
доктор медицинских наук, профессор, Белоруссия
Jun-Young Paeng
доктор медицинских наук, профессор, Корея
Jinichi Sakamoto
доктор медицинских наук, профессор, Япония
Дустмухамедов Дильшод Махмудович
доктор медицинских наук, доцент, Узбекистан
Ризаев Элёр Алимджанович
доктор медицинских наук, доцент, Узбекистан
Камалова Феруза Рахматиллаевна
доктор медицинских наук, доцент, Узбекистан
Абдувакилов Жахонгир Убайдулла угли
доктор медицинских наук, доцент, Узбекистан
Зоиров Тулкин Элназарович
доктор медицинских наук, доцент, Узбекистан
Мамедов Умиджон Суннатович
доктор медицинских наук, доцент, Узбекистан
Раимкулова Дилноза Фарходдиновна
PhD, доцент, Узбекистан
Юнусходжаева Мадина Камалитдиновна
доцент, Узбекистан Билалов Эркин Назимович
доктор медицинских наук, профессор, Узбекистан Билалов Эркин Назимович
доктор медицинских наук, профессор, Узбекистан Новиков Вадим Михайлович
доктор медицинских наук, профессор, Украина Новиков Вадим Михайлович
доктор медицинских наук, профессор, Украина Бекжанова Ольга Есеновна
доктор медицинских наук, профессор, Узбекистан Бахритдинова Фазилат Арифовна
доктор медицинских наук, профессор, Узбекистан Шомуродов Кахрамон Эркинович
доктор медицинских наук, доцент, Узбекистан Шамсиев Жахонгир Фазлиддинович
доктор медицинских наук, доцент, Узбекистан Юсупалиходжаева Саодат Хамидуллаевна
доктор медицинских наук, доцент, Узбекистан Вахидов Улугбек Нуритдитнович
доктор медицинских наук, доцент, Узбекистан Jinichi Sakamoto
доктор медицинских наук, профессор, Япония Муртазаев Саидмуродхон Саидаълоевич
доктор медицинских наук, доцент, Узбекистан Дустмухамедов Дильшод Махмудович
доктор медицинских наук, доцент, Узбекистан Шукурова Умида Абдурасуловна
доктор медицинских наук, доцент, Узбекистан Ризаев Элёр Алимджанович
доктор медицинских наук, доцент, Узбекистан Хасанова Лола Эмильевна
доктор медицинских наук, доцент, Узбекистан Камалова Феруза Рахматиллаевна
доктор медицинских наук, доцент, Узбекистан Абдувакилов Жахонгир Убайдулла угли
доктор медицинских наук, доцент, Узбекистан Зоиров Тулкин Элназарович
доктор медицинских наук, доцент, Узбекистан Мамедов Умиджон Суннатович
доктор медицинских наук, доцент, Узбекистан Раимкулова Дилноза Фарходдиновна
PhD, доцент, Узбекистан 3 3 ɸɖɮɸɮɖ Fazilat A. Bahritdinova
Doctor of Medical Sciences, Professor, Uzbekistan Kakhramon E. Shomurodov
Doctor of Medical Sciences, Docent, Uzbekistan Ulugbek N. Vakhidov
Doctor of Medical Sciences, Docent, Uzbekistan Jinichi Sakamoto
Doctor of Medicine, Professor, Japan Dilshod M. Dustmukhamedov
Doctor of Medical Sciences, Docent, Uzbekistan Saidmurodkhon S. Murtazaev
Doctor of Medical Sciences, Docent, Uzbekistan Saidmurodkhon S. Murtazaev
Doctor of Medical Sciences, Docent, Uzbekistan Rizaev Elyor Alimdjanovich
Doctor of Medical Sciences, Docent, Uzbekistan Umida A. Shukurova
Doctor of Medical Sciences, Docent, Uzbekistan Umida A. Shukurova
Doctor of Medical Sciences, Docent, Uzbekistan Umida A. Shukurova
Doctor of Medical Sciences, Docent, Uzbekistan Kamalova Feruza Raxmatillaevna
Doctor of Medical Sciences, Docent, Uzbekistan Lola E. Khasanova
Doctor of Medical Sciences, Docent, Uzbekistan Lola E. Khasanova
Doctor of Medical Sciences, Docent, Uzbekistan Jakhongir U. Abduvakilov
Doctor of Medical Sciences, Docent, Uzbekistan Tulkin E. Zoirov
Doctor of Medical Sciences, Docent, Uzbekistan 4
ɮɮɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\№2 | 2023
1. Ризаев Жасур Алимджанович, Агзамова Сайёра Саидаминовна, Туляганов Нозим Алишерович
РЕЗУЛЬТАТЫ РЕТРОСПЕКТИВНОГО АНАЛИЗА СОЧЕТАННЫХ ТРАВМ СРЕДНЕЙ ЗОНЫ ЛИЦА……………………6
2. Ризаев Элёр Алимджанович, Бузрукзода Жавохирхон Даврон
ИЗУЧЕНИЕ МОРФОЛОГИЧЕСКОЙ КАРТИНЫ ПРИ ДЕНТАЛЬНОЙ ИМПЛАНТАЦИИ…………………………………..13
3. Nasretdinova Maxzuna Taxsinovna, Normirova Nargiza Nazarovna, Baxronov Bezod Shavkatovich,
Normuradov Nodir Alisherovich
MUVOZANAT FUNKSIYASI BUZILGANDA VESTIBULYAR ANALIZATORNI BAHOLASH……………………………..17
4. Исламова Нилуфар Бустановна, Назарова Нодира Шариповна
СУРУНКАЛИ ТАРҚАЛГАН ПАРОДОНТИТ БИЛАН КАСАЛЛАНГАН ПОСТМЕНОПАУЗА
ДАВРИДАГИ АЁЛЛАРНИНГ ПАРОДОНТ ТЎҚИМАСИНИНГ ДАВОЛАШ САМАРАДОРЛИГИ ОШИРИШ……………20
5. Рахматова Дилнора Саиджоновна
БОЛАЛАР ОРАСИДА ТИШ КАРИЕСИ КАСАЛЛИГИ ТЕКШИРИЛАЁТГАН БОЛАЛАРНИНГ
КЛИНИК ХУСУСИЯТЛАРИ………………………………………………………………………………………………………25
6. Хамракулова Наргиза Орзуевна
СУРУНКАЛИ СИНУСИТНИНГ УЗОҚ МУДДАТЛИ ШАКЛЛАРИ БЎЛГАН БЕМОРЛАРНИ
БОШҚАРИШ ХУСУСИЯТЛАРИ………………………………………………………………………………………………….29
7. Насретдинова Махзуна Тахсиновна, Раупова Камола, Лутфуллаева Гульноза, Нормурадов Нодир Алишерович
ОЦЕНКА ЭФФЕКТИВНОСТИ ЛЕЧЕНИЯ ПАЦИЕНТОВ С НЕЙРОСЕНСОРНОЙ ТУГОУХОСТЬЮ……………………..31
8. Терехов Алексей Борисович, Нэстасе Корнелиу Иванович
СОВРЕМЕННАЯ КОНЦЕПЦИЯ В ДИАГНОСТИКЕ И ЛЕЧЕНИИ КАРИЕСА КОРНЯ………………………………………36
9. Ризаев Жасур Алимджанович, Шодмонов Ахрорбек А, Раджабий Музаянна Азиз кизи
ОЦЕНКА ТОЧНОСТИ УСТАНОВКИ ДЕНТАЛЬНЫХ ИМПЛАНТАТОВ…………………………………………………….40
10. Ахмедов Хуршид Камалович
ОРТОПЕДИК ДАВОЛАНИШДАН ОЛДИН БЕМОРЛАРДА ОҒИЗ БЎШЛИҒИДАГИ ЦИТОКИН
ҲОЛАТИНИНГ ПАРАМЕТРЛАРИ АНИҚЛАШ УСУЛЛАРИ………………………………………………………………….44
11. Қурбонов Дилшод Фарходович, Хабибова Назира Насуллоевна
ҲОМИЛАДОР АЁЛЛАР СТОМАТОЛОГИК САЛОМАТЛИГИНИ АНИҚЛАШ КЎРСАТКИЧЛАРИ………………………47
12. Эронов Ёқуб Қуватович
ИМКОНИЯТИ ЧЕКЛАНГАН БОЛАЛАРДА ПАРОДОНТ ТЎҚИМАСИНИНГ МИКРОБИОЛОГИК
ТАДҚИҚОД НАТИЖАЛАРИ СОЛИШТИРМА ТАҲЛИЛЛАРИ……………………………………………………………….52
13. Атоева Максад Амановна
РАСПРОСТРАНЕННОСТЬ, ИНТЕНСИВНОСТЬ И ОСОБЕННОСТИ КЛИНИЧЕСКОГО ТЕЧЕНИЯ
ЗАБОЛЕВАНИЙ ПАРОДОНТА У ЛИЦ МОЛОДОГО ВОЗРАСТА…………………………………………………………….55
14. Нурова Шоҳсанам Норпўлотовна
АЁЛЛАРДА ЭСТРОГЕН ЕТИШМОВЧИЛИГИ ОҚИБАТИДА ТИШ-ЖАҒ ТИЗИМИДАГИ
ЎЗГАРИШЛАР БЎЙИЧА УМУМИЙ ТАВСИФ………………………………………………………………………………….58
15. Идиев Ойбек Элмуродович, Ибрагимова Феруза Икромовна
БОШ МИЯ ФАЛАЖИ БИЛАН КАСАЛЛАНГАН БОЛАЛАРДА ЧАЙНОВ МУШАКЛАРИНИНГ
ЭЛЕКТРОМИОГРАФИК КЎРСАТКИЧЛАРИНИ АНИҚЛАШ…………………………………………………………………61
16. ɸɖɮɸɮɖ Астанов Отабек Миржонович
ЧАККА-ПАСТКИ ЖАҒ БЎҒИМИ КАСАЛЛИКЛАРИДА КОМПЛЕКС ДАВОЛАШ
УСУЛЛАРИНИНГ САМАРАДОРЛИГИНИ БАҲОЛАШ………………………………………………………………………..66
17. Ражабов Отабек Асрорович
МЕТАЛЛОКЕРАМИК ПРОТЕЗИ БЎЛГАН БЕМОРЛАР ГУРУҲИДА ДАВОЛАШДАН ОЛДИНГИ
ВА КЕЙИНГИ ИММУНОЛОГИК ТАДҚИҚОТЛАР ТАҲЛИЛИЙ НАТИЖАЛАРИ………………………………………….69
18. Taylakova Dildora Ibragimovna
PREVENTION AND TREATMENT OF DENTAL FLUOROSIS IN CHILDREN…………………………………………………72
19. Ташева Гулчехра Сулямановна
ОДОНТОГЕН ЯЛЛИҒЛАНИШ БИЛАН КАСАЛЛАНГАН БОЛАЛАРНИ КОМПЛЕКС ДАВОЛАШ УСУЛЛАРИ………..76
20. Фозилов Уктам Абдураззоқович
ЮҚОРИГИ ЖАҒНИНГ ТОРАЙИШИ ҲИСОБИГА ЮЗАГА КЕЛГАН ПАТОЛОГИК ОКЛЮЗИЯНИ
ДАВОЛАШ УСУЛЛАРИНИ ТАКОМИЛЛАШТИРИШ…………………………………………………………………………80
daWXcYS`[X¬ɹ
10. Ахмедов Хуршид Камалович 13. Атоева Максад Амановна 14. Нурова Шоҳсанам Норпўлотовна
Ё 16. Астанов Отабек Миржонович
Ў 17. Ражабов Отабек Асрорович 18. Taylakova Dildora Ibragimovna 5
ɮɮɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\ №2 | 2023 АННОТАЦИЯ Ц
Сегодня пациенты с одонтогенными воспалительными заболеваниями составляют 10-20% стоматологических заболеваний. А 30-70%
одонтогенных воспалительных заболеваний - это абсцессы и флегмоны, совершенствование диагностики этих заболеваний и
эффективное лечение считается одной из актуальных проблем. Важно разработать специальные меры лечения детей с этим заболеванием. Ключевые слова: одонтогенные воспалительные заболевания, абсцессы и флегмоны. Ташева Гулчехра Сулямановна
Бухоро давлат тиббиёт институти Ташева Гулчехра Сулямановна Ташева Гулчехра Сулямановна
Бухарский государственный медицинский институт Бухарский государственный медицинский институт http://dx.doi.org/10.5281/zenodo.8033098 http://dx.doi.org/10.5281/zenodo.8033098 АННОТАЦИЯ Ц
Бугунги кунда одонтоген яллиғланиш касалликлари билан оғриган беморлар стоматологик касалликларни 10 – 20 % ташкил этади. Одонтоген яллиғланиш касалликларини 30 - 70 % ни эса абцесс ва флегмоналар ташкил этиб, ушбу касалликларга ташхис қўйишни
мукаммаллаштириш ва самарали даволаш долзарб муаммолардан бири бўлиб ҳисобланади. Ушбу касаллик билан касалланган болаларда
махсус даволаш чораларини ишлаб чиқиш муҳим аҳамиятга эга. у
р
р
у
Калит сўзлар: одонтоген яллиғланиш касалликлари, абцесс ва флегмоналар. 1-жадвал 1 жадва
Одонтоген яллиғланиш асоратлари билан касалланган болаларда ёши ва жинсига қараб, гурухлараро тақсимланиши. Гурухлар
Ўғил
болалар сони
Қиз
болалар
сони
0-6
ёшлилар сони
6-12
ёшлилар сони
12-18
ёшлилар сони
Жами
сони
I
28
21
20
19
10
49
II
29
24
16
27
10
53
III
12
8
2
9
9
20 д
иш асоратлари билан касалланган болаларда ёши ва жинсига қараб, гурухлараро тақсимланиши. Одонтоген яллиғланиш асоратлари билан касалланган болаларда ёши ва жинсига қараб, гурухл фаоллиги ва самарадорлигини одонтоген яллиғланиш касалликлар
асоратларини олдини олишни таъминлайди. Қўллаш усуллари,
бактериофаг воситаси қон ва лимфа ичига кириб, яллиғланиш
медиаторларини
бужмайтирувчи
таъсир
кўрсатади. Бактериофагнинг асосий қисми буйраклар орқали чиқарилади,
сийдик йўлларида дезинфекциялаш таъсирига эга, қолганлари эса
ошқозон
-
ичак тракти
орқали чиқарилиши
аниқланган. Бактериофаг одонтоген яллиғланиш касалликларига антисепти ва
антибактериал таъсир хусусиятига эга. Одонтоген
яллиғланиш
асоратлари
билан
касалланган
болаларда I гурухда ўғил болалар 28 нафарни, қиз болалар эса 21
наафрни ташкил этди. Бу гурухда ўғил болаларда қиз болаларга
нисбатан касалланиш тенденцияси юқорилигида дарак беради. II
ва III гурухларда ҳам ўғил болаларда касалланиш юқориги
аниқланди. Бактириофаг воситасини одонтоген яллиғланиш
касалликларига таъсир этиш механизмлари: ANNOTATION С. Счаде, Э. С. 19819, Э. Булл. ). Тадқиқот материали ва усуллари: Одонтоген яллиғланиш
билан касалланган болаларни комплекс даволашда 122 нафар
одонтоген яллиғланиш билан мурожаат қилган бемор болалар
олинди. Илмий изланишимизда беморларни 3 гурухга бўлиб
ўрганилди. I гурух одонтоген яллиғланиш касаллиги асорати тиш – жағ
тизими абцесс ва флегмонаси билан касалланган 49 нафар бемор
болаларда текширув олиб борилди ва комплекс даволанди. I гурух одонтоген яллиғланиш касаллиги асорати тиш – жағ
тизими абцесс ва флегмонаси билан касалланган 49 нафар бемор
болаларда текширув олиб борилди ва комплекс даволанди. II гурух одонтоген яллиғланиш касаллиги асорати тиш – жағ
тизими периостити билан касалланган 53 нафар бемор болаларда
текширув олиб борилди ва комплекс даволанди. II гурух одонтоген яллиғланиш касаллиги асорати тиш – жағ
тизими периостити билан касалланган 53 нафар бемор болаларда
текширув олиб борилди ва комплекс даволанди. Бизга маълумки, одонтоген яллиғланиш патологияларининг
тарқалиши пасайишга мойил эмас, даволаш жараёни машаққатли
ва узоқ давом этади ва асоратларнинг частотаси доимий равишда
юқорилиги аниқланган. Одонтоген яллиғланиш жараёнларининг
ўчоғи организмнинг гетеро- ва аутосенсибилизациясининг манбаи
ҳисобланади
ва
маҳаллий
ҳамда
умумий
патологиянинг
ривожланишдаги этакчи омилларидан биридир. III гурух одонтоген яллиғланиш касаллиги асорати тиш – жағ
тизими одонтоген остеомилети билан касалланган 20 нафар бемор
болаларда текширув олиб борилди ва комплекс даволанди. Тадқиқот натижалари ва муҳокамалар Текширилган бемор
болаларда одонтоген яллиғланиш касалликлар асоратларини
ананавий даволаш усулидан тубдан фарқ қиладиган, Бактериофаг
дори воситаси орқали комплекс даволашда касалликнинг тузалиш
даврини камайтиришга эришилди. Болалар орасида одонтоген яллиғланиш касалликларининг
тарқалиши ўткир йирингли - яллиғланиш касалликларининг оғир
шакллари ва грипп, тонзиллит, юқумли гепатит ва бошқалар каби
умумий
юқумли
касалликларининг
асорати
натижасида
ривожланади. Одонтоген яллиғланиш касалликлари асоратлари билан
касалланган бемор болаларни ёши, жинс бўйича гурухлараро
тақсимланиши 1-жадвалда келтирилган. Одонтоген йирингли-яллиғланиш жараёнларининг боришини
эрта ташҳислаш стоматологияда кенг тарқалганлиги ва оғир
асоратларни ривожланишга олиб келади. ANNOTATION Today, patients with odontogenic inflammatory diseases account for 10-20% of dental diseases. And 30-70% of odontogenic inflammatory
diseases are abscesses and phlegmons, improving the diagnosis of these diseases and effective treatment is considered one of the urgent problems. It is important to develop special treatment measures for children with this disease. Key words: odontogenic inflammatory diseases, abscesses and phlegmons. В.В., Коринская Н.Н., 1996; Ушаков Р. Б. 1995; Каваи Т. ва
бошқалар, 1998 ва бошқалар). Кириш. Стоматологиянинг энг долзарб муаммоларидан бири
ўткир одонтоген яллиғланиш жараёнларидир (Губин М.А., 2013;
Балин В.Н., 2000; Карнаухов А.Т., 1999). Одонтоген яллишланиш
касалликларига қарши курашнинг янги усулларини ишлаб
чиқишга қарамасдан, яллиғланиш касалликлари билан касалланган
болалар сони, айниқса тез-тез касал бўлган болаларда кўпайиш
тенденциясига
эга. (Робустова
Т.Г. ва
бошқалар,
2011;
Шаргородский А.Г., 2010 ва бошқалар). Айни пайтда, маълумки, болаларда тиш – жағ тизими ўткир
яллиғланиш
жараёнлари
кўпинча
тананинг
иммунологик
реактивлигининг пасайиши билан ривожланади ва касалликнинг
кечиши ва асоратлар эҳтимоли асосан иммунитетнинг дастлабки
параметрлари билан белгиланади. Болаларда одонтоген яллиғланишларнинг кечиши боланинг
тиш – жағ тизими ва тўқималарининг нисбий этуклиги,
иммунитетнинг
номукаммаллиги,
лимфа
тўқималарининг
кўплиги,
тишлар
тузилишининг
анатомик
ва
физиологик
хусусиятларининг мавжудлиги сабабли бир қатор хусусиятларга
эга ҳамда жағлар, шикастланишнинг осонлиги ва табиий ҳимоя
тўсиқларининг микробларга ўтказувчанлигини ошириш ҳам
муҳим аҳамиятга эгадир. Маълумки, тиш – жағ тизимининг яллиғланиш касалликларини
ташхислаш ва даволаш болалар стоматологиясининг асосий
муаммоларидан
биридир. Бир
қатор
муаллифларнинг
маълумотларига кўра, тиш
–жағ тизимининг
яллиғланиш
касалликлари
болалик
давридаги
барча
жарроҳлик
касалликларининг 21 % тиш касалликларининг 52 % ташкил
қилади. Кўплаб
эпидемиологик
тадқиқотлар
натижалари
шуни
кўрсатадики, ҳар бир бола йилига ўртача 3 дан 5 гача ўткир
респиратор вирусли инфекциялар (ЎРВИ) эпизодларидан азият
чекади. Ўткир респираторли инфекцияларнинг энг юқори Тиш – жағ тизими йирингли-яллиғланиш жараёнларининг
этиологияси, патогенези, олдини олиш ва даволашнинг кўплаб
масалалари ҳали ҳам ҳал этилмаган бўлиб, бу тадқиқотчиларнинг
уларга доимий қизиқиши ва эътиборини тушунтиради (Рогинский 76
ɮɮɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\ №2 | 2023 даражаси ёш болалар, мактабгача ёшдаги болалар ва кичик мактаб
ўқувчиларида кузатилади. Ҳаётнинг биринчи 3 ёшидаги болалар
10 ёш ва ундан катта ёшдаги болаларга қараганда йил давомида
ЎРВИ билан 2-2,5 марта тез-тез касаланиш кузатилади. Такрорий
респиратор инфекциялар тананинг функционал ҳолатининг
бузилишига олиб келади, мослашув этишмовчилигини келтириб
чиқариши ва сурункали патологиянинг ривожланишига олиб
келиши мумкин [Ключников С.О. ва бошқ., 2017]. Болаларда одонтоген яллиғланиш касалликларининг кечиши ва
тарқалиши қуйидаги омилларга боғлиқ. 1. тиш кариесининг бирламчи ва иккиламчи профилактикаси,
шунингдек,
тиш
кариесининг
мураккаб
шаклларини
даволашнинг этарли даражада эмаслиги. 2. ANNOTATION бирламчи
стоматологик
касалликларни
ўз
вақтида
даволанмаслиги
ҳамда
оғиз
бўшлиғи
шахсий
гигиена
қоидаларига риоя қилмаслик. 3. одонтоген
микрофлоранинг
антибиотикларга
сезувчанлигининг пасайиши. 3. одонтоген
микрофлоранинг
антибиотикларга
сезувчанлигининг пасайиши. Бу тиш – жағ тизими одонтоген йирингли яллиғланиш
жарроҳликдаги муаммонинг долзарблигини кўрсатади. Қайта
респиратор
инфекциялари
бўлган
болаларда
жарроҳлик
касалликлари курси мураккаб иммунитет механизмлари ва
уларнинг ошқозон ости бези, гипофиз, қалқонсимон без ва буйрак
усти гормонлари билан ўзаро таъсири билан белгиланади. Маълумки, кўпинча одонтоген яллиғланиш касалликлари оғиз
бўшлиғининг нормал микрофлорасининг патологик микрофлорага
ўтишидан келиб чиқади. Одонтоген йирингли яллиғланишлар
ривожланиши
аслида
иммун
тизимининг
компенсацион
имкониятларининг пасайиши туфайли юзага келади. Иммунитет тизимининг бостирилиши, метаболик касалликлар,
оғриқ
сезувчанлигининг
пасайиши
кўплаб
жарроҳлик
касалликларининг клиник кўринишини ва курсини ўзгартиради, бу
жиддий диагностика хатоларига олиб келиши ва жарроҳлик
патологиясининг натижасига салбий таъсир кўрсатиши мумкин. (Галимов О.В. ва бошқалар, 2018; Заитсева Э.Л., 2018;
Пиаггесиетал, 2018). Иммунитет тизимининг бостирилиши, метаболик касалликлар,
оғриқ
сезувчанлигининг
пасайиши
кўплаб
жарроҳлик
касалликларининг клиник кўринишини ва курсини ўзгартиради, бу
жиддий диагностика хатоларига олиб келиши ва жарроҳлик
патологиясининг натижасига салбий таъсир кўрсатиши мумкин. (Галимов О.В. ва бошқалар, 2018; Заитсева Э.Л., 2018;
Пиаггесиетал, 2018). Триггер механизми юқумли касалликлар, масалан, грипп,
тонзиллит ва бошқалар туфайли келиб чиққан вақтинчалик
иммуносупрессия
бўлиши
ҳам
одонтоген
яллиғланиш
касалликларини ривожланишини келтириб чиқаради. Тадқиқотнинг
мақсади
одонтоген
яллиғланиш
билан
касалланган
болаларни
комплекс
даволаш
усулларини
такомиллаштиришдан иборат. Тез-тез касал бўлган болаларда одонтоген яллиғланиш
касалликлари ва уларнинг асоратлари аниқ микросиркуляция
бузилиши, микротромблар, дистрофик ва некротик жараёнларнинг
мавжудлиги,
яллиғланиш
компонентининг
репаративдан
устунлиги,
ҳужайралар
кўпайишини
ҳосил
қилиш
билан
тавсифланган
маълум
хусусиятларга
эга. Лейкоцитларнинг
фагоцитлар
фаоллиги,
тўлиқ
бўлмаган
фагоцитоз,
яра
тўқималарининг микробиал ифлосланишининг юқори даражаси,
умумий ва маҳаллий иммунологик реактивликнинг пасайиши (Н. И. Камзалакова, 2000, А. Ю. Токмакова, 2003, Д. С. Счаде, Э. С. 19819, Э. Булл. ). Тез-тез касал бўлган болаларда одонтоген яллиғланиш
касалликлари ва уларнинг асоратлари аниқ микросиркуляция
бузилиши, микротромблар, дистрофик ва некротик жараёнларнинг
мавжудлиги,
яллиғланиш
компонентининг
репаративдан
устунлиги,
ҳужайралар
кўпайишини
ҳосил
қилиш
билан
тавсифланган
маълум
хусусиятларга
эга. Лейкоцитларнинг
фагоцитлар
фаоллиги,
тўлиқ
бўлмаган
фагоцитоз,
яра
тўқималарининг микробиал ифлосланишининг юқори даражаси,
умумий ва маҳаллий иммунологик реактивликнинг пасайиши (Н. И. Камзалакова, 2000, А. Ю. Токмакова, 2003, Д. С. Счаде, Э. С. 19819, Э. Булл. ). Тез-тез касал бўлган болаларда одонтоген яллиғланиш
касалликлари ва уларнинг асоратлари аниқ микросиркуляция
бузилиши, микротромблар, дистрофик ва некротик жараёнларнинг
мавжудлиги,
яллиғланиш
компонентининг
репаративдан
устунлиги,
ҳужайралар
кўпайишини
ҳосил
қилиш
билан
тавсифланган
маълум
хусусиятларга
эга. Лейкоцитларнинг
фагоцитлар
фаоллиги,
тўлиқ
бўлмаган
фагоцитоз,
яра
тўқималарининг микробиал ифлосланишининг юқори даражаси,
умумий ва маҳаллий иммунологик реактивликнинг пасайиши (Н. И. Камзалакова, 2000, А. Ю. Токмакова, 2003, Д. 2 – жадвал д
Одонтоген яллиғланиш касалликлар асоратларини анавий даволаш ва Бактериофаг дори воситаси орқали комплекс
даволашни гурухлараро таққослашнинг таҳлилий натижалари
Гурухлар
Ананавий
даволаш
Соғайиш
тенденсияси
Бактериофаг
орқали комплекс
даволаш
Соғайиш тенденсияси
I
Антибактериал
воситалар
7 кун
Бактериофаг
5 кун
II
Антибактериал
воситалар
5 кун
Бактериофаг
3 кун
III
Антибактериал
воситалар
14 кун
Бактериофаг
10 кун Одонтоген яллиғланиш касалликлар асоратларини анавий даволаш ва Бактериофаг дори воситаси
даволашни гурухлараро таққослашнинг таҳлилий натижалари ланиш касалликлар асоратларини анавий даволаш ва Бактериофаг дори воситаси орқали комплекс
даволашни гурухлараро таққослашнинг таҳлилий натижалари Бактириофаг воситасини одонтоген яллиғланиш
касалликларига таъсир этиш механизмлари: Бактериофаг-бу
гомологик
бактериянинг
ҳужайра
мембранасида адсорбсияланган, ҳужайра ичига кириб, уни лизисга
учратади. Бактериянинг ўзига хос вирусига таъсир кўрсатиб,
таркибига бактерияларнинг селектив, фақат вирусли фаглари -
Staphylococcus aureusга тасир қилади. Бактериофаг юқори Жароҳат майдонига
бактериофаг
воситасини қўллаш
зарарланган
тўқима
ҳажмига
қараб
индивидуал
равишда
белгиланади. Жарохат майдонидан йирингни олиб ташлаганидан
кейин оғиз бўшлиғига пуркаш тавсия қилинади. Беморларда 77
ɮɮɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\ №2 | 2023 кейинги
қатнов
кунларда
бактериофаг
дренаж
ёрдамида
инфекцияланган бўшлиққа қўйилади. кейинги
қатнов
кунларда
бактериофаг
дренаж
ёрдамида
инфекцияланган бўшлиққа қўйилади. операциядан кейинги 40 мл миқдоридаги юқумли асоратларни
олдини
олишда
ишлатилади. Одонтоген
яллиғланиш
касалликларини даволашда бактериофаг дозаси беморларнинг
аҳволига қараб белгиланади. Агар стафилококк бактериофаг
препаратини қўллашдан олдин яраларни даволаш учун кимёвий
антисептиклар ишлатилган бўлса, ярани стерил 0,9% натрий
хлорид эритмаси билан яхшилаб ювиш тавсия этилди. Илмий
тадқиқотимиз давомида ушбу усулни қўллаш тан нархи арзон
қўллаш қулай, даволаш самарадорлиги юқори ҳисобланади. Ушбу жараён кунига 1 марта, 3-5 кун давомида амалга
оширилади. Оғиз бўшлиғи дренажланган бўлса, бактериофаг
кунига 2 марта 5-10 мл юборилади. Бактериофаг намланган
турундни ювиш, томизиш, юбориш учун ишлатилади. Оғиз
бўшлиғи йирингли яллиғланиш ҳолатларида даволаш учун восита
чайиш учун ишлатилади ва бир вақтнинг ўзида буюрилади. Стоматит ва сурункали умумий периодонтитни даволашда восита
кунига 3-4 марта 10-20 мл дозада оғиз ювиш воситаси шаклида
қўлланилади, шунингдек, пиобактериофаг билан сингдирилган
турунданинг периодонтал чўнтакларига 5-10 дақиқа давомида
қўлланилади. Профилактика мақсадида бактериофаг операциядан
кейинги ва одонтоген яллиғланиш даволашда, шунингдек, тиш –
жағ тизими ва оғиз бўшлиғи касалликларини олдини олишда, Одонтоген яллиғланиш касалликлар асоратларини ананавий
даволаш усулидан тубдан фарқ қиладиган, Бактериофаг дори
воситаси орқали комплекс даволашда касалликнинг соғайиш
тенденсиясининг самарали натижага эришилганлиги 2 – жадвалда
келтирилган. Хулосалар Ху
Одонтоген йирингли яллиғланиш касалликлари асоратлари
билан оғриган учала гурухдаги беморларни Бактериофаг дори
воситаси билан комплекс даволашда беморларни тузалиш кунини
биринчи гурухда 7 кундан 5 кунга, иккинчи гурухда 5 кундан 3 р
кунга, учунчи гурухда эса 14 кундан 10 кунга камайтиришга
эришилди. Беморларни комплекс даволаш курсидан сўнг чайнов
самарадорлик ҳолатини тўлиқ тиклашга эришилди. Фойдаланилган адабиётлар у
3. Азимов М. Диагностика и патогенетические аспекты терапии воспалительных заболеваний челюстно-лицевой области:
Автореф. дис. ... д-ра мед. наук: 14.00.21. - Ташкент, 1991.-316 с. р ф
р
у
4. Алешина О.Е. Дифференциальная диагностика гнойных и негнойных вос-палительных процессов челюстно-лицевой области у
детей: Автореф. дис. ... канд. мед. наук; 14.00.21. - М., 2002. - 28 с. 4. Алешина О.Е. Дифференциальная диагностика гнойных и негнойных вос-палительных процессов челюстно-лицевой области у
детей: Автореф. дис. ... канд. мед. наук; 14.00.21. - М., 2002. - 28 с. имизация комплексного лечения гнойно-воспалительных заболеваний челюстно-лицевой области: Дис.... канд
/ ИГМУ. - Иркутск, 1997.- 131 с. 5. Алешкин И.Г. Оптимизация комплексного лечения гнойно-воспалительных заболеваний челюстно-
мед. наук: 14.00.21 / ИГМУ. - Иркутск, 1997.- 131 с. 5. Алешкин И.Г. Оптимизация комплексного лечения гнойно-воспалительных заболеваний челюстно-лицевой области: Дис.... канд. мед. наук: 14.00.21 / ИГМУ. - Иркутск, 1997.- 131 с. 6
Бажанов Н Н
Итоги обсуждения классификации острых одонтогенных воспалительных процессов / Н Н Бажанов М М 6. Бажанов Н.Н. Итоги обсуждения классификации острых одонтогенных воспалительных проц
Соловьев, В.В. Рогинский // Стоматология. - 1990. -№ 3. - С. 87-89. 7. Бажанов Н.Н. Фагоцитоз при острых воспалительных заболеваниях челюстно лицевой области / Н.Н. Бажанов, М.В. Ларская, М.Т. Александров // Специализированная медицинская помощь и современные проблемы ее интеграции: Тез. докл. Всесоюзн. конф. -
М., 1986 - С. 143-146. 7. Бажанов Н.Н. Фагоцитоз при острых воспалительных заболеваниях челюстно лицевой области / Н.Н. Бажанов, М.В. Ларская, М.Т. Александров // Специализированная медицинская помощь и современные проблемы ее интеграции: Тез. докл. Всесоюзн. конф. -
М., 1986 - С. 143-146. ,
8. Баиров Г.А. Гнойная хирургия детей / Г.А. Баиров, Л.М. Рошаль. - Л.: Медицина, 1991. - С. 72-74,237. 9
Б
ф
й
б
й
б
/ Н Н Б
Е П 8. Баиров Г.А. Гнойная хирургия детей / Г.А. Баиров, Л.М. Рошаль. - Л.: Медицина, 1991. - С. 72-74,237. 9. Бактериальная микрофлора при одонтогенных острых гнойных заболева¬ниях челюстно-лицевой области / Н.Н. Бажанов, Е.П. Пашков, М.С. Култа- ев va boshq. // Стоматология. - 1985. - № 1. - С. 31-32. Баиров Г.А. Гнойная хирургия детей / Г.А. Баиров, Л.М. Рошаль. - Л.: Медицина, 1991. - С. 72-74,237. Бактериальная микрофлора при одонтогенных острых гнойных заболева¬ниях челюстно-лицевой области / Пашков, М.С. Култа- ев va boshq. // Стоматология. - 1985. - № 1. - С. 31-32. 10. Баркаган З.С. Геморрагические заболевания и синдромы / З.С. Баркаган. - М., 1988.-60 с. 10. Баркаган З.С. Геморрагические заболевания и синдромы / З.С. Баркаган. 17.
Rizaev Jasur Alimjanovich, Ahrorova Malika Shavkatovna, Kubaev Aziz Saydalimovich, Hazratov Alisher Isamiddinovich, Morphological
Changes in the Oral Mucous Membrane in Patients with COVID-19, American Journal of Medicine and Medical Sciences, Vol. 12 No. 5,
2022, pp. 466-470. doi: 10.5923/j.ajmms.20221205.04. Фойдаланилган адабиётлар - М., 1988.-60 с. Д
у
у
р
у
Д
12. Кубаев А. и др. Comparative analysis of methods for treating depressed frontal sinus fractures //Журнал стоматологии и
краниофациальных исследований. – 2020. – Т. 1. – №. 1. – С. 25-28 13. Ризаев Ж., Кубаев А., Бузрукзода Ж. Современный подход к комплексной реабилитации пациентов с приобретенными дефектами
верхней челюсти (обзор литературы) //Журнал стоматологии и краниофациальных исследований. – 2021. – Т. 2. – №. 3. – С. 77-
83. 14. Rizaev Elyor Alimdjanovich, & Buzrukzoda Javokhirkhon Davron. (2023). HEALING WITH THE USE OF TITANIUM THREADS OF
CONTROLLED BONE RESORPTION. American Journal of Interdisciplinary Research and Development, 16, 9–14. Retrieved from
http://ajird.journalspark.org/index.php/ajird/article/view/649 p
j
j
p
g
p p j
15. Rizaev, E. A., & Buzrukzoda, J. D. (2022). OPTIMIZATION OF GUIDED BONE REGENERATION IN CONDITIONS OF JAW BONE
ATROPHY. Applied Information Aspects of Medicine (Prikladnye informacionnye aspekty mediciny), 25(4), 4-8. 16. Rizaev J.A., Akhrorova M.Sh., Kubaev A.S., Khazratov A.I. Clinical and immunological aspects of the relationship of the oral cavity and
covid-19, TJE - Thematic journal of Education, 7, 3-15, 2022 p
j
j
p
g
p p j
15. Rizaev, E. A., & Buzrukzoda, J. D. (2022). OPTIMIZATION OF GUIDED BONE REGENERATION IN CONDITIONS OF JAW BONE
ATROPHY. Applied Information Aspects of Medicine (Prikladnye informacionnye aspekty mediciny), 25(4), 4-8. ATROPHY. Applied Information Aspects of Medicine (Prikladnye informacionnye aspekty mediciny), 25(4), 4-8. 16. Rizaev J.A., Akhrorova M.Sh., Kubaev A.S., Khazratov A.I. Clinical and immunological aspects of the relationship of the oral cavity and
covid-19, TJE - Thematic journal of Education, 7, 3-15, 2022 16. Rizaev J.A., Akhrorova M.Sh., Kubaev A.S., Khazratov A.I. Clinical and immunological aspects of the relationship of the oral cavity and
covid-19, TJE - Thematic journal of Education, 7, 3-15, 2022 78
ɮɮɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\№2 | 2023
17.
Rizaev Jasur Alimjanovich, Ahrorova Malika Shavkatovna, Kubaev Aziz Saydalimovich, Hazratov Alisher Isamiddinovich, Morphological
Changes in the Oral Mucous Membrane in Patients with COVID-19, American Journal of Medicine and Medical Sciences, Vol. 12 No. 5,
2022, pp. 466-470. doi: 10.5923/j.ajmms.20221205.04.
ɮɮɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\ №2 | 2023 №2 | 2023 17. Rizaev Jasur Alimjanovich, Ahrorova Malika Shavkatovna, Kubaev Aziz Saydalimovich, Hazratov Alisher Isamiddinovich, Morphological
Changes in the Oral Mucous Membrane in Patients with COVID-19, American Journal of Medicine and Medical Sciences, Vol. 12 No. 5,
2022, pp. 466-470. doi: 10.5923/j.ajmms.20221205.04. 17. Rizaev Jasur Alimjanovich, Ahrorova Malika Shavkatovna, Kubaev Aziz Saydalimovich, Hazratov Alisher Isamiddinovich, Morphological
Changes in the Oral Mucous Membrane in Patients with COVID-19, American Journal of Medicine and Medical Sciences, Vol. 12 No. 5,
2022, pp. 466-470. doi: 10.5923/j.ajmms.20221205.04. 79 90
ISSN 2181-0966
Doi Journal 10.26739/2181-0966
Yfc`S^dea_Sea^aV[[[
]cS`[agSi[S^o`nh[dd^XWaUS`[\
ea_`a_Xc
ɮɮɖɹ
ɮ
ɖɹɮ
ɮɹɮɮɹ
Контакт редакций журналов. www.tadqiqot.uz
ООО Tadqiqot город Ташкент,
улица Амира Темура пр.1, дом-2.
Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz
Тел: (+998-94) 404-0000
Editorial staff of the journals of www.tadqiqot.uz
Tadqiqot LLC The city of Tashkent,
Amir Temur Street pr.1, House 2.
Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz
Phone: (+998-94) 404-0000 90
Контакт редакций журналов. www.tadqiqot.uz
ООО Tadqiqot город Ташкент,
улица Амира Темура пр.1, дом-2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz
Тел: (+998-94) 404-0000
Editorial staff of the journals of www.tadqiqot.uz
Tadqiqot LLC The city of Tashkent,
Amir Temur Street pr.1, House 2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz
Phone: (+998-94) 404-0000 Контакт редакций журналов. www.tadqiqot.uz
ООО Tadqiqot город Ташкент,
улица Амира Темура пр.1, дом-2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz
Тел: (+998-94) 404-0000
|
https://openalex.org/W1530997907
|
https://www.scielo.br/j/rbgo/a/7x9GNGBL5zP4DJd775Rf3XQ/?lang=pt&format=pdf
|
Portuguese
| null |
O arrependimento após a esterilização cirúrgica e o uso das tecnologias reprodutivas
|
Revista brasileira de ginecologia e obstetrícia
| 2,007
|
cc-by
| 3,314
|
O arrependimento após a esterilização
cirúrgica e o uso das tecnologias reprodutivas Repentance after surgical sterilization and the use
of reproductive technologies Repentance after surgical sterilization and the use
of reproductive technologies Editorial As informações mais recentes que temos sobre o uso de métodos anticoncepcionais
para o território nacional são os dados da Pesquisa Nacional sobre Demografia e Saúde1
conduzida em 1996, que apontavam para alta prevalência de esterilização feminina (40,1%)
entre as mulheres de 15 a 49 anos em união sexual (casada ou em coabitação). O segun
do método mais usado, em proporção bastante inferior, 21,4%, era o anticoncepcional
hormonal oral. Em estudo comparativo entre o Estado de São Paulo e o Brasil, de 1986
e 19962, observam-se algumas tendências: até os 30 anos de idade, o anticoncepcional
hormonal oral é prevalente e, a partir dessa idade, a esterilização feminina cresce. A
esterilização feminina aumenta com o número de filhos e diminui com a escolaridade. A vasectomia era, naquela época, cinco vezes menos utilizada que a laqueadura, embora
já se percebesse uma tendência de aumento de uso de métodos masculinos. As altas taxas de esterilização feminina no país chamaram a atenção, na década de
80 e 90, de estudiosos de várias áreas: demografia, saúde pública, ginecologia/obstetrícia
e sociologia. Até mesmo uma Comissão Parlamentar de Inquérito (CPI)3 foi criada para
entender como ocorria essa desenfreada oferta. Vários estudos conduzidos no país4-9 ten
taram entender como e por que a esterilização feminina fazia (e ainda faz) tanto sucesso,
apesar de ser, na época, um procedimento que não estava legalmente regulamentado. Por isso, os Conselhos de Medicina afirmavam não ser ético realizar a esterilização, visto
que o médico poderia sofrer as conseqüências previstas no parágrafo 3º do Artigo 129
do Código Penal, que trata de lesões corporais, perda de órgãos ou funções10. Apesar
das dúvidas sobre a sua legalidade, a oferta da esterilização, tanto feminina quanto
masculina, tornou-se uma prática no setor privado11. As mulheres obtinham a esterili
zação clandestinamente pelo pagamento “por fora”, na hora do parto, freqüentemente
por meio de uma cirurgia cesariana12. Em Ribeirão Preto, cerca de 60% das mulheres
esterilizadas durante uma cesariana, em 1998, pagaram pelo procedimento13. Outros
estudos tentaram entender como a esterilização feminina foi usada como moeda de troca
no país, favores políticos e votos14. Desnecessário dizer que durante esse processo ocorre
acentuada queda na taxa de fecundidade no país. Elisabeth Meloni Vieira Elisabeth Meloni Vieira Professora Associada do Departamento de Medicina Social da Faculdade de Medicina de Ribeirão Preto da Universidade de São
Paulo – USP – Ribeirão Preto (SP), Brasil. Correspondência:
Elisabeth Meloni Vieira
Rua Bernardino de Campos, 50, apto.1101 – Centro
CEP 14015-130 – Ribeirão Preto/SP
E-mail: bmeloni@fmrp.usp.br
Recebido
28/05/2007
Aceito com modificações
01/06/2007 O arrependimento após a esterilização
cirúrgica e o uso das tecnologias reprodutivas A noção de
direito reprodutivo como direito humano desenvolveu-se ao longo do século XX, sendo um fenômeno recente,
relacionado às mudanças no papel das mulheres e seu valor na sociedade, às mudanças de consciência das mulheres
sobre seu papel e valor e ao desenvolvimento das tecnologias médicas. Embora a Declaração Universal dos Direitos
Humanos não mencionasse os Direitos Reprodutivos, a partir de 1968 a reprodução humana tornou-se objeto de
preocupação. Os direitos reprodutivos foram definitivamente incorporados na Conferência Internacional sobre
População e Desenvolvimento do Cairo, em 1994: “Os direitos reprodutivos baseiam-se no reconhecimento do direito básico de todos os casais e indivíduos decidirem livre
e responsavelmente sobre o número, espaçamento e quando devem ter os filhos e de terem acesso a informação sobre a
forma como fazê-lo, bem como o direito de se beneficiarem de saúde sexual e reprodutiva do mais alto nível. Também
incluem o direito de todos tomarem decisões sobre a reprodução sem discriminação, coerção nem violência.” (p.58)19. Desde 1988, no Brasil, o planejamento familiar é um direito do cidadão, e é definido como um conjunto
de ações de regulação da fertilidade, que objetiva garantir direitos iguais de constituição (limitação ou au
mento de prole) pela mulher, pelo homem ou pelo casal. Deve ser parte integrante do atendimento à saúde,
com ações preventivas e educativas no acesso igualitário às informações e meios de anticoncepção cientifi
camente aceitos. É dever do Estado assegurar o livre exercício do planejamento familiar. Mas foi somente a
partir de 1997 que a oferta da esterilização cirúrgica voluntária passou a ser regulamentada no Brasil, com a
aprovação da Lei 9.263, regulamentando o parágrafo 7o do Artigo 226 da Constituição Federal. A lei 9.263
foi aprovada em janeiro de 1996, mas o Artigo 10o, sobre a esterilização cirúrgica, permaneceu vetado pelo
Presidente até agosto de 1997. Composta de 25 Artigos e organizada em três Capítulos, a Lei aborda vários
aspectos do planejamento familiar, estabelecendo que nenhum programa deva visar a metas demográficas. Fornece diretrizes claras para questões institucionais, alocando no Sistema Único de Saúde (SUS) as ações de
planejamento familiar, como parte integrante da atenção à saúde reprodutiva, incluindo as atividades básicas
de assistência à concepção e contracepção, o atendimento pré-natal, a assistência ao parto, ao puerpério e ao
neonato, o controle das doenças sexualmente transmissíveis e o controle e prevenção do câncer cérvico-uterino,
do câncer de mama e de pênis. O arrependimento após a esterilização
cirúrgica e o uso das tecnologias reprodutivas i A insuficiência do Estado em implantar e fornecer um planejamento reprodutivo
adequado, que já havia sido desenhado no Programa de Atenção Integral à Saúde da
Mulher (Paism) em 1984, entrou em primeiro lugar na lista de explicações para a altís
sima prevalência de esterilização feminina. Também se chamou atenção para o caráter de Vieira EM dissociação entre sexo e reprodução no uso da esterilização5, com a ação legitimadora do médico que confirma para
a mulher que seu desejo de contracepção é válido, sério e deve ser considerado15, explicando, então, a preferência
cultural da esterilização. Discutiu-se muito seu uso em uma situação quase obrigatória após duas cesáreas (outra
preferência nacional)16. Berquó (1993) alertou para uma cultura da esterilização16. Nesse contexto, destaca-se o
papel da medicalização, que tomou lugar no vácuo de uma política pública que não foi implementada17. Podemos olhar melhor para a alta prevalência da esterilização feminina no contexto maior da oferta de métodos
contraceptivos pelo serviço público. O relatório técnico de uma missão do Fundo de População das Nações Unidas
(UNFPA)18 ao Brasil, em 1994, revelou vários problemas relativos ao planejamento familiar no país: a imensa
lacuna entre a demanda por contraceptivos e o acesso a eles, a falta de insumos nos serviços públicos de saúde, a
ausência de gerenciamento logístico, a falta de capacidade técnica das equipes. Uma análise detalhada sobre as
características dos serviços prestados na área de saúde reprodutiva apontava para várias deficiências: predominância
de apenas dois métodos anticoncepcionais, leque de escolhas reduzido, os métodos oferecidos demonstram medi
calização excessiva, predominância do setor privado (compra de pílulas e ofertas de cirurgias), acesso tardio (quase
50% das mulheres começavam a fazer contracepção depois do primeiro filho), uso inadequado de contraceptivos,
alta proporção de gravidez indesejada, falta de informação e desigualdade no acesso17. Acrescenta-se a isso uma questão de gênero importante, pois são as mulheres que mais contribuem com a
contracepção, já que a participação masculina, embora crescente, ainda não atinge os níveis de outros países, onde
parece haver maior equilíbrio de gênero, principalmente no que se refere à esterilização. A oferta de métodos contraceptivos em programas de saúde baseia-se nos direitos reprodutivos. 226
Rev Bras Ginecol Obstet. 2007; 29(5):225-9 226
Rev Bras Ginecol Obstet. 2007; 29(5):225-9 O arrependimento após a esterilização
cirúrgica e o uso das tecnologias reprodutivas O arrependimento após a esterilização é uma séria questão médica, porque, na maioria dos casos, a escolha
da laqueadura é eletiva, não se pode garantir o retorno à fertilidade e existem alternativas contraceptivas mais
reversíveis do que a esterilização que podem ser utilizadas com grande eficácia. O arrependimento já foi estimado
entre 11 e 15% das mulheres brasileiras esterilizadas24. Reconhecendo a dificuldade de medir com precisão o arrependimento, Marcil-Gratton25 usou um gradiente
de sentimentos de arrependimento para classificar as mulheres em alguns grupos: 1. mulheres que nunca sentiram arrependimento; i 2. mulheres que tentariam ter outro filho se não estivessem esterilizadas; i
3. mulheres que conversaram com o médico sobre a reversão do procedimento; 4. mulheres que solicitaram a reversão da laqueadura para seus médicos. Alguns fatores como idade jovem, falta de estabilidade no relacionamento conjugal e morte de filho são apon
tados em todo o mundo como predisponentes ao arrependimento23. No Brasil, além de ter motivos diferentes para
satisfação com o número de filhos, destaca-se a idade jovem ao ter o primeiro filho como fator para o arrependi
mento, visto que predispõe à laqueadura jovem. Observou-se, também, que o pagamento atua como obstáculo ao
arrependimento, de maneira que este pode aumentar após a oferta de esterilização gratuita pelo SUS6. Dessa forma, é importante garantir que a escolha da esterilização seja feita de forma consciente e infor
mada pelos indivíduos e que estes recebam aconselhamento e orientação sobre todos os métodos anticon
cepcionais, inclusive os reversíveis, tal como prevê a Lei 9.263. Tem sido o caso do município de Ribeirão
Preto, que, desde 1999, oferece a esterilização masculina e feminina pelo SUS com adequado suporte de
aconselhamento realizado por profissionais de saúde capacitados. Mesmo neste caso não existem garantias de
que não haja arrependimento entre os esterilizados, pois ele pode ocorrer mesmo após a escolha informada,
como já foi explorado por vasta literatura em estudos realizados em diversos países23. A oferta conjunta da
esterilização, tanto feminina quanto masculina, pelo sistema público representa uma novidade em termos
de serviços de contracepção no Brasil, pois concentra, em um mesmo serviço, a oferta das duas cirurgias
que anteriormente eram oferecidas por especialistas de duas áreas distintas e, aparentemente, sem conexões:
ginecologistas e urologistas. O arrependimento após a esterilização
cirúrgica e o uso das tecnologias reprodutivas Estabelece, ainda, que é dever do Estado assegurar o livre exercício do plane
jamento familiar e que a esterilização voluntária é somente permitida em duas situações: • em homens e mulheres com capacidade civil plena e maiores de 25 anos ou, pelo menos, dois filhos vivos, desde
que observado o prazo mínimo de 60 dias entre a manifestação da vontade e o ato cirúrgico, período pelo qual
será propiciado à pessoa interessada acesso a serviço de regulação da fecundidade, incluindo aconselhamento por
equipe multidisciplinar, visando desencorajar a esterilização precoce; • em homens e mulheres com capacidade civil plena e maiores de 25 anos ou, pelo menos, dois filhos vivos, desde
que observado o prazo mínimo de 60 dias entre a manifestação da vontade e o ato cirúrgico, período pelo qual
será propiciado à pessoa interessada acesso a serviço de regulação da fecundidade, incluindo aconselhamento por
equipe multidisciplinar, visando desencorajar a esterilização precoce; • risco à vida ou à saúde da mulher ou do futuro concepto, testemunhado em relatório escrito e assinado por
dois médicos. Para desvincular a esterilização feminina das cesarianas, a lei veda a esterilização cirúrgica em mulher
nos períodos de parto ou aborto, exceto nos casos de comprovada necessidade, por cesarianas anteriores. Prevê
ainda fiscalização e controle pelo SUS das instituições que realizam planejamento familiar. Várias portarias e
resoluções no nível federal e estadual, estabelecendo formas de controle, foram publicadas20,21. Em seu capítulo II,
a Lei 9.263 prevê penalidades, definindo como crime as esterilizações em desacordo com a lei. Embora a regulamentação legal dos direitos assegurados na Constituição Brasileira seja fundamental para
garantir os direitos reprodutivos e melhorar a qualidade de vida das pessoas, apenas a regulamentação não
seria suficiente para criar situações para o exercício dos direitos reprodutivos, como afirmam alguns autores22. Quando os mecanismos legais não são suficientes, então outros aspectos sociais, materiais e institucionais da
sociedade precisam ser desenvolvidos para assegurá-los6. É o caso da oferta do planejamento familiar de boa
qualidade que necessita de investimento na capacitação de profissionais, na compra de insumos e de bom
desenvolvimento técnico e tecnológico para se efetivar. Em sua ausência, há situações como testemunhamos neste estudo relevante realizado por pesquisadores da Uni
versidade de Brasília apresentado nesta edição de RBGO23. Este nos informa sobre um contingente de 98 mulheres
arrependidas que procuraram um serviço de reprodução assistida para conceber após terem sido esterilizadas. O arrependimento após a esterilização
cirúrgica e o uso das tecnologias reprodutivas É importante notar, em concordância com alguns estudos6,23, que apenas uma pequena parcela de mulheres
arrependidas estaria disposta a se submeter a essas técnicas para conceber e uma parcela menor ainda atinge seus
objetivos. Em estudo realizado no Brasil, entre 79 mulheres arrependidas, apenas 19 (24%) procuraram por infor
mação sobre reversão26. No caso do estudo em questão, das 98 mulheres, apenas 8% tiveram o sucesso almejado. Das 20 mulheres submetidas à reversão da laqueadura, seis (30%) tiveram o bebê a termo, e, entre oito mulheres
submetidas às técnicas de fertilização in vitro com transferência de embrião, duas (25%) obtiveram sucesso. Chama Vieira EM Vieira EM atenção que para 22 mulheres estava indicada fertilização in vitro com transferência de embrião, mas infere-se que
elas fazem parte das que desistiram do procedimento. O uso das tecnologias reprodutivas, cirurgia de recanalização tubária ou transferência de embriões não é inó
cuo, e pode causar dor e sofrimento. Existem riscos de gravidez tubária, abortamento, óbito neonatal e gravidez
múltipla. O mais estarrecedor é que entre as 98 mulheres estudadas, 83,6% afirmaram não terem tido informação
suficiente a respeito do caráter definitivo da laqueadura tubária. Ou seja, uma decisão definitiva como a esterilização
cirúrgica foi tomada sem a informação necessária. Essa distorção fere os direitos reprodutivos. A sociedade deve
aperfeiçoar suas regulamentações e normas, de forma a garantir o bem-estar dos indivíduos e os limites de respon
sabilidade profissional. O currículo médico na área deve incluir discussões sobre bioética e direitos reprodutivos,
para evitar situações como essas. É necessário também ao médico ter consciência do processo de medicalização,
pois a sociedade pode colocar em suas mãos decisões e responsabilidades enormes. li p
p
p
Refletindo recentemente sobre um texto de Bateman27, que afirma que a atuação médica na manipulação
das tecnologias reprodutivas difere de outros procedimentos médicos já atuantes sobre o campo da não-doença,
separando definitivamente o sexo da reprodução e colocando para a medicina questões éticas, morais e políticas
jamais pensadas pela humanidade, apontamos para a medicalização como processo de tomada do objeto do co
nhecimento do campo do não-médico para o médico. Para Conrad28, a medicalização contém, em si, uma questão
conceitual, ou seja: o problema é definido em termos médicos, usa-se uma linguagem médica para descrevê-lo,
adota-se um referencial teórico médico para entendê-lo ou usa-se a intervenção médica para tratá-lo. Sociedade Civil Bem-Estar Familiar no Brasil (Bemfam). Pesquisa
nacional sobre demografia e saúde – Pnds 1996. Rio de Janeiro:
Bemfam/Macro; 1997.
1. O arrependimento após a esterilização
cirúrgica e o uso das tecnologias reprodutivas É um processo
sociocultural que pode ou não envolver a profissão, conduz a um controle médico social ou tratamento médico, ou
é resultado de uma expansão intencional da profissão médica. No caso de mulheres cirurgicamente esterilizadas e
tratadas com tecnologias reprodutivas, pode-se colocar o problema desta medicalização, provavelmente, como a
de expansão não intencional da prática médica, mas que coloca em jogo um dos princípios éticos essenciais a essa
prática revelado pelo aforisma primum non nocere (primeiro, não ferir). Na análise dessa situação, outras questões poderiam ser incluídas: em que condições se dão essas escolhas repro
dutivas? Houve aconselhamento? Houve consciência dos sujeitos envolvidos nessa decisão? Deve aconselhar o proce
dimento de esterilização o indivíduo que receberá pagamento por ele? Devem ter acesso às tecnologias reprodutivas
todos os indivíduos que se arrependeram de um procedimento tão definitivo quanto a esterilização cirúrgica?l Há necessidade de maior debate sobre a bioética e suas condições de desenvolvimento, promovendo a reflexão
dos profissionais sobre sua prática que, provavelmente, se realizou fundada em boas intenções. É necessário que o
entendimento e o desenvolvimento de tecnologias educacionais de aconselhamento, orientação, conversa e amplo
acesso a métodos sejam esgotados antes de uma decisão definitiva como essa. Para tal, o profissional médico ne
cessita de outros profissionais para compartilhar seu trabalho. A ausência de uma estrutura de múltiplos olhares
profissionais que apóie os envolvidos nos processos de decisão os abandona à própria sorte em tomadas de decisões
complexas e difíceis. A ausência de regulamentações sobre as tecnologias deixa médicos e requerentes à margem
de um processo justo e igualitário, em termos de direitos e escolhas. Referências Osis MJD. Laqueadura e representações acerca da sexualidade
e do papel reprodutivo [tese]. São Paulo: Universidade de São
Paulo; 2001. 7. Vieira EM, Badiani R, Fabbro ALD, Rodrigues Junior AL. Características
do uso de métodos anticoncepcionais no Estado de São Paulo. Rev Saúde Pública. 2001;36(3):263-70. 2. Serruya S. Mulheres esterilizadas: submissão e desejo. Belém:
Naea/Afpa/Uepa; 1996. 8. Minella LS. A produção científica sobre esterilização feminina no
Brasil nos anos 80 e início dos anos 90: um debate em aberto. Rev Bras Estud Popul. ����������������
1998;15(1):3-22. 9. Barroso C. Esterilização no Brasil. Brasília: Comissão de Estudos
de Direitos da Reprodução/MS; 1988. 3. Berquó E. A esterilização feminina hoje. Ciênc Tecnol. 1989;88(5):598-610. 4. Berquó E. A esterilização feminina hoje. Ciênc Tecnol. 1989;88(5):598-610. 4. Conselho Regional de Medicina do Estado de São Paulo
(Cremesp). Aspectos éticos que envolvem a esterilização. Ética
Méd. 1988;1(1):67. 10. Barroso C. Esterilização feminina: liberdade e opressão. Rev
Saúde Pública. 1984;18(2):170-80. 5. Berquó E, Cavenaghi SM. Direitos reprodutivos de mulheres e
homens face à nova legislação brasileira sobre esterilização
voluntária. Cad Saúde Pública. 2003;19 Supl 2:441-53. 11. Vieira EM. Regret after female sterilization among low-income women
in São Paulo, Brazil [thesis]. Exeter: University of Exeter�������
; 1994. 6. 228
Rev Bras Ginecol Obstet. 2007; 29(5):225-9 Vieira EM, Ford NJ. The provision of female sterilization in São
Paulo, Brazil: a study among low income women. Soc Sci Med. 1996;42(10):1427-32. 12. Resolução SS-5, de 11 de janeiro de 2000. Dispõe sobre
Estabelece critérios para a efetivação dos procedimentos de
esterilização no âmbito do Estado de São Paulo. Diário Oficial
do Estado de São Paulo, Poder Executivo, São Paulo jan. 2000;
Seç.I (110): p.13. 21. Vieira EM, Ford NJ. Provision of female sterilization in Ribeirão Preto,
São Paulo, Brazil. Cad Saúde Pública. 2004;20(5):1201-10. 13. Freedman LP, Isaacs SL. Human rights and reproductive choice. Stud Fam Plann. �����������������
1993;24(1):18-30. 22. Caetano AJ. Sterilization for votes in the Brazilian Northeast: the
case of Pernambuco [thesis]. Austin: University of Texas; 2000. 14. Cunha ACR, Wanderley MS, Garrafa V. Fatores associados ao
futuro reprodutivo de mulheres desejosas de gestação após ligadura
tubária. Rev Bras Ginecol Obstet. 29(5):230-4. 23. Vieira EM. A medicalização do corpo feminino. Rio de Janeiro:
Fiocruz; 2002. 15. Berquó E. Brasil, um caso exemplar: anticoncepção e partos cirúrgicos
à espera de uma ação exemplar. Estud Fem. 1993;1(2):366-81. 16. Vieira EM. O arrependimento após a esterilização feminina: é
possível prevenir? �������������������������������
Reprod Clim. 1999;14(4):178-84. Referências 24. Vieira EM. Políticas públicas e contracepção no Brasil. In: Berquó
E, organizador. Sexo e vida: panorama de saúde reprodutiva no
Brasil. Campinas: Unicamp; 2003. p. 151-96. 17. Marcil-Gratton N. Sterilization regret among women in metropolitan
Montreal. �������������������������������������
Fam Plann Perspect. 1988;20(5):222-7. 25. United Nations Population. Contraceptive requirements and
logistics management needs in Brazil. New York: FNUAP; 1995. (Technical Report, 21). 18. 26. Vieira EM. Arrependimento após a esterilização feminina. Cad Saúde Pública. 1998;14 Supl 1:59-68. 26. Bateman S. When reproductive freedom encounters medical
responsibility: changing conceptions of reproductive choice. In:
Vayena E, Rowe PJ, Griffin PD, editors. Current practices and
controversies in assisted reproduction. Report of a WHO meeting. Geneva: World Health Organization; 2002. p. 320-32. 27. Nações Unidas. Relatório da Conferência Internacional
sobre População e Desenvolvimento; 5-13 set 1994; Cairo. Brasília: Comissão Nacional de População e Desenvolvimento/
FNUAP; 1994. 19. Ministério da Saúde. Secretaria de Assistência à Saúde. Portaria
n.o 144 de novembro de 1997. Dispõe sobre..... Diário Oficial
da União, Brasília (DF) 20 nov. 1997;(227). 20. Conrad P. Medicalization and social control. Annu Rev Sociol. 1992;18:209-32. 28.
|
https://openalex.org/W3174794728
|
https://www.e3s-conferences.org/articles/e3sconf/pdf/2021/54/e3sconf_sdemr2021_03025.pdf
|
English
| null |
Mining Region Economy Servization (on example of Kemerovo Region-Kuzbass)
|
E3S web of conferences
| 2,021
|
cc-by
| 3,152
|
Mining
Region
Economy
Servization
(on
example of Kemerovo Region-Kuzbass)
Olga Esina1, Natalia Tereshchenko1,*, and Tatiana Pakshina2
1 Siberian Federal University, 660075 Lida Prushinskaya St. 2, Krasnoyarsk, Russia
2 Vitebsk State Technological University, 210038, 72 Moskovsky av., Vitebsk, Belarus Olga Esina1, Natalia Tereshchenko1,*, and Tatiana Pakshina2 1 Siberian Federal University, 660075 Lida Prushinskaya St. 2, Krasnoyarsk, Russia
2 Vitebsk State Technological University, 210038, 72 Moskovsky av., Vitebsk, Belarus Abstract. The social-and-economic development of the regions is largely
determined by the state and specific features of the service industries’
development. The high-quality level of transport, housebuilding,
communications, education and science, health care and other sectors of this
industry creates the preconditions for sustainable growth of all spheres of
the national economy. The article defines the development dynamics in the
service sector in Kuzbass, its significance in the formation of the gross
regional product, analyzes the average per capita volumes and the structure
of service sector revenue in the Kemerovo region in comparison with the
indicators in the Russian Federation and Siberian Federal District. Based on
the research carried out, the specific features and trends in the development
of the service sector in the region have been identified. 1 Introduction The formation of the regional economy structure occurs under the influence of a number of
objective factors, such as historical, climatic, infrastructural, technological conditions of
development. However, regardless of the cumulative impact of many constituent indicators,
one of the most dynamically developing sectors of economy is the service sector. In modern conditions of economic development and globalization processes, the service
sector significantly affects both the volume of economy and the structure of the gross
domestic product. According to the information resource CIAWordFactbook [1], the share
of the service sector in the formation of the gross product in 19 countries with the largest
GDP, accounts for from 45.4% in India to 80.2% in the UK. Russia ranks the 6th by this
indicator and over the past decade the share of the service sector in the country's GDP ranges
from 70.66% in 2014 to 62% in 2017 [2]. At the same time, all other values for this indicator
arranged by the years are within the specified interval. In the context of the federal districts of the Russian Federation, the share of the service
sector in the gross regional product ranges from 42% in Urals Federal District to 75% in
Central Federal District (Fig. 1). E3S Web of Conferences 278, 03025 (2021)
SDEMR-2021 E3S Web of Conferences 278, 03025 (2021)
SDEMR-2021 https://doi.org/10.1051/e3sconf/202127803025 © 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/). * Corresponding author: tereshchenko.65@mail.ru 2 Materials and Methods The issues of research and modeling of the service sector development are described in the
works of both foreign and domestic researchers. The issues of research and modeling of the service sector development are described in the
works of both foreign and domestic researchers. A number of scientists (Kitova O.V., Savinova V.M., Dyakonova L.P., Bruskin S.N.,
Beshmelnitskiy A.A., Danko T.P., Sekerin V.D.) [3] offer methodological approaches to the
study of the service sector through the creation of situational centers. They focus on assessing
social-and-economic indicators at different levels, planning and factor analysis in real time
and forecasting the development of the service sector using a variety of qualitative and
quantitative methods. Many foreign and domestic scientists (Razumova, E., Ilienko, O., [4] Dalevska N.,
Khobta V., Kwilinski A., Kravchenko S. [5], Dmitrieva M., Strekaleva T., Suslov D.,
Tereshchenko N. [6], Demchenko, S.K., Zlotnikov M.S., Melnikova T.A., Demchenko O.S. [7,8]) propose a methodological tool in the field of analysis and economic modeling of the
service sector as a whole and for individual constituent industries. Generalization and critical analysis of the works of domestic and foreign authors allows
determining a number of basic characteristics of the service sector as a central sector of the
economy at all levels. Firstly, the service sector is an indicator of the social-and-economic development of the
region. The civilizational approach to the study of the society development (theories of D. Bell, E. Tafler, A. Lesch) puts forward the state of the service sector as the main
characteristics of industrial and post-industrial society. As this sector of economy determines
the transformed resources in the form of knowledge, information, storage systems and data
processing and key intelligent technologies. Thus, a high or constantly increasing level of
development of the service sector testifies to the successful social and economic development
of the regions. g
Secondly, the service sector acts as the main factor in the development of production. By
improving the occupancy and content of service industries, the formation and interaction of
the main classical factors of production takes place, since it is with the use of intelligent
technologies that individual and collective skills, production and exchange technologies are
developed, the significance of the results achieved increases. Thirdly, the service sector can be viewed as an indicator of ensuring the quality of life of
the population. * Corresponding author: tereshchenko.65@mail.ru E3S Web of Conferences 278, 03025 (2021) E3S Web of Conferences 278, 03025 (2021)
SDEMR-2021 https://doi.org/10.1051/e3sconf/202127803025 Fig. 1. The share of the service sector by Federal Districts of the Russian Federation in 2019, %. Fig. 1. The share of the service sector by Federal Districts of the Russian Federation in 2019, %. Thus, we can say that the structural composition of the regional economy has a significant
impact on its social-and-economic development, while the service sector, along with the
extractive industries and the production sector, plays an important role in this process. 2 Materials and Methods A number of basic characteristics identified by UNESCO as part of assessing
the life quality, such as safety, health, education, rational nutrition, are formed with the direct
participation of certain service industries. Therefore, the higher the level of development of 2 https://doi.org/10.1051/e3sconf/202127803025 E3S Web of Conferences 278, 03025 (2021)
SDEMR-2021 a given sector of economy, the more qualitative characteristics are determined by the standard
of living of the population. a given sector of economy, the more qualitative characteristics are determined by the standard
of living of the population. As already noted, the development of the service sector is influenced by many factors that
determine its state in various territories. The object of our research is Kuzbass. The Kemerovo region is one of the technologically
advanced mining regions of the Russian Federation, in connection with which the service
sector in Kuzbass has a certain specificity, which is advisable to consider through a number
of indicators: - the share of the service sector in the formation of the gross regional product; - the dynamics of the revenues’ volume in service industries for a number of years;
the average per capita revenue in service industries per 1000 inhabitants; - the average per capita revenue in service industries per 1000 inhabitants;
- the structural changes in main types of services provided to the population. - the structural changes in main types of services provided to the population. The Russian practice of statistical research reflects the results of the activities of economic
sectors in accordance with the Russian National Classifier of Economic Activities Types,
which introduced a number of changes that do not imply a clear separation of three main
sectors of economy: extractive industries and agriculture, manufacturing and services. In this
regard, the structuring of statistical data provided by the Federal State Statistics Service [2]
makes it possible to determine the share of participation of the above groups of industries in
the formation of the gross domestic product and gross regional products of the objects of
research. 3 Results and Discussion A comparative characteristic of the service sector importance in the economy of the Russian
Federation, Siberian Federal District and the Kemerovo region is shown in Figure 2. Fig. 2. Dynamics of the share of the service sector in the GDP of the Russian Federation and the GRP
of Siberian Federal District and the Kemerovo region in 2008 – 2019, %. The data analysis shows that in the Russian Federation in the period from 2008 to 2019
the share of service industries accounted for from 71.01% in 2013 to 62% in 2017. In recent
years, there has been a slowdown in the rate of increase in the volume of services, and, Fig. 2. Dynamics of the share of the service sector in the GDP of the Russian Federation and the G
of Siberian Federal District and the Kemerovo region in 2008 – 2019, %. Fig. 2. Dynamics of the share of the service sector in the GDP of the Russian Federation and the GRP
of Siberian Federal District and the Kemerovo region in 2008 – 2019, %. The data analysis shows that in the Russian Federation in the period from 2008 to 2019
the share of service industries accounted for from 71.01% in 2013 to 62% in 2017. In recent
years, there has been a slowdown in the rate of increase in the volume of services, and,
accordingly, its share in the gross domestic product. A similar situation is observed in the 3 E3S Web of Conferences 278, 03025 (2021)
SDEMR-2021 https://doi.org/10.1051/e3sconf/202127803025 economy of Siberian Federal District. Here the interval of participation of the service sector
in the formation of the gross regional product ranges from 58.4% in 2013 to 53.1% in 2017. The share of the service sector in the formation of the gross regional product in the Kemerovo
region, ranges from 50.7% in 2014 to 41.2% in 2017 and is slightly lower than in the Russian
Federation and Siberian Federal District. economy of Siberian Federal District. Here the interval of participation of the service sector
in the formation of the gross regional product ranges from 58.4% in 2013 to 53.1% in 2017. The share of the service sector in the formation of the gross regional product in the Kemerovo
region, ranges from 50.7% in 2014 to 41.2% in 2017 and is slightly lower than in the Russian
Federation and Siberian Federal District. 3 Results and Discussion Thus, it can be noted that the importance of the service sector in the economy of Kuzbass
is not so significant, although it accounts for more than a third of the enterprises revenues in
the region, which is explained by the focus of the region on the mining and manufacturing
sectors. Comparative characteristics of the dynamics of the change chain rates in the revenue of
the subjects in the service sector, presented in Figure 3, shows that in all three analyzed
regions there is a positive change in this indicator, with the exception of 2017, when there
was a decrease in Siberian Federal District (86.75%) and in the Kemerovo region (99.44%). Fig. 3. Dynamics of revenue in service industries in the Russian Federation, Siberian Federal District
and the Kemerovo region in 2010-2019, %. Fig. 3. Dynamics of revenue in service industries in the Russian Federation, Siberian Federal District
and the Kemerovo region in 2010-2019, %. At the same time, it should be noted that the largest increase in the revenue of service
sector entities was observed in 2010 for all research objects. The rate of increase in the
volume of service industries in the Kemerovo Region is comparable to the average Russian
and the indicators of Siberian Federal District, but we can note some lag in the last 4 years. Thus, we can note the positive dynamics of the development of the service industries in
Kuzbass, which, however, is taking place at a slower pace than in the Russian Federation and
Siberian Federal District. One of the characteristics of the service sector development is the average per capita
revenue of industries per 1000 residents, which makes it possible to form an estimate of the
consumption of services both in dynamics and in comparison by regions. Table 1. GDP of the Russian Federation and GRP of Siberian Federal District and the Kemerovo
region from the service industries per 1000 residents of the region, million rubles. (Compiled
according to [2]). 2009
2011
2013
2015
2017
2019
The Russian
Federation
142.9
254.56
316.85
351.29
323.9
326.6 Table 1. GDP of the Russian Federation and GRP of Siberian Federal District and the Kemerovo
region from the service industries per 1000 residents of the region, million rubles. (Compiled
according to [2]). 3 Results and Discussion 4 E3S Web of Conferences 278, 03025 (2021)
SDEMR-2021 https://doi.org/10.1051/e3sconf/202127803025 Siberian Federal
District
100.5
134.5
167.8
191.8
213.1
218.3
The Kemerovo
region
90.4
120.3
128.9
146.1
160.9
166.4 Analysis of the data summarized in Table 1 makes it possible to reveal the positive
dynamics of the development of this indicator for almost all objects of study and in all
periods, however, its cost characteristic is ambiguous. It should be noted that in the
Kemerovo region in 2019 the volume of services per 1000 residents amounted to 0.51 points
from the national average and 0.76 points from the indicator of Siberian Federal District. Thus, every resident of Kuzbass was provided services for about 13 thousand rubles per
month (including utilities, medical, educational, communication services, transport, etc.),
despite the fact that the national average of this figure was about 27 thousand rubles, and in
Siberian Federal District it was about 18 thousand rubles. Thus, we can conclude that the development of the service sector in the region lags behind
the average Russian and subject indicators and creates an opportunity for setting up and
developing of business in the relevant sectors of economy. As it was noted earlier, there is no special form of accounting for services, including by
type of activity in the State Statistics. Certain information is presented in the information and
analytical system "Statistics" [9] and statistical collections of the Higher School of
Economics [10]. Generalization of the data from the above sources allows making a judgment
about the consumption structure of services by the population of the Kemerovo region in
Table 2. Table 2. Dynamics of the structure of revenue from the provision of services in 2000-2019 in the
Kemerovo region (compiled according to [9, 10]). Services
2000
2010
2019
Deviations
2010
from
2000
2019
from
2010
2019
from
2000
Domestic services
19.36
8.60
11.34
-10.76
2.74
-8.02
Veterinary services
0.17
0.13
0.14
-0.04
0.01
-0.03
Housing services
6.92
8.24
9.46
1.32
1.22
2.54
Utilities services
14.90
23.38
23.58
8.48
0.20
8.68
Medical services
4.38
6.51
7.12
2.13
0.61
2.74
Legal services
1.09
1.84
1.09
0.75
-0.75
-
Sanitary and wellness
2.88
1.90
1.40
-0.98
-0.05
-1.48
Educational services
5.62
6.78
7.12
1.16
0.34
1.50
Transport services
28.10
13.20
12.85
-14.90
-0.35
-15.25
Tourist and excursion
0.38
3.93
3.97
3.55
0.04
3.59 Table 2. 3 Results and Discussion Dynamics of the structure of revenue from the provision of services in 2000-2019 in th
Kemerovo region (compiled according to [9, 10]). 5 5 https://doi.org/10.1051/e3sconf/202127803025 E3S Web of Conferences 278, 03025 (2021)
SDEMR-2021 Hotels, motels
-
1.41
1.10
1.41
-0.31
1.10
Communications
8.78
20.30
16.02
11.52
-4.25
7.24
Cultural institutions
1.24
1.43
1.57
0.19
0.14
0.33
Physical culture and
sports
0.26
0.76
1.10
0.50
0.34
0.84
Other services
5.92
1.58
2.15
-4.34
0.57
-3.77
Total
100.0
100.0
100.0
-
-
- Analysis of the data in the table allows determining that over the past twenty years, the
consumption structure of services by the population of Kuzbass has changed significantly. If
in 2000 the main share was accounted for transport (28.1%), domestic (19.36%) and utilities
(14.9%) services, then in 2019, the share of communication (16.02%) and utilities (23.56%)
services is significantly increasing, at the same time, the share of transport services remains
significant (12.85%) too. Veterinary services (0.14%), legal services (1.09%), services of
hotels and motels, and services of physical culture and sports (1.10%) have insignificant
shares in the structure of revenues from the provision of services. Thus, a thesis about the possibility of entrepreneurship development in these service
sectors arises. 4 Conclusion The service sector is one of the most important sectors of economy, determining many
aspects of the national economy development. The studies carried out made it possible to
determine a number of specific features of the service sector development on the territory of
the Kemerovo region – Kuzbass, which may be summarized in the following provisions: g
y
g
- There is lower importance of the service sector in the formation of the gross regional product
than in the Russian Federation and Siberian Federal District. - There is lower importance of the service sector in the formation of the gross regional product
than in the Russian Federation and Siberian Federal District. - There are slower rates of development of this sector of economy in comparison with the
average Russian and subject indicators. - There are slower rates of development of this sector of economy in comparison with the
average Russian and subject indicators. - There is a significant lag in the volume of services per 1000 residents from the average in
the country and Siberian Federal District. - There is poor development of certain branches of the service sector related to the
development of health, legal support, culture, recreation. - There is poor development of certain branches of the service sector related to the
development of health, legal support, culture, recreation. All of the above allows making the conclusion that business development in this territory
has great opportunities in the service sector. All of the above allows making the conclusion that business development in this territory
has great opportunities in the service sector. It should be noted that the “Strategy for the social-and-economic development of the
Kemerovo region – Kuzbass for the period up to 2035” [11] pays considerable attention to
the development of the service industries. The main principles are high quality and reliable
provision of a new standard of living and meeting the needs of residents. In this regard, four
main development priorities have been formed, ensuring professional excellence, high
quality of life, and a green economy for residents. The presented provisions will undoubtedly
contribute to the development of the service sector in Kuzbass. 6 6 https://doi.org/10.1051/e3sconf/202127803025 E3S Web of Conferences 278, 03025 (2021)
SDEMR-2021 References 1. С.I.A. theWorldFactbook. URL: https://www.cia.gov/library/publications/the-world-
factbook/. 2. Rosstat, Official Website. URL: www.gks.ru 3. O. Kitova, V. Savinova, L. Dyakonova, S. Bruskin, European Research Studies Journal,
20(4), 275-283 (2017) 4. E. Razumova, O. Ilienko, V. Ihnatiuk, Baltic journal of economic studies, 4(2), 191-197
(2018) 5. N. Dalevska, V. Khobta, A. Kwilinski, S. Kravchenko, Entrepreneurship and
Sustainability Issues, 6(4), 1839-1860 (2019) 6. M. Dmitrieva, T. Strekaleva, D. Suslov, N. Tereshchenko, Proceedings The 32nd IBIMA
conference (Seville, Spain, 2018) 7. S.K. Demchenko, M.S. Zlotnikov, T.A. Melnikova, O.S. Demchenko, International
Journal of Civil Engineering and Technology, 10(2), 1877-1884 (2019) 8. S. Zhironkin, S. Demchenko, G. Kayachev, E. Taran, O. Zhironkina, E3S Web Conf. 105, 03008 (2019) 9. Information and analytical system "Statistics". URL: www.ias-stat.ru/ 10. HSE, Statistical collections. URL: https://www.hse.ru/primarydata/ 11. Strategy of socio-economic development of the Kemerovo region-Kuzbass for the period
up to 2035 (AKO, Kemerovo, 2019) 7
|
https://openalex.org/W4313238144
|
https://jhoonline.biomedcentral.com/counter/pdf/10.1186/s13045-022-01395-0
|
English
| null |
FTL004, an anti-CD38 mAb with negligible RBC binding and enhanced pro-apoptotic activity, is a novel candidate for treatments of multiple myeloma and non-Hodgkin lymphoma
|
Journal of hematology & oncology
| 2,022
|
cc-by
| 3,698
|
Zhang et al.
Journal of Hematology & Oncology (2022) 15:177
https://doi.org/10.1186/s13045-022-01395-0 Zhang et al. Journal of Hematology & Oncology (2022) 15:177
https://doi.org/10.1186/s13045-022-01395-0 Zhang et al. FTL004, an anti‑CD38 mAb with negligible
RBC binding and enhanced pro‑apoptotic
activity, is a novel candidate for treatments
of multiple myeloma and non‑Hodgkin
lymphoma Guangbing Zhang1†, Cuiyu Guo1†, Yan Wang1†, Xianda Zhang2†, Shuang Liu1,2, Wen Qu3, Chunxia Chen4,
Lingli Yan4, Zhouning Yang2, Zhixiong Zhang1, Xiaohua Jiang1, Xiaofeng Chen1, Hong Liu1, Qinhuai Lai1,
Xian Wei1, Ying Lu1, Shengyan Zhao1, Han Deng1, Yuxi Wang1,5, Lin Yu3, Hongbin Yu6, Yu Wu6, Zhaoming Su1,
Pengyu Chen2, Ziqing Ren2, Meng Yu2, Feng Qu2, Yong Luo7, Lantu Gou1, Qing Li2,8, Ying Huang2†,
Fanxin Ma1,2† and Jinliang Yang1,9*† Abstract Anti-CD38 monoclonal antibodies (mAbs), daratumumab, and isatuximab have represented a breakthrough in the
treatment of multiple myeloma (MM). Recently, CD38-based mAbs were expected to achieve increasing poten-
tial beyond MM, which encouraged us to develop new anti-CD38 mAbs to meet clinical needs. In this study, we
developed a novel humanized anti-CD38 antibody, FTL004, which exhibited enhanced pro-apoptotic ability and
negligible binding to red blood cells (RBCs). FTL004 presented a better ability to induce direct apoptosis independ-
ent of Fc-mediated cross-linking against lymphoma and MM cell lines as well as primary myeloma cells derived from
MM patients. For instance, FTL004 induced RPMI 8226 cells with 55% early apoptosis cells compared with 20% in
the isatuximab-treated group. Of interest, FTL004 showed ignorable binding to CD38 on human RBCs in contrast to
tumor cells, even at concentrations up to 30 μg/mL. Furthermore, with an engineered Fc domain, FTL004 displayed
stronger antibody-dependent cellular cytotoxicity (ADCC) against CD38+ malignant cells. In vivo MM and non-Hodg-
kin lymphoma tumor xenograft models showed that FTL004 possessed an effective anti-tumor effect. Cryo-electron
microscopy structure resolved two epitope centers of FTL004 on CD38: one of which was unique while the other © The Author(s) 2022. Open Access This arti
permits use, sharing, adaptation, distribution
original author(s) and the source, provide a li
other third party material in this article are in
to the material. If material is not included in t
regulation or exceeds the permitted use, you
licence, visit http://creativecommons.org/lice
mmons.org/publicdomain/zero/1.0/) applies
†Guangbing Zhang, Cuiyu Guo, Yan Wang, and Xianda Zhang equally
contributors
†Ying Huang, Fanxin Ma, and Jinliang Yang contributed equally to the article
as Co-senior authors
*Correspondence: jinliangyang@scu.edu.cn
1 State Key Laboratory of Biotherapy and Cancer Center/Collaborative
Innovation Center for Biotherapy, West China Hospital, Sichuan University,
3‑17 People Road, Chengdu, Sichuan 610041, People’s Republic of China
Full list of author information is available at the end of the article †Guangbing Zhang, Cuiyu Guo, Yan Wang, and Xianda Zhang equally
contributors †Ying Huang, Fanxin Ma, and Jinliang Yang contributed equally to the article
as Co-senior authors *Correspondence: jinliangyang@scu.edu.cn 1 State Key Laboratory of Biotherapy and Cancer Center/Collaborative
Innovation Center for Biotherapy, West China Hospital, Sichuan University,
3‑17 People Road, Chengdu, Sichuan 610041, People’s Republic of China
Full list of author information is available at the end of the article CORRESPONDENCE Open Access To the editor CD38 is a type II transmembrane glycoprotein with mul-
tiple functions and expressed lower on normal blood cells
and higher on hematologic tumor cells [1, 2]. Anti-CD38
mAbs were effective in some hematological malignan-
cies, especially multiple myeloma (MM) [3]. Although
both daratumumab and isatuximab have significantly
improved the outcome of patients with MM, incomplete
responses and on-target/off-tumor effects emerge dur-
ing the treatment [4, 5]. Herein, we described FTL004,
a novel humanized IgG1κ anti-CD38 mAb possessing
novel properties. Then, the CD38/FTL004 complex structure was deter-
mined using Cryo-electron microscopy at 3.86 Å reso-
lution (Fig. 1h, i). Of note, there seem to be two epitope
centers of FTL004, one of them includes from Pro52 to
His56 and the other one is 91TQTV94. Superimposition
of the CD38/daratumumab (PDB code 7DHA) complex
and CD38/isatuximab (PDB code 4CMH) complex onto
the CD38/FTL004 complex revealed that the epitopes of
FTL004 share no common residue of CD38 with dara-
tumumab, whereas one of the epitopes (91TQTV94) of
FTL004 partly overlaps with that of isatuximab (Fig. 1j). FTL004 exhibited similar affinities to CD38 (KD of
2.55, 3.84, and 1.2 nM, respectively) in surface plasmon
resonance (Fig. 1a) and to CD38-positive cell lines in flow
cytometry (Fig. 1b), compared with daratumumab or
isatuximab. However, these antibodies bound differently
to blood cells. Daratumumab or isatuximab bound to
RBCs to different extents from healthy donors, while the
binding of FTL004 to RBCs was virtually undetectable
(Fig. 1c). This property should result in a more favorable
pharmacokinetic of FTL004 by circumventing binding to
CD38 on circulating RBCs [6] and a better safety profile
by minimizing side effects associated with RBC binding. Moreover, FTL004 did not agglutinate RBCs in indirect
antiglobulin tests (Fig. 1d), which could avoid the inter-
ferences with blood transfusion that are observed in
other anti-CD38 mAbs [7, 8]. Next, to evaluate the tumor-killing capacity of
FTL004, pro-apoptotic activity was first analyzed. We
found that FTL004 had a superior ability to induce
direct apoptosis independent of cross-linking rea-
gents against CD38-positive cell lines (Fig. 2a) and
primary MM cells (Fig. 2b) than isatuximab. Particu-
larly, the highest apoptosis rate of FTL004 on pri-
mary MM cells was detected to be up to 60%. On the
other hand, the Fc domain of FTL004 was engineered
(S240D/I333E mutation) to elevate the affinity to
human Fcγ receptors, leading to the enhancement of
antibody-dependent cellular cytotoxicity (ADCC) [10]. (See figure on next page.)
Fig. 1 Distinct binding profile of FTL004 to CD38+ tumor cells, RBCs, and immune cells. a Affinity analysis of FTL004 to human CD38 as measured
by Biacore 8 K (KD = 2.55 × 10–9 mol/L). b EC50 values for anti-CD38 mAbs binding to diverse cells. DARA, daratumumab; ISA, isatuximab. c Binding
of FTL004, daratumumab or isatuximab to freshly isolated RBCs from healthy donors. d Indirect antiglobulin tests for CD38 mAbs to the same three
healthy donors RBCs above. e The percentage of binding of CD38 mAbs to normal PBMCs (n = 4), including to CD45+ CD3+ T cells, CD3+ CD4+ T
cells, CD3+ CD8+ T cells, CD45+ CD19+ B cells and CD3-CD56+ NK cells. Data show means ± SD and unpaired Student’s t tests were used to
determine the statistical significance between daratumumab versus FTL004. *P < 0.05, **P < 0.01 and ***P < 0.001. f Flow cytometry dot plots
showing the binding of anti-CD38 mAbs to NK cells and CD8+ T cells. g PBMCs from normal donors (n = 6) were pretreated with 1 μg/mL FTL004
or isatuximab for 3 days. Flow cytometry was used to determine the percentage of CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells
in lymphocytes. Data were then normalized to isotype controls and fold changes (means ± SD) are shown. Isotype cells were used as controls in
settings. h The overall structure of the CD38/FTL004 Fab complex. CD38 and the heavy and light chains of FTL004 are colored gray, purple, and
green, respectively. i Stereoscopic view of the direct hydrogen bonds, salt bridge, in the interface between CD38 and FTL004. j Comparison of the
binding of FTL004, daratumumab, and isatuximab. Surface representation of the epitopes of FTL004 (green), daratumumab (blue), isatuximab (red)
and superposition part of FTL004 and isatuximab (yellow) on CD38 (gray) © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Page 2 of 6 partly overlapped with that of isatuximab. Taken together, FTL004 distinguishes it from other CD38 targeting mAbs
and represents a potential candidate for the treatment of MM and non-Hodgkin lymphoma. Keywords: Multiple myeloma, Monoclonal antibodies, CD38, Red blood cells, Direct apoptosis partly overlapped with that of isatuximab. Taken together, FTL004 distinguishes it from other CD38 targeting mAbs
and represents a potential candidate for the treatment of MM and non-Hodgkin lymphoma. Keywords: Multiple myeloma, Monoclonal antibodies, CD38, Red blood cells, Direct apoptosis partly overlapped with that of isatuximab. Taken together, FTL004 distinguishes it from other CD38 targeting mAbs
and represents a potential candidate for the treatment of MM and non-Hodgkin lymphoma. Keywords: Multiple myeloma, Monoclonal antibodies, CD38, Red blood cells, Direct apoptosis reflected that the epitope of FTL004 binding on CD38
may be distinct. Although anti-CD38 mAbs require a
high threshold for CD38 expression to induce cell death,
we must be wary of its possible off-target effects. PBMCs
coculture assays showed that there was no depletion in
T and B cells, indicating the limited off-tumor toxicity of
FTL004 (Fig. 1g). But as expected, NK cells, with higher
expression of CD38, were reduced to an acceptable level
[9].h To the editor In ADCC bioassays with Jurkat-Lucia-CD16a-NFAT
cells [11], FTL004 showed a distinctly higher intensity
of ADCC, with EC50 values of 2–8 ng/mL, compared For other immune cells, FTL004 bound to most sub-
population cells of peripheral blood mononuclear cells
(PBMCs) in a comparable intensity with daratumumab
or isatuximab (Fig. 1e). Unexpectedly, FTL004 exhib-
ited a slightly higher binding to CD8+ T cells and a
much higher binding to NK cells (Fig. 1f). These events Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Page 3 of 6 Page 3 of 6
Zhang et al. Journal of Hematology & Oncology (2022) 15:177
Fig. 1 (See legend on previous page.) Fig. 1 (See legend on previous page.) Fig. 1 (See legend on previous page.) Fig. 1 (See legend on previous page.) Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Page 4 of 6 Fig. 2 Anti-tumor efficacy of FTL004. a Induction of apoptosis by FTL004, daratumumab, or isatuximab in various cell lines, cells were treated
with anti-CD38 mAbs 1.5 μg/mL for 24 h. Data represent means of triplicates and SD. Statistical significance between FTL004 and hIgG groups
was determined by unpaired Student’s t tests. *P < 0.05; **P < 0.01, ***P < 0.001. b Apoptosis induction by FTL004 in primary MM cells. c, d ADCC
reporter bioassays to various CD38+ tumor cell lines (c) and MM primary cells (d) of FTL004, daratumumab, or isatuximab. Data represent means
of triplicates and SD. e–g Representative dose–response curve examples for ADCC activity (e), ADCP activity (f), and CDC activity (g) of FTL004 or
daratumumab to CHO-CD38+ cells. Data show mean ± SD of three experiments. h Anti-tumor efficacy of FTL004 in lymphoma cell Ramos, MM
cell H929, MM1S xenograft models. NOD-SCID mice bearing xenografts were treated with administration of PBS, daratumumab, or FTL004. Tumor
burdens were monitored. Each point on the graph represents the average tumor volume Fig. 2 Anti-tumor efficacy of FTL004. a Induction of apoptosis by FTL004, daratumumab, or isatuximab in various cell lines, cells were treated
with anti-CD38 mAbs 1.5 μg/mL for 24 h. Data represent means of triplicates and SD. Statistical significance between FTL004 and hIgG groups
was determined by unpaired Student’s t tests. *P < 0.05; **P < 0.01, ***P < 0.001. b Apoptosis induction by FTL004 in primary MM cells. Consent for publication
Not applicable. Consent for publication
Not applicable. References 1. Chini CCS, Peclat TR, Warner GM, Kashyap S, Espindola-Netto JM, de
Oliveira GC, et al. CD38 ecto-enzyme in immune cells is induced
during aging and regulates NAD(+) and NMN levels. Nat Metab. 2020;2(11):1284–304. Competing interests
Q Qing Li, Ying Huang, Fanxin Ma, Xianda Zhang, Shuang Liu, Zhouning Yang,
Pengyu Chen, Ziqing Ren, Meng Yu, and Feng Qu are employees at Sound
Biopharmaceuticals Co., Ltd. No potential conflicts of interest were disclosed
by the other authors. Acknowledgements The authors thank all of the patients and healthy donors who consented to
this study. They also thank Yiran Tao (West China-California Research Center for
Predictive Intervention Medicine, West China Hospital, Sichuan University) for
FACS data acquisition and analysis. To the editor c, d ADCC
reporter bioassays to various CD38+ tumor cell lines (c) and MM primary cells (d) of FTL004, daratumumab, or isatuximab. Data represent means
of triplicates and SD. e–g Representative dose–response curve examples for ADCC activity (e), ADCP activity (f), and CDC activity (g) of FTL004 or
daratumumab to CHO-CD38+ cells. Data show mean ± SD of three experiments. h Anti-tumor efficacy of FTL004 in lymphoma cell Ramos, MM
cell H929, MM1S xenograft models. NOD-SCID mice bearing xenografts were treated with administration of PBS, daratumumab, or FTL004. Tumor
burdens were monitored. Each point on the graph represents the average tumor volume Fig. 2 Anti-tumor efficacy of FTL004. a Induction of apoptosis by FTL004, daratumumab, or isatuximab in various cell lines, cells were treated
with anti-CD38 mAbs 1.5 μg/mL for 24 h. Data represent means of triplicates and SD. Statistical significance between FTL004 and hIgG groups
was determined by unpaired Student’s t tests. *P < 0.05; **P < 0.01, ***P < 0.001. b Apoptosis induction by FTL004 in primary MM cells. c, d ADCC
reporter bioassays to various CD38+ tumor cell lines (c) and MM primary cells (d) of FTL004, daratumumab, or isatuximab. Data represent means
of triplicates and SD. e–g Representative dose–response curve examples for ADCC activity (e), ADCP activity (f), and CDC activity (g) of FTL004 or
daratumumab to CHO-CD38+ cells. Data show mean ± SD of three experiments. h Anti-tumor efficacy of FTL004 in lymphoma cell Ramos, MM
cell H929, MM1S xenograft models. NOD-SCID mice bearing xenografts were treated with administration of PBS, daratumumab, or FTL004. Tumor
burdens were monitored. Each point on the graph represents the average tumor volume Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Page 5 of 6 with daratumumab and isatuximab (Fig. 2c). Consistent
results were obtained in primary MM cells (Fig. 2d). Abbreviations
Ab M
l mAb: Monoclonal antibody; MM: Multiple myeloma; RBCs: Red blood cells;
PBMCs: Peripheral blood mononuclear cells; ADCC: Antibody-dependent
cellular cytotoxicity. Received: 24 October 2022 Accepted: 20 December 2022 Received: 24 October 2022 Accepted: 20 December 2022 Funding
h
k This work was supported by the National Natural Science Foundation of
China (81,872,907, 32,070,049, 32,222,040), Science and Technology Plan
Project of Sichuan Province (Provincial Academy and Provincial University
Cooperation Project) (2020YFSY0025), Sichuan Science and Technology
Program (2021YJ0454, 2019YFS0003), Ministry of Science and Technology
of China (MoST) (2021YFA1301900), Sichuan University start-up funding
(20822041D4057), the CAMS Innovation Fund for Medical Sciences (CIFMS)
(2021-I2M-5-075), and the National Major Scientific and Technological Special
Project for Significant New Drugs Development (2018ZX09201018-021,
2017ZX09302010). 5. Ofran Y. Daratumumab: new indications revolving around “off-targets.”
Haematologica. 2021;106(12):3032–3. 6. Fedyk ER, Idamakanti N, Chen J, Estevam J, Palumbo A. The binding of
CD38 therapeutics to red blood cells and platelets subverts depletion of
target cells. Blood. 2019;134(Supplement_1):3136. 7. Werle E, Ziebart J, Wasmund E, Eske-Pogodda K. Daratumumab interfer-
ence in pretransfusion testing is overcome by addition of daratumumab
Fab fragments to patients’ plasma. Transfus Med Hemother. 2019. https://
doi.org/10.1159/000495773. 7. Werle E, Ziebart J, Wasmund E, Eske-Pogodda K. Daratumumab interfer-
ence in pretransfusion testing is overcome by addition of daratumumab
Fab fragments to patients’ plasma. Transfus Med Hemother. 2019. https://
doi.org/10.1159/000495773. 8. Lancman G, Arinsburg S, Jhang J, Cho HJ, Jagannath S, Madduri D, et al. Blood transfusion management for patients treated with anti-CD38
monoclonal antibodies. Front Immunol. 2018;9:2616. 8. Lancman G, Arinsburg S, Jhang J, Cho HJ, Jagannath S, Madduri D, et al. Blood transfusion management for patients treated with anti-CD38
monoclonal antibodies. Front Immunol. 2018;9:2616. Author details 1 State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innova-
tion Center for Biotherapy, West China Hospital, Sichuan University, 3‑17
People Road, Chengdu, Sichuan 610041, People’s Republic of China. 2 Sound
Biopharmaceuticals Co., Ltd., Tianfu International Bio‑Town, Huigu Dong 2nd
Road 8, Chengdu, Sichuan 610200, People’s Republic of China. 3 Department
of Clinical Laboratory, Mianyang Central Hospital, Mianyang, People’s Republic
of China. 4 Department of Transfusion, West China Hospital, Sichuan University,
Chengdu, People’s Republic of China. 5 Department of Clinical Research Man-
agement, West China Hospital, Sichuan University, Chengdu, People’s Republic
of China. 6 Department of Hematology, West China Hospital, Sichuan Univer-
sity, Chengdu, People’s Republic of China. 7 Department of Head and Neck
Oncology, West China Hospital, Sichuan University, Chengdu, People’s Repub-
lic of China. 8 West China School of Public Health, Sichuan University, Chengdu,
People’s Republic of China. 9 Research Unit of Gene and Immunotherapy,
Chinese Academy of Medical Sciences, Chengdu, People’s Republic of China. In summary, FTL004 is a novel CD38 mAb that owns
unique properties. Although the mechanism by which
FTL004 binds differently to tumor cells versus RBCs
remains unclear, it is expected that FTL004 owns lim-
ited on-target/off-tumor effects. The results above sug-
gest that FTL004 is a therapeutic Ab with high potential
for the treatment of MM and non-Hodgkin lymphoma. Author contributions 2. Mehta K, Shahid U, Malavasi F. Human CD38, a cell-surface pro-
tein with multiple functions. FASEB J Off Publ Fed Am Soc Exp Biol. 1996;10(12):1408–17. ZGB, GCY, ZXD, MFX, HY, LQ, GLT, and YJL were involved in study conception
and design; ZGB, WY, LS, YZN, ZZX, JXH, and WYX contributed to the develop-
ment of methodology; QW, LQH, YLL, CCX, YL, YHB, WY, and SZM participated
in acquisition of data; LH, CPY, RZQ, YM, and QF were involved in analysis and
interpretation of data; ZGB, WX, LY, ZSY, CXF, DH, and LY wrote and reviewed
the manuscript. All authors read and approved the final manuscript. 3. van de Donk NW, Janmaat ML, Mutis T, van Lammerts Bueren JJ, Ahmadi
T, Sasser AK, et al. Monoclonal antibodies targeting CD38 in hematologi-
cal malignancies and beyond. Immunol Rev. 2016;270(1):95–112. 3. van de Donk NW, Janmaat ML, Mutis T, van Lammerts Bueren JJ, Ahmadi
T, Sasser AK, et al. Monoclonal antibodies targeting CD38 in hematologi-
cal malignancies and beyond. Immunol Rev. 2016;270(1):95–112. 4. Bhatnagar V, Gormley NJ, Lola L, et al. FDA approval summary: dara-
tumumab for treatment of multiple myeloma after one prior therapy. Oncologist. 2017. https://doi.org/10.1634/theoncologist.2017-0229. Ethics approval and consent to participate To investigate further, we examined the ADCC
of FTL004 with healthy PBMCs. PBMCs exhibited
enhanced ADCC activity against CHO-CD38+ cells
in the presence of FTL004, with threefold higher
than daratumumab (Fig. 2e). Meanwhile, FTL004
induced stronger ADCP with 74% phagocytosed CHO-
CD38+ cells in an EC50 value of 40 ng/mL (Fig. 2f). However, FTL004 induced a lower CDC than daratu-
mumab of CHO-CD38+ cells (Fig. 2g). In vivo studies
were further carried out, and FTL004 was examined
in Ramos, H929, and MM1S-bearing NOD-SCID mice
(Fig. 2h). FTL004 treatment resulted in significant inhi-
bition of tumor growth compared to PBS treatment. The human subject studies were approved by the Ethnic Committee of West
China Hospital of Sichuan University. All patients provided written informed
consent. Availability of data and materials The datasets used and/or analyzed during the current study are available from
the corresponding author upon reasonable request. 9. Naeimi Kararoudi M, Nagai Y, Elmas E, de Souza Fernandes Pereira M, Ali
SA, Imus PH, et al. CD38 deletion of human primary NK cells eliminates
daratumumab-induced fratricide and boosts their effector activity. Blood. 2020;136(21):2416–27. Page 6 of 6 Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Zhang et al. Journal of Hematology & Oncology (2022) 15:177 10. Jebamani P, Sriramulu DK, Jung ST, Lee S-G. Structural study on the
impact of S239D/I332E mutations in the binding of Fc and FcγRIIIa. Biotechnol Bioprocess Eng. 2021;26(6):985–92. 11. Hong Y, Guo H, Wei M, Zhang Y, Fang M, Cheng T, et al. Cell-based
reporter assays for measurements of antibody-mediated cellular cytotox-
icity and phagocytosis against SARS-CoV-2 spike protein. J Virol Methods. 2022;307: 114564. 11. Hong Y, Guo H, Wei M, Zhang Y, Fang M, Cheng T, et al. Cell-based
reporter assays for measurements of antibody-mediated cellular cytotox-
icity and phagocytosis against SARS-CoV-2 spike protein. J Virol Methods. 2022;307: 114564. Publisher’s Note
S
i
N
i Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from: •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from: •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from:
|
https://openalex.org/W2033987423
|
http://conservancy.umn.edu/bitstream/11299/174058/1/JTLU_vol8_no2_pp171-189.pdf
|
English
| null |
Has Mexico City’s shift to commercially produced housing increased car ownership and car use?
|
Journal of transport and land use
| 2,015
|
cc-by
| 8,940
|
http://jtlu.org
vol. 8 no. 2 [2015] pp. 171–189 http://jtlu.org
vol. 8 no. 2 [2015] pp. 171–189 Article history: Abstract: Mexico City’s principal form of housing production has
shifted over the past two decades. More households now purchase
houses in large commercially built housing developments than move
into informal settlements. Looking at 1500 households in two subur-
ban municipalities from a 2007 metropolitan travel survey, this study
is the first to quantify differences in car ownership and car use across
households in informal settlements and commercial housing develop-
ments. Accounting for income, proxies for wealth, household com-
position, and geography, households living in commercial housing
developments are likely to own more cars and drive more than similar
households in neighboring informal settlements. A test for residen-
tial self-selection finds no unobserved correlations across households
that own cars and live in commercial housing developments, suggest-
ing that the included controls do a good job of capturing the effects
of residential self-selection or that the effects are limited. Something
about the local land use and design of new commercial housing devel-
opments appears conducive to car ownership and use. Differences be-
tween the two settlement types, including more parking, wider streets,
less-connected street-grids, and less accessible transit stops in commer-
cial settlements, likely play a role. Article history:
Received: December 28, 2013
Received in revised form:
August 25, 2014
Accepted: February 16, 2015
Available online: May 29, 2015 Introduction 1 At 35,000 feet—a typical cruising altitude for a long-distance commercial flight—Los Héroes Tecámac
looks a bit like an urban airport. Two long, thick, near-perpendicular, gray lines intersect like massive
runways providing access from northwest to southeast and northeast to southwest. To the north are the
unmistakable tan and green rectangles of agricultural land. To the east and west are the equally unmis-
takable curving black, gray, and tan specks and lines of informal urbanization that is so characteristic of
Mexico City’s peripheral neighborhoods and many like them around the world (Figure 1). Located al-
most 20 miles to the northeast of the historic center, Los Héroes, despite its somewhat unusual appear-
ance from the air, is typical of contemporary housing developments in Mexico City on the ground. Over the past two decades, Mexico City’s principal form of new housing production has shifted from
an informal process, where households built their own properties generally in informal settlements on Copyright 2015 Erick Guerra
http://dx.doi.org/10.5198/jtlu.2015.714
ISSN: 1938-7849 | Licensed under the Creative Commons Attribution – Noncommercial License 3.0 Copyright 2015 Erick Guerra
http://dx.doi.org/10.5198/jtlu.2015.714
ISSN: 1938-7849 | Licensed under the Creative Commons Attribution – Noncommercial License 3.0 Has Mexico City’s shift to commercially produced housing
increased car ownership and car use? Erick Guerra
University of Pennsylvania
erickg@design.upenn.edu Copyright 2015 Erick Guerra
http://dx.doi.org/10.5198/jtlu.2015.714
ISSN: 1938-7849 | Licensed under the Creative Commons Attribution – Noncommercial License 3.0 py g
http://dx.doi.org/10.5198/jtlu.2015.714
ISSN: 1938-7849 | Licensed under the Creative Commons Attribution – Noncommercial License 3.0 The Journal of Transport and Land Use is the official journal of the World Society for Transport and Land Use
(WSTLUR) and is published and sponsored by the University of Minnesota Center for Transportation Studies. This paper is also published with sponsorship from WSTLUR and the Institutes of Transportation Studies at the
University of California, Davis, and the University of California, Berkeley. 172 JOURNAL OF TRANSPORT AND LAND USE 8.2 the periphery, to a formal process, where private developers acquire large peripheral lots, build new hous-
ing speculatively, and sell completed homes in massive developments to households that qualify for
publicly subsidized mortgages (Monkkonen 2011a, 2011b, Pardo and Velasco Sánchez 2006). What
look like thick gray lines from the air are rows of identical one-, two-, and three-story attached houses
and condominiums along intersecting rectangular grids of arterial roads and linear culs-de-sac. Small
parking spaces separate the building facades from the sidewalk and street. Figure 1: Aerial view of Los Héroes and surroundings in Ecatepec and Tecamac, State of Mexico
(Google Earth 02-08-2013) Figure 1: Aerial view of Los Héroes and surroundings in Ecatepec and Tecamac, State of Mexico
(Google Earth 02-08-2013) Changes in public policy, rather than demand, primarily drove this shift in housing production. Since
Mexico’s 1994 economic crisis and currency devaluation, the central government has pursued financial
liberalization reforms including the reform of Infonavit, a national housing provident fund that requires
private-sector employers to pay 5 percent of salaried workers’ wages into the fund, which subsidizes
loans for qualified low- to moderate- income workers to purchase newly built, privately produced homes
(Monkkonen 2011a, Pardo and Velasco Sánchez 2006). Between 1995 and 2005, public agencies pro-
vided 75 percent of all housing loans by value—and even more by volume—in Mexico. Infonavit
accounted for 81 percent of these publicly financed loans (Monkkonen 2011b). Smaller but similar
housing funds for low-income workers with informal employment, government workers, and the state-
owned oil company (Pemex) also contributed to the increase in commercial housing production. The
legalization of sales of communally owned farmland, a simplification of housing construction regula-
tions, and active political engagement with private developers also facilitated the rapid shift in housing
production (Monkkonen 2011a). Copyright 2015 Erick Guerra
http://dx.doi.org/10.5198/jtlu.2015.714
ISSN: 1938-7849 | Licensed under the Creative Commons Attribution – Noncommercial License 3.0 In 1980, commercial developers produced just 15 percent of housing in the Mexico City me-
tropolis (Dowall and Wilk 1989). By 2010, they produced the majority, particularly in fast-growing Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 173 suburban municipalities like Tecamac. By the early 21st century, Mexico’s largest commercial housing
builders—Geo, Homex, Javer, Sadasi, and Urbi—were among the world’s largest housing producers by
volume. In 2010, Geo produced and sold more homes per year than the combination of D.R. Horton
and the Pulte Group, the two largest housing producers by volume in the United States. All five of the
above-mentioned Mexican companies would have made recent top-10 lists of the largest US housing
producers (Hanley Wood LLC 2013, Nava 2011). Over the same period as the shift in housing production occurred, car ownership and use have
increased steadily. Between 2000 and 2010, the number of registered cars increased an average of 14
percent annually (INEGI 2013). Between 1994 and 2007, total vehicle kilometers traveled (VKT) on
an average weekday increased from 24 to 32 million (Guerra 2014, INEGI 2007, 1994). Most of this
growth has occurred in suburban municipalities outside of the Federal District of Mexico City where the
shift in housing production has been so pronounced. In general, the new commercial housing develop-
ments are denser and closer to the city center than recently constructed housing in informal settlements
(Monkkonen 2011c) but have less connected local street networks, and each housing unit comes with
a single, exterior parking space. Although many residents convert parking into commercial retail spaces
or residential additions over time, the default state has moved from one in which households expend
land, energy, and resources to build a parking space to one in which they must expend time and energy
to convert the parking to something else. This paper provides the first empirical analysis of whether the shift in Mexico’s housing policy is
contributing to the rapid growth in car ownership and VKT in Mexico City. Are households that live
in new commercial housing developments more likely to own and drive cars than similar households in
nearby informal settlements? To what extent do differences in car ownership and use relate to local land
use and design, or unobserved differences between households, such as a preference for driving and thus
a preference to locate in new, more car-friendly neighborhoods? Copyright 2015 Erick Guerra
http://dx.doi.org/10.5198/jtlu.2015.714
ISSN: 1938-7849 | Licensed under the Creative Commons Attribution – Noncommercial License 3.0 Given the level of public involvement in housing production and the public desire to reduce emis-
sions and congestion, the answers to these questions matter. If the increased driving of households in
commercial housing developments is related to local land use or design, then the public sector has the
opportunity to reduce car use by redesigning commercial housing developments and requiring new
construction to follow design guidelines. If, by contrast, differences are primarily related to regional
accessibility or varying household preferences, local design and land-use changes will have little to no
influence on car use. The answers also matter outside of Mexico City. There is a long and lively debate over whether and
to what extent local land use and design influence travel behavior (Boarnet and Crane 2001, Brown-
stone 2008, Crane 2000, 1996, Ewing and Cervero 2010, Handy, Cao, and Mokhtarian 2005, Pickrell
1999). One point of particular contention is the extent to which residential self-selection influences the
observed relationships with urban design and land use (for a discussion, see Mokhtarian and Cao 2008). In Mexico City, however, the recent and dramatic change in housing type may provide something akin
to a natural experiment. The new housing product is uniform, giving households little chance to select
amenities based on preferences. Furthermore, due to the housing subsidy, households are most likely
choosing between living in a peripheral informal settlement or a peripheral commercial housing devel-
opment based on loan eligibility rather than travel preferences. Since the new developments have the
same levels of regional transit- and car-accessibility as the informal neighborhoods around them, differ-
ences in travel behavior that are related to the built environment stem from neighborhood rather than
regional differences. If local land use and design influence travel in peripheral Mexico City, they also
likely influence travel in the fast-growing peripheral neighborhoods of many other cities where popula-
tion, suburbanization, and car ownership are increasing rapidly. JOURNAL OF TRANSPORT AND LAND USE 8.2 JOURNAL OF TRANSPORT AND LAND USE 8.2 174 2
Research framework and methods
2.1
Study area and data 2 Study area and data 2.1 This study focuses on two suburban municipalities: Ixtapaluca and Tecamac. Both grew rapidly between
1990 and 2010—a period that coincides with the rapid shift from informal to formal housing produc-
tion. Ixtapaluca, located about 30 kilometers east of Mexico City’s historic center, grew at an average
annual rate of 12 percent, adding 330,000 new residents between 1990 and 2010. At an average an-
nual growth rate of 10 percent, Tecamac’s population tripled from 120,000 to 360,000 residents over
the same period. Figure 2 maps metropolitan population growth by municipality between the last two
decennial censuses and shows the location of Ixtapaluca and Tecamac. Large-scale, high-density private
commercial housing developments accounted for a substantial portion of the growth in both munici-
palities. Figure 2: Mexico City’s average annual population growth by municipality Figure 2: Mexico City’s average annual population growth by municipality Based on 2007 and 2008 satellite imagery from Google Earth, and first-hand knowledge of the new
development sites, I drew geographic shapefiles around commercial housing developments and matched
them with existing Census Tract shapefiles from the National Institute of Geography and Statistics
(INEGI). Figure 3 shows the location of commercial housing developments in urbanized areas of Ix-
tapaluca and Tecamac. Each Census Tract then received a score ranging from 0 to 1 indicating the pro-
portion of the tract that fell within a commercial housing development around the time of the 2007
metropolitan household travel survey (INEGI 2007). Figure 5 shows the distribution of the proportion 175 Has Mexico City’s shift to commercially produced housing increased car ownership and car use? of each Census Tract that overlapped the drawn shapefiles of commercial housing developments. Two-
thirds of Census Tracts contain either no commercial housing developments or only commercial hous-
ing developments. I then matched these data to household location, trip origin, and trip destination data
from the 2007 metropolitan household travel survey. Figure 3: Location of large-scale commercial developments in Ixtapaluca and Tecamac Figure 3: Location of large-scale commercial developments in Ixtapaluca and Tecamac JOURNAL OF TRANSPORT AND LAND USE 8.2 car ownership and use. For example, commercial housing residents are less likely to live in poverty since
the new housing finance program has minimum income requirements. This is likely associated with
higher car ownership and use. Computer ownership and home ownership are included as proxies for
wealth, since income captures earnings, rather than total assets. Owning a computer may also correlate
with lifestyle preferences that favor car ownership and use. p
Table 1: Proposed control variables and expected correlation with whether
a household lives in a commercial housing development, car ownership, and
car use p
Table 1: Proposed control variables and expected correlation with whether
a household lives in a commercial housing development, car ownership, and
car use a household lives in a commercial housing development, car ownership, and
car use
Variable
Commercial Housing
(Treatment Group)
Car Ownership
and Use
Car ownership
(+)
NA
Average daily VKT
(+)
NA
Household income
(+)
(+)
Owns home
(+)
(+)
Owns a computer
(+)
(+)
Average age of household
(-)
(?)
Presence of children
(?)
(+)
Distance to downtown
(?)
(-)
Distance to highway
(?)
(+)
Distance to Metro
(?)
(-)
Local population density
(+)
(-)
Source: 2007 Metropolitan Mexico City Household Travel Survey (INEGI
2007) and author’s calculations 2.2
Research hypotheses This study tests two overarching hypotheses about the relationship between living in a commercial hous-
ing development and owning and using cars. H1: Households living in commercial housing developments are more likely to own and use cars than
similar households in nearby informal settlements. Commercial housing developments tend to have more parking, wider streets, and a more discon-
nected street grid than nearby informal housing developments. As a result, residents are more likely to
own and use cars than those in informal settlements. Limiting the sample to two municipalities with
similar levels of regional accessibility and including municipal-level fixed effects ensures that any differ-
ences found across households relates to local rather than regional differences. gf
Table 1 presents a list of control variables and their likely association with housing type and car
ownership and use. Even within Ixtapaluca and Tecamac, there are likely some systematic differences
between households in formal developments and those in informal ones. Many are also correlated with 176 JOURNAL OF TRANSPORT AND LAND USE 8.2 Written formally, the null hypothesis is H0: E( Yi | Di , Xi ) = E( Yi | Xi ) Written formally, the null hypothesis is H0: E( Yi | Di , Xi ) = E( Yi | Xi ) Where E is an expectation function; Where E is an expectation function; Y is a variable indicating whether a household owns one or more cars and the number of VKT generated
on the weekday of the household travel survey; D is a dummy variable that indicates whether a household lives in a commercial housing development; X is the vector of demographic and geographic control variables; and
i denotes a household. H2: The relationship between commercial housing and car ownership and VKT is independent of un-
identified differences between households in the two types of housing Households tend to choose to live in neighborhoods that meet their transportation preferences. (For a
discussion, see Cervero 2007, Chatman 2009, or Mokhtarian and Cao 2008.) As a result, households
that favor transit often move into locations with good transit access, while households that wish to own
a car tend to move to locations with available parking. To the extent that these preferences vary within
the population and are independent of observed socioeconomic controls, they may bias estimates of the Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 177 relationship between the built environment and travel behavior, particularly in cross-sectional studies. What may appear to be a causal relationship between living in dense neighborhoods and walking, for
example, could be partially or even entirely caused by people with a strong preference for walking (and
who would have walked in any environment) selecting to the densest, most walkable neighborhoods. In a review of 38 empirical studies, Cao, Mohktarian, and Handy (2009) found that this residential
self-selection bias tends to lead to overestimates of the relationship between the built environment and
travel, but that it only accounts for a portion of the correlation. To the extent that the built environ-
ment allows people to exercise preferences (if, for example, people who prefer to walk would not do so
as much unless they could select into denser neighborhoods), the relationship is causal and not biased
by residential self-selection. Given an undersupply or oversupply of specific neighborhood types, self-
selection may even lead to underestimates of the influence of the built environment on travel (Chatman
2009, Levine 2006). Written formally, the null hypothesis is H0: E( Yi | Di , Xi ) = E( Yi | Xi ) In Mexico City, the recent and dramatic change in housing type may provide something akin to
a natural experiment in regard to residential self-selection. Instead of households choosing to live in a
housing unit based on neighborhood preferences, the primary determinant of whether a household
chooses to move into a new housing development is whether its members qualify for a subsidized home
and are purchasing a new house. Since the commercial housing developments are all similar, there is
little opportunity to select based on urban design or land-use preferences. Since the subsidy makes the
housing cheaper relative to market alternatives, there is little incentive to purchase market-rate housing. By limiting the sample to households in two peripheral municipalities, the study design also limits po-
tential selection bias to local rather than regional residential preferences, such as preference to live near
downtown or near a Metro station. I therefore hypothesize that the effects of residential self-selection
will be weak or unobservable, when conditioned on income and other socioeconomic attributes that
correlate with living in a commercial housing development and owning and driving cars. g
g
g
g
Mohtktarian and Cao (2008) and Cao, Mohtkarian, and Handy (2009) identify nine principal
methods to identify and control for residential self-selection bias. A subset is applicable to this analysis,
which relies on cross-sectional data and does not include a variable indicating residential preference. I
use two such methods. The first is an instrument variable approach. For examples of applications to
address residential self-selection bias, see Boarnet and Sarmiento (1998) or Vance and Hedel (2007). In theory, a good instrument is one that exerts influence on the outcome of interest (car ownership and
use), but only through its influence on the treatment of interest (residence type) (Wooldridge 2010). In practice, finding good instruments is difficult (Angrist and Pischke 2008, Bound, Jaeger, and Baker
1995), and in LaLonde’s (1986) landmark study of econometric methods, using them failed to replicate
experimental results of the influence of worker training programs on employment—presumably a much
more straightforward relationship than that between residential selection, the built environment, and
travel behavior. For instruments, I used whether households had members who worked in public-sector, adminis-
trative, or professional jobs. Written formally, the null hypothesis is H0: E( Yi | Di , Xi ) = E( Yi | Xi ) In theory, these job types make households more likely to qualify for Info-
navit loans but no less likely to own a car or drive outside of their effect on wealth and income, which
are included as controls. In practice, the instrument variable approach produced inconsistent parameter
estimates depending on which job types were included as instruments and what other controls were
included. I chose not to report the results. The second approach in this study models car ownership and residential location simultaneously
with a nested logit model, an approach used by Cervero (2007) and Salon (2009). Each household faces
six different discrete choices: whether to live in a commercial housing development and whether to own
zero, one, or two or more cars. If residential self-selection or other unobserved household attributes JOURNAL OF TRANSPORT AND LAND USE 8.2 178 influence the correlation between living in a commercial development and owning a car, then nesting
these choices and estimating a parameter for the nest should produce an estimate that ranges from zero
to one, but is statistically different from one. The closer the estimate is to zero, the stronger the unob-
served correlation between living in a commercial development and owning a car. For example, a nest
value of 0.4 implies an unobserved correlation of 0.6, and a value of 0.2, a correlation of 0.8. Cervero
(2007) found a statistically significant unobserved correlation across households that live near rail transit
and use it of almost 0.75. If the nest value is either greater than or not statistically different from one, as
in Salon’s (2009) study of car ownership and residential location within New York, it indicates that the
nesting parameter should not be included in the estimation. 2.3 This section provides sources, means, and standard deviations for each of the independent and depen-
dent variables modeled. It also describes the choice of expectation function used for car ownership and
car use. As discussed in Section 2.1, the borders of commercial housing developments and Census Tracts
do not match precisely (Figure 4). Most Census Tracts, however, have no commercial housing develop-
ments or only have commercial housing developments (Figure 5). Furthermore, since Census Tracts
contain non-residential land uses, including roads, vacant land, and open space, the Census Tracts with
a high percentage of commercial housing developments often have no other residential uses. Although
somewhat of a simplification, I treat households residing in Census Tracts with 50 percent or more land
comprised of commercial housing developments as residents of commercial housing developments. This
may weaken the statistical relationships tested, but otherwise is unlikely to bias parameter estimates. Figure 4: Matching and overlap of Census Tracts and commercial housing developments Figure 4: Matching and overlap of Census Tracts and commercial housing developments Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 179 Figure 5: Histogram of households’ residential location by the proportion of the Census Tract that is comprised of commercial
housing developments Figure 5: Histogram of households’ residential location by the proportion of the Census Tract that is comprised of commercial
housing developments Table 2 presents the dependent and independent variables, based on whether households are assumed to
live in traditional informal settlements or commercial housing developments. As expected, residents of commercial housing developments tend to own more cars and drive more
frequently than households in informal settlements. On average, households in commercial housing
developments in Tecamac and Ixtapaluca own 56 percent more cars and generate 160 percent more
weekday VKT than residents in traditional, informal housing developments. They also tend to have
higher incomes, though only by 17 percent, and higher rates of home and computer ownership. The
distribution of residents’ income is also much tighter, presumably because households must meet mini-
mum and maximum income requirements to qualify for a loan. In terms of geographic location, those
in commercial housing developments tend to live in more densely populated neighborhoods that are
marginally closer to Mexico City’s historic center, the Metro system, and regional highways than those
in traditional informal developments. JOURNAL OF TRANSPORT AND LAND USE 8.2 JOURNAL OF TRANSPORT AND LAND USE 8.2 180 Table 2: Sample means and standard deviations of independent and dependent variables by whether house-
holds reside in a Census Tract that is primarily comprised of commercial housing developments
Traditional Development
(Control Group)
Commercial Housing*
(Treatment Group)
Mean
SD
Mean
SD
Average number of cars1
0.39
0.60
0.61
0.64
Has one or more cars1
34%
48%
54%
50%
Has two or more cars1
4%
19%
7%
26%
Average daily VKT2
6.0
20.1
15.6
33.2
Monthly income (in pesos)1
$7431
$11,909
$8678
$6426
People in household1
4.5
2.0
4.0
1.4
Household has children1
56%
50%
62%
49%
Owns house1
81%
39%
90%
30%
Average age of adults1
36.1
7.8
35.7
5.9
Has a computer1
23%
42%
43%
50%
A member is college-educated1
45%
77%
70%
80%
A member works in public sector1
1%
12%
2%
14%
A member works in administration1
13%
40%
28%
52%
A member works in professional sector1
19%
47%
37%
61%
People per hectare in Census Tract3
107
74
204
100
Kilometers to Zocalo4
33.4
6.0
31.1
2.9
Accessibility score2
61,026
32,767
69,541
31,312
Kilometers to highway4
1.2
0.9
0.9
0.6
Kilometers to Metro4
16.8
7.3
13.8
2.9
In Ixtapaluca1
54%
50%
68%
47%
Total Households (N)
1070
-
456
-
*indicates that a household lives in a Census Tract that is 50 percent or more comprised of commercial housing *indicates that a household lives in a Census Tract that is 50 percent or more comprised of commercial housing
developments. Sources: 1) 2007 Metropolitan Mexico City Household Travel Survey (INEGI, 2007); 2) Travel
survey and author calculations; 3) 2005 Census; and 4) Network-distance based calculation using household
travel survey and road network shapefiles. To estimate household car-ownership levels, I fit binomial logit, multinomial logit, and nested logit
models. Only 5 percent of all households owned two or more cars. Sixty percent owned none. In the
multinomial models, households choose between no cars, one car, or two-or-more cars. The nested logit
model accounts for unobserved correlations between households that own a single car and those that
own two or more. The nest, however, is not statistically different from one, and I opt to report the unor-
dered multinomial logit model. To estimate VKT, I fit a left-censored Tobit model, which is commonly
applied to joint discrete and continuous data including VKT. 2.3 This conforms to Monkkonen’s (2011c) findings about com-
mercial housing developments in 128 of Mexico’s urban areas. A higher proportion of households in
commercial developments were in Ixtapaluca than Tecamac, where several massive commercial housing
developments have opened since 2007. 3
New commercial housing development, car ownership, and car use 3 After controlling for household income, age, composition, and location, the odds that the average
household owns a single car are 61 percent higher in a commercial housing development than in a tra-
ditional neighborhood. The odds of owning two or more cars are 87 percent higher. Wealthier house-
holds that own their homes and a computer and have college-educated members are also significantly
more likely to own cars. Neither the average age of adults in the household nor the number of people in
the household is statistically correlated with a higher probability of car ownership. By contrast, house-
holds with one or more children are more likely to own a car. With the exception of the fixed effect for
whether a household lives in Ixtapaluca, none of the geographic controls is statistically significantly as-
sociated with higher or lower car ownership. I opted to drop distance from the Metro, distance from
Mexico City’s historic center, and the accessibility score from the model since they were not significant
and they are highly correlated with one another and the municipal fixed effect. This does not indicate
that regional accessibility does not matter, but that there is insufficient variation within each municipal-
ity to estimate the correlation with car ownership. Table 3: Multinomial logit model of car ownership in Tecamac and Ixtapaluca
Estimate
Std. Error
Odds-ratio
Commercial housing development: 1 cars
0.48
0.15
1.61
**
Commercial housing development: 2+ cars
0.62
0.32
1.87
. JOURNAL OF TRANSPORT AND LAND USE 8.2 Three quarters of households did not have
any members who drove on the survey day and thus produced no VKT. For a complete description of
the model forms and estimation procedures, see Wooldridge (2010). Due to a long-tailed distribution, I
log-transform the positive VKT data. This produces a better model fit and more homoscedastic residual
plots. Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 181 Due to non-linearity, logit model coefficients and odds ratios only provide behavioral information about
a theoretical average household. Table 4 shows the car ownership levels that the model predicts assuming
that either no households or all households live in a commercial housing development. If no households
live in commercial housing developments, the model predicts that 38 percent of households would own JOURNAL OF TRANSPORT AND LAND USE 8.2 more cars, compared to 48 percent if all households lived in commercial housing developments. one or more cars, compared to 48 percent if all households lived in commercial housing developments. Table 4: Predicted car ownership rates based on no, some, or all households living in
commercial housing developments
No cars
One car
Two-plus cars
No commercial housing
61.9%
33.6%
4.4%
Existing conditions (31%
commercial housing)
58.6%
36.3%
5.0%
All commercial housing
51.7%
42.2%
6.0% Table 4: Predicted car ownership rates based on no, some, or all households living in
commercial housing developments g
p
No cars
One car
Two-plus cars
No commercial housing
61.9%
33.6%
4.4%
Existing conditions (31%
commercial housing)
58.6%
36.3%
5.0%
All commercial housing
51.7%
42.2%
6.0% Living in a commercial housing development also correlates with producing statistically significantly
more VKT per weekday than living in a traditional, informal neighborhood (Table 5). On average, a
household in a commercial development generates four times more latent VKT (the exponent of 1.408
equals 4.088) than a similar household in an informal settlement. The Tobit regression, however, pri-
marily predicts negative values since two-thirds of households produced no VKT on the survey day. This
coefficient must therefore be interpreted with caution. At the mean predicted latent VKT of -2.479, the
model does not expect a change in actual VKT from a household moving from an informal settlement
to a commercial housing development, since latent VKT would still be negative. An ordinary least
squares regression on the subset of households with VKT above zero produces a weaker, though still
statistically significant result: Households in commercial developments generate 80 percent more VKT
(with a T-value of 4.2) than households in traditional neighborhoods. The Tobit model also finds strong
positive correlations between car use and households’ income, educational attainment, and computer
ownership. Higher population densities and residence in Ixtapaluca are associated with fewer weekday
VKT Table 5: Type I Tobit model of log of households’ weekday VKT in Tecamac and Ixtapaluca Table 5: Type I Tobit model of log of households’ weekday VKT in Tecamac and Ixtapal
Estimate
Std. 3
New commercial housing development, car ownership, and car use Log of income (in pesos): 1 car
0.58
0.10
1.79
***
Log of income (in pesos): 2+ cars
1.29
0.21
3.63
***
Owns home: 1 car
0.39
0.18
1.48
*
Owns home: 2+ cars
1.54
0.74
4.65
*
Has a computer: 1 car
0.80
0.14
2.22
***
Has a computer: : 2+ cars
1.39
0.29
4.01
***
College education: 1 car
0.24
0.08
1.27
**
College education: 2+ cars
0.66
0.15
1.93
***
Number of household members: 1 car
-0.03
0.04
0.97
Number of household members: 2+ cars
0.04
0.09
1.05
Average age of adults: 1 car
0.02
0.06
1.02
Average age of adults: 2+ cars
0.07
0.18
1.07
Average age of adults squared: 1 car
0.00
0.00
1.00
Average age of adults squared: 2+ cars
0.00
0.00
1.00
In Ixtapaluca: 1 car
-0.55
0.13
0.58
***
In Ixtapaluca: 2+ car
- 0.71
0.28
0.49
*
Population per hectare: 1 car
0.00
0.00
1.00
Population per hectare: 2+ cars
0.00
0.00
1.00
Constant: 1 car
-6.61
1.37
***
Constant: 2+ cars
-18.42
3.74
***
Significance codes: ***0.001, **0.01 , *0.05, .0.1, McFadden R2: 0.128, N = 1478 Table 3: Multinomial logit model of car ownership in Tecamac and Ixtapaluca Due to non-linearity, logit model coefficients and odds ratios only provide behavioral information about
a theoretical average household. Table 4 shows the car ownership levels that the model predicts assuming
that either no households or all households live in a commercial housing development. If no households
live in commercial housing developments, the model predicts that 38 percent of households would own 182 JOURNAL OF TRANSPORT AND LAND USE 8.2 JOURNAL OF TRANSPORT AND LAND USE 8.2 Error
Commercial housing development
1.408
0.340
***
Log of monthly income (in pesos)
1.310
0.215
***
Owns home
0.047
0.402
Has a computer
1.165
0.310
***
College education
0.898
0.174
***
People in household
0.023
0.089
Household has children
0.769
0.315
**
Average age of adults
-0.187
0.144
Average age squared
0.002
0.002
In Ixtapaluca
-1.088
0.288
***
People per hectare in Census Tract
-0.003
0.002
*
Constant
-11.435
3.075
***
Significance codes: ***0.001, **0.01 , *0.05, .0.1, McFadden R2: 0.063 To demonstrate the stability of the estimated average effect of living in a commercial housing develop-
ment on car ownership and VKT and the relationship to the control variables, Table 6 provides coeffi-
cient estimates from four additional multinomial logit and Tobit models with fewer controls, as well as
the coefficients with full controls as presented in Tables 4 and 5. Without any controls, households in
commercial housing developments have more than twice the odds of owning one or more cars than
those in traditional informal neighborhoods. Introducing controls for income, wealth, household com- Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 183 position, and geographic features, the odds drop to 50 percent to 60 percent higher. Similarly, predicted
VKT drops when controlling for household income, wealth, and demographics. The correlation, how-
ever, strengthens when geographic variables are introduced. Commercial housing developments tend to
have higher population densities, which are associated with fewer weekday VKT per household. The
association between living in a commercial housing development and owning two or more cars is not
always statistically different from zero with controls included, which is likely due to the small sample of
households with two-or-more cars. In none of the models is it statistically different from the relationship
to owning one car. Table 6: Relationship between living in commercial housing development and household car ownership and weekday VKT
Multinomial logit model
Tobit model
Controls
One car
Two-plus cars
Log of VKT
Income and wealth
Household composition
Geography
0.73***
0.89***
1.67***
(No)
(No)
(No)
0.44**
0.40
0.96***
(Yes)
(No)
(No)
0.40**
0.37
0.96***
(Yes)
(Yes)
(No)
0.48**
0.62. 1.41***
(Yes)
(Yes)
(Yes)
Significance codes: ***0.001, **0.01 , *0.05, .0.1 p between living in commercial housing development and household car ownership and weekday VKT Significance codes: ***0.001, **0.01 , *0.05, .0.1 3.1 The nested logit model of residential location and car ownership produces generally similar estimates of
the relationship between socioeconomic control variables and car ownership (Table 7). It also indicates
that a number of the control variables are indeed associated with whether households live in commercial
housing developments. For example, smaller, wealthier households with computers are more likely to
live in commercial housing developments. As expected, households with members who work in profes-
sional or administrative jobs are more likely to live in commercial housing developments. Government
employment is not statistically correlated, likely because government employees qualify for a separate
fund, the Housing Fund of the Institute of Social Security of State Workers. The model includes two
nest parameters, one for car ownership and one for the combination of car ownership and residence in
a commercial housing development. The car-ownership nest of 0.83 indicates an unobserved 17 per-
cent correlation across households that own cars. However, the value is not statistically different from
one. The nest for car ownership and commercial residence is greater than one, which indicates that the
choices are not correlated and should not be nested. Again, however, the value is not statistically dif-
ferent from one. Residential self-selection does not appear to play a significant role in influencing the
car-ownership parameter. This supports the hypothesis that the rapid change in housing production has
limited the effects of residential self-selection. The rich set of socioeconomic control variables may also
do a good job of capturing the effects of residential self-selection as found in studies by Brownstone and
Golob (2009) and Bhat and Guo (2007). 184 JOURNAL OF TRANSPORT AND LAND USE 8.2 Table 7: Nested logit model of joint decision of how many cars to own and whether to live in a com-
mercial housing development
Estimate
Std. Error
Odds-ratio
Car ownership
Log of monthly income: 1 car
0.587
0.093
1.799
***
Log of monthly income: 2+ cars
1.223
0.235
3.397
***
Owns home: 1 car
0.438
0.177
1.550
*
Owns home: 2+ cars
1.622
0.856
5.064
. 3.1 Has a computer: 1 car
0.794
0.140
2.213
***
Has a computer: 2+ cars
1.353
0.331
3.870
***
College education: 1 car
0.234
0.083
1.264
**
College education: 2+ cars
0.633
0.172
1.884
***
People in household: 1 car
-0.030
0.038
0.970
People in household: 2+ cars
0.019
0.099
1.019
Average age of adults: 1 car
0.018
0.066
1.018
Average age of adults: 2+ cars
0.057
0.174
1.058
Average age squared: 1 car
0.000
0.001
1.000
Average age squared: 2+ cars
0.000
0.002
1.000
In Ixtapaluca: 1 car
-0.605
0.130
0.546
***
In Ixtapaluca: 2+ cars
-0.779
0.298
0.459
**
Population per hectare: 1 car
0.001
0.001
1.001
Population per hectare: 2+ cars
-0.001
0.002
0.999
Lives in commercial housing development
Log of monthly income
0.178
0.103
1.195
. Has a computer
0.421
0.136
1.524
**
College education
0.007
0.083
1.007
People in household
-0.322
0.051
0.725
***
Average age of adults
0.340
0.075
1.405
***
Average age squared
-0.005
0.001
0.995
***
In Ixtapaluca
0.566
0.136
1.761
***
Professional job
0.562
0.164
1.755
***
Administrative job
0.770
0.164
2.161
***
Public sector job
0.050
0.424
1.051
Nests
Car ownership and commercial development
1.246
0.662
Car ownership
0.831
0.240
Significance codes: ***0.001, **0.01 , *0.05, .0.1, McFadden R2: 0.118, N = 1478, Constants not
reported to conserve space Table 7: Nested logit model of joint decision of how many cars to own and whether to live in a com-
mercial housing development 4
Discussion 4 There is a clear and statistically significant positive correlation between living in commercial housing
developments and owning and using cars in Tecamac and Ixtapaluca, two suburban municipalities of
Mexico City. Given the rapid shift in housing production from an informal process to a formal one,
whether, how, and to what extent this relationship is causal is of great policy concern. Congestion, in Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 185 particular, has large social and economic costs; the average one-way commute lasted 72 minutes for
residents of the two municipalities in 2007 (INEGI 2007). Furthermore, due to the federal role in fi-
nancing mortgages for commercial housing developments, the central government has unusually strong
control over the design, location, and regulation of future commercial housing developments like those
in Ixtapaluca and Tecamac. While this study cannot definitively quantify a causal relationship, it does
suggest that one exists. This section discusses the evidence for the positive relationship being causal and
then considers why commercial housing might produce higher rates of car ownership and use. There are two principal reasons that living in commercial housing developments is likely to corre-
late with higher car ownership and use. Either the developments attract households with a stronger pref-
erence for driving and car ownership or there is something about the design and location of the develop-
ments that is conducive to driving and car ownership. The descriptive statistics and regression results
strongly indicate that commercial housing developments attract wealthier households with observed
preferences for driving. Including income, home ownership, and computer ownership in the statistical
models reduces the average correlation between residing in a commercial housing development and car
ownership and weekday driving by around 40 percent. There may also be unobserved variables that cor-
relate with car ownership and driving as well as living in a commercial housing development, and bias
estimates of the relationship between commercial housing development and car ownership and driving. Possibilities include personal preference, job location, and social status above and beyond what might
be captured by income, home ownership, educational attainment, and computer ownership. However,
the results from the nested logit model suggest that this is not the case. 4
Discussion Furthermore, the strength of the
observed relationship and the rapid and exogenous shift in housing production—since selection into a
commercial development is largely determined by whether a household qualifies for a loan, rather than
residential preference—both suggest that there is something about the commercial housing develop-
ments that encourages higher rates of car ownership and use. Since the analysis draws data from two similar suburban municipalities and includes a fixed effect
for one of them, any of the observed relationship between the physical form of commercial housing
developments and car ownership and use is related to local features, like design or local transit access,
rather than regional ones, like accessibility to job centers. There are also likely to be differences related
to regional accessibility, as found in a study of car use in metropolitan Mexico City (Guerra 2014), but
this study would not capture them. Unfortunately, the analysis cannot provide insight into which aspects of commercial housing devel-
opments correlate most strongly with higher car ownership or use. There is simply not enough variation
in the design and land use of commercial housing developments. Furthermore, this shortcoming is com-
mon in studies of the relationship between the built environment and travel, either because a variable
like neighborhood population density serves as a proxy (intentional or not) for other measures (see for
example Brownstone and Golob 2009), or the measure of the built environment is a qualitative dummy
variable for a neighborhood type (see for example Handy, Cao, and Mokhtarian 2005). Given the evidence that there may be a causal link between the local features of commercial hous-
ing developments and car ownership and car use, it is worth considering how peripheral commercial
housing developments differ from nearby informal neighborhoods. Both tend to contain small, fairly
uniform, cement housing units on small lots. Both are primarily constructed on former communal
farmland. In Tecamac and Ixtapula, commercial developments tend to be more densely populated. Per-
haps the most apparent difference is in how these neighborhoods develop over time. Informal neighbor-
hoods grow organically over time, with residents and entrepreneurs building housing units and shops to
suit their needs. By contrast, residents move into fully constructed single-use residential neighborhoods
in commercial housing developments, although residents often convert units or parking spaces into JOURNAL OF TRANSPORT AND LAND USE 8.2 186 shops over time. 4
Discussion With limited access points, furthermore, informal transit, which serves around half of
all metropolitan trips (INEGI 2007), is generally less well-integrated into commercial housing develop-
ments than informal neighborhoods. Geo built the 11,000-unit Villas de Santa Barbara in Ixtapaluca
with just three access points (Peralta and Hofer 2006). Rather than providing stations at the center of a
10,000-unit development, informal transit operators will generally pick up and drop off passengers at
the edges. Ample parking likely plays a particularly important role. Roadways are wider than in informal
settlements allowing for more on-street parking and each unit has an off-street parking space. Many
households would not voluntarily choose to expend time, land, and money to create a parking space
from scratch—indeed many take the time and effort to convert parking into some other use—but the
default suburban housing product in Mexico City now comes with parking, when previously it did not. If indeed, “[m]inimum parking requirements are truly a great planning disaster—perhaps the greatest
of all time [in the United States] (Shoup 2005, p. 218),” the potential costs in rapidly motorizing met-
ropolitan areas like Mexico City are alarming. More than three in four children born today will live in a
city in a developing country (United Nations Population Division 2007). Most will live in newly form-
ing neighborhoods in rapidly motorizing suburban locations like Tecamac and Ixtapaluca. The parking
policies adopted by these metropolitan areas will have tremendous economic, social, and environmental
costs, both locally and globally. 5
Conclusion While it is Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 187 unclear precisely how Infonavit or housing production will respond to the growing and often conflicting
interests and priorities of private homebuilders, workers, and the public sector, it is clear that they are
changing. This study finds that, from the perspective of limiting the growth in car ownership and use,
the local design and land use of commercial housing developments matter. In addition to promoting
more centralized, vertical development, policymakers should pay more attention to designing and retro-
fitting peripheral commercial housing developments in ways that do less to encourage driving. Theory,
previous findings, and differences with informal settlements suggest that fewer parking spaces, more
connected street grids, and better local transit access would help. 5
Conclusion 5 This paper examined the correlation between living in a commercial housing development and owning
and driving a car across 1500 households in two suburban municipalities in Mexico City. Accounting
for income, proxies for wealth, household composition, and geography, residents of an average house-
hold living in commercial housing developments are 61 percent more likely to own a car and 87 percent
more likely to own two or more cars than those of a similar household in a neighboring informal settle-
ment. They also generate significantly more private vehicle travel on an average weekday. While some
of the differences in car ownership and use may relate to unobserved household preferences, the rapid
shift in housing production and the results of a nested logit model of car ownership and residential lo-
cation suggest that something about the new commercial housing developments is more conducive to
car ownership and use. Since the sample only includes households from two similar suburban munici-
palities and the models include regional controls, the observed statistical correlations likely stem from
differences in local land use and design. The shift in housing policy has almost certainly increased car
ownership and use. In the wake of the Great Recession, this policy is starting to change. Hundreds of thousands of new
homes sit vacant throughout Mexico. Not only have new homes not been purchased, each year Infona-
vit and other lenders foreclose on tens of thousands of homes (Juarez 2013). Homes in the most periph-
eral new developments have been particularly vulnerable. Housing fund administrators have responded
by cutting lending and reorienting the fund’s portfolio to the renovation and resale of existing proper-
ties. Meanwhile, the national homebuilders have hit rock bottom. Geo and Urbi, both publicly traded,
failed to report second quarter earnings in 2013 and were suspended from trading on the Mexican stock
exchange (Purnell 2013). Days earlier, Homex, which reported earnings, experienced the steepest drop
in value of any highly traded company on the Bloomberg World Index (Levin 2013). Workers’ unions
are demanding better, larger housing in more central locations, greater leniency for unemployed work-
ers with mortgages, and expanded access to credit for poorer households. The new administration of
President Peña Nieto has made expanding access to credit and reorienting lending to support vertical
construction in more central locations public-policy priorities (La Crónica de Hoy 2013). References Angrist, J. D., and J. S. Pischke. 2008. Mostly Harmless Econometrics: An Empiricist’s Companion, 1st ed. Princeton, NJ: Princeton University Press. Bhat, C. R., and J. Y. Guo. 2007. A comprehensive analysis of built environment characteristics on
household residential choice and auto ownership levels. Transportation Research Part B: Methodologi-
cal 41: 506–526. doi:10.1016/j.trb.2005.12.005 Boarnet, M., and R. Crane. 2001. The influence of land use on travel behavior: Specification and es-
timation strategies. Transportation Research Part A: Policy and Practice 35: 823–845. doi:10.1016/
S0965-8564(00)00019-7 Boarnet, M., and S. Sarmiento. 1998. Can land-use policy really affect travel behavior? A study of
the link between non-work travel and land-use characteristics. Urban Studies 35: 1155–1169. doi:10.1080/0042098984538 Bound, J., D. A. Jaeger, and R. M. Baker. 1995. Problems with instrumental variables estimation when
the correlation between the instruments and the endogenous explanatory variable is weak. Journal of
the American Statistical Association 90: 443–450. doi:10.1080/01621459.1995.10476536 Brownstone, D. 2008. Key Relationships Between the Built Environment and VMT (Special Report No. 298), Driving and the Built Environment: The Effects of Compact Development on Motorized
Travel, Energy Use, and CO2 Emissions. Washington, DC: Transportation Research Board. y
p
p
p
298), Driving and the Built Environment: The Effects of Compact Development on Motorized
Travel Energy Use and CO2 Emissions Washington DC: Transportation Research Board Brownstone, D., and T. F. Golob. 2009. The impact of residential density on vehicle usage and energy
consumption. Journal of Urban Economics 65: 91–98. doi:10.1016/j.jue.2008.09.002 Cao, X., P. Mokhtarian, and S. Handy. 2009. Examining the impacts of residential self‐selec-
tion on travel behavior: A focus on empirical findings. Transport Reviews 29: 359–395. doi:10.1080/01441640802539195 Cervero, R. 2007. Transit-oriented development’s ridership bonus: A product of self-selection and pub-
lic policies. Environment and Planning A 39: 2068–2085. Chatman, D. 2009. Residential choice, the built environment, and nonwork travel: Evidence using new
data and methods. Environment and Planning A 41: 1072–1089. Crane, R. 1996. On form versus function: Will the new urbanism reduce traffic, or increase it? Journal
of Planning Education and Research 15: 117 –126. doi:10.1177/0739456X9601500204hl Crane, R. 2000. The influence of urban form on travel: An interpretive review. Journal of Planning Lit-
erature 15: 3–23. doi:10.1177/08854120022092890 Dowall, D. E., and D. Wilk. 1989. Population growth, land development, and housing in Mexico City
(Working Paper No. 502). University of California at Berkeley, Institute of Urban and Regional
Development, Berkeley. Ewing, R., and R. Cervero. 2010. References Travel and the built environment: A meta-analysis. Journal of the
American Planning Association 76: 265–294. doi:10.1080/01944361003766766
G
E
4 Th b l
d
M
C
h
l
h
h Ewing, R., and R. Cervero. 2010. Travel and the built environment: A meta-analysis. Journal of the
American Planning Association 76: 265–294. doi:10.1080/01944361003766766 g
Guerra, E. 2014. The built environment and car use in Mexico City is the relationship changing o Guerra, E. 2014. The built environment and car use in Mexico City is the relationship changing over 188 JOURNAL OF TRANSPORT AND LAND USE 8.2 Handy, S., X. Cao, and P. Mokhtarian. 2005. Correlation or causality between the built environment
and travel behavior? Evidence from Northern California. Transportation Research Part D: Transport
and Environment 10: 427–444. doi:10.1016/j.trd.2005.05.002 Hanley Wood, LLC. 2013. 2010 Builder 100 [WWW Document]. URL: http://www.builderonline. com/builder100/2010.aspx. INEGI. 1994. Encuesta Origen–Destino de los Viajes de los Residentes de la Zona Metropolitana del Valle de
México 1994. Mexico City: Instituto Nacional de Estadística, Geografía e Informática. INEGI. 2007. Encuesta Origen–Destino de los Viajes de los Residentes de la Zona Metropolitana del Valle de
México 2007. Mexico City: Instituto Nacional de Estadística, Geografía e Informática. INEGI. 2013. Instituto Nacional de Estadística y Geografía [WWW Document]. URL: http://www. inegi.org.mx/. Juarez, E. 2013. Sedatu busca atacar abandono de vivienda. El Economista. Juarez, E. 2013. Sedatu busca atacar abandono de vivienda. El Economista. La Crónica de Hoy. 2013. Ordena Peña Nieto Revisar Requisitos Para Otorgar Créditos de Vivienda. Mex-
ico City: La Crónica de Hoy. LaLonde, R. J. 1986. Evaluating the econometric evaluations of training programs with experimental
data. The American Economic Review 76(4): 604–620. Levine, J. 2006. Zoned Out: Regulation, Markets, and Choices in Transportation and Metropolitan Land
Use. Washington, DC: RFF Press. in, J. 2013. Homex posts world’s worst selloff on Mexican homebuilder’s loss. Bloomberg Business. Mokhtarian, P., and X. Cao. 2008. Examining the impacts of residential self-selection on travel be-
havior: A focus on methodologies. Transportation Research Part B: Methodological 42: 204–228. doi:10.1016/j.trb.2007.07.006 Monkkonen, P. 2011a. The housing transition in Mexico expanding access to housing finance. Urban
Affairs Review 47: 672–695. doi:10.1177/1078087411400381 f
Monkkonen, P. 2011b. Housing finance reform and increasing socioeconomic segregation in Mex-
ico. International Journal of Urban and Regional Research 36: 757–772. doi:10.1111/j.1468-
2427.2011.01085.x Monkkonen, P. 2011c. Do Mexican cities sprawl? Housing-finance reform and changing patterns of
urban growth. Urban Geography 32: 406–423. doi:10.2747/0272-3638.32.3.406 Nava, R. 2011. Vance, C., and R. Hedel. 2007. The impact of urban form on automobile travel: Disentangling causa-
tion from correlation. Transportation 34: 575–588. doi:10.1007/s11116-007-9128-6 Wooldridge, J. M. 2010. Econometric Analysis of Cross Section and Panel Data, second edition. Cam-
bridge, MA: The MIT Press. Has Mexico City’s shift to commercially produced housing increased car ownership and car use? Vance, C., and R. Hedel. 2007. The impact of urban form on automobile travel: Disentangling causa-
tion from correlation. Transportation 34: 575–588. doi:10.1007/s11116-007-9128-6
Wooldridge, J. M. 2010. Econometric Analysis of Cross Section and Panel Data, second edition. Cam-
bridge, MA: The MIT Press. Vance, C., and R. Hedel. 2007. The impact of urban form on automobile travel: Disentangling causa-
tion from correlation. Transportation 34: 575–588. doi:10.1007/s11116-007-9128-6
Wooldridge, J. M. 2010. Econometric Analysis of Cross Section and Panel Data, second edition. Cam- References Geo es líder en venta de casas con crédito Infonavit [WWW Document]. BMV Traders. URL: http://bmvtraders.com/2011/01/geo-es-lider-en-venta-de-casas-con-credito-infonavit/. Pardo, M. del C., and E. Velasco Sánchez. 2006. El Proceso de Modernización en el Infonavit 2001-2006:
Estrategia, redes y liderazgo. Mexico, DF: Colegio de México. Peralta, B. G., and A. Hofer. 2006. Housing for the working class on the periphery of Mexico City: A
new version of gated communities. Social Justice 33: 129–141. Pickrell, D. 1999. Transportation and land use. In Transportation Economics and Policy Handbook, edited
by J. Gomez-Ibanez, W. B. Tye, and C. Winston. Washington, DC: Brookings Institution Press. Purnell, N. 2013. Homex jumps after government pledges support: Mexico City mover. Bloomberg
Business. Salon, D. 2009. Neighborhoods, cars, and commuting in New York City: A discrete choice approach. Transportation Research Part A: Policy and Practice 43: 180–196. doi:10.1016/j.tra.2008.10.002 Shoup, D. 2005. The High Cost of Free Parking, illustrated edition. Chicago, IL: American Planning
Association. United Nations Population Division. 2007. World Urbanization Prospects: The 2007 Revision Popula-
tion Database [WWW Document]. URL: http://esa.un.org/unup/ (accessed 8.25.11). 189
|
https://openalex.org/W4243915947
|
https://www.researchsquare.com/article/rs-35320/latest.pdf
|
English
| null |
Relationship of attitudes toward uncertainty and preventive health behaviors with breast cancer screening participation
|
Research Square (Research Square)
| 2,020
|
cc-by
| 8,697
|
Relationship of attitudes toward uncertainty and
preventive health behaviors with breast cancer
screening participation Miho Satoh
(
miho.sth@mail.com
)
Yokohama Shiritsu Daigaku
https://orcid.org/0000-0001-8939-5595 Fukushima Kenritsu Ika Daigaku Abstract Background: Early detection of breast cancer is effective for prolonging survival, but the participation rate
in breast cancer screening among target Japanese women remains low. This study examined the
relationships between tendencies in decision-making under conditions of uncertainty, health behaviors,
demographics, and breast cancer screening participation in Japanese women. Methods: The study population consisted of the 2,945 households that completed the 2017 Keio
Household Panel Survey (KHPS). Data were obtained from the KHPS for women aged 40 years or older. Breast cancer screening participation in the past year, risk aversion, time preference, health behaviors
(e.g., smoking, alcohol consumption, and medical treatment received in the past year), and demographic
variables were analyzed. Results: Data from 708 women were analyzed. Among the respondents, 28.8% had attended breast
cancer screening in the past year. Factors found to significantly contribute to breast cancer screening
participation included higher risk aversion (odds ratio [OR], 2.34; 95% confidence interval [CI] = 1.03-5.32;
p = 0.043), medical treatment received in the past year (OR, 1.56; 95% CI = 1.06-2.30; p = 0.026), higher
self-rated health (OR, 1.47; 95% CI = 1.18-1.83; p = 0.001), living above the poverty line (OR, 2.31; 95% CI =
1.13-4.72; p = 0.022), and having children (OR, 1.57; 95% CI = 1.02-2.42; p = 0.042). Factors not
significantly associated with screening participation were smoking (OR, 0.20; 95% CI = 0.10-0.42; p <
0.000), alcohol consumption (OR, 0.56; 95% CI = 0.37-0.86; p = 0.007), being self-employed (OR, 0.22; 95%
CI = 0.10-0.46; p < 0.000), and being unemployed (OR, 0.48; 95% CI = 0.26-0.90; p = 0.022). No significant
relationship was observed between time preference and screening participation. Conclusions: The results indicate that women who recognize the actual risk of developing breast cancer
or have high awareness of breast cancer prevention tend to participate in breast cancer screening. Barriers to screening participation are not working for an organization that encourages screening and low
income. Conclusions: The results indicate that women who recognize the actual risk of developing breast cancer
or have high awareness of breast cancer prevention tend to participate in breast cancer screening. Barriers to screening participation are not working for an organization that encourages screening and low
income. Research article License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Version of Record: A version of this preprint was published at BMC Women's Health on April 21st, 2021. See the published version at https://doi.org/10.1186/s12905-021-01317-1. Page 1/20 Page 1/20 Page 1/20 Background Breast cancer is the most common cancer among women worldwide [1]. In Japan, it is the leading cause
of cancer mortality among women aged 30 to 64 years [2]. Both the number of women who are
diagnosed with breast cancer and the number who die of the disease is increasing; 14,653 per 100,000
population died of breast cancer in 2018 in Japan [2]. Early detection and early treatment of breast cancer can lead to favorable prognosis [3, 4]. The 5-year net
survival rate for breast cancer in the United Kingdom is reported to be 97.9% for stage 1 disease and
89.6% for stage 2, but this decreases to 72.0% for stage 3 disease and 26.2% for stage 4 [5]. In the United
States, the 5-year survival rate is 98.8% for localized female breast cancer but 27.4% for female breast
cancer with distant metastases [6]. It is therefore critical to detect breast cancer early. Page 2/20 Page 2/20 Mammography is the most effective breast cancer screening tool and can help to decrease breast cancer
mortality by allowing earlier treatment [7-9]. Since 2004, the Japan Ministry of Health, Labour and Welfare
has recommended mammography screening for women aged 40 years or over every 2 years [10]. In the
United States in 2016, 72.5% of women aged 40 years or over had undergone mammography screening
within the past 2 years [11]; in Western industrialized countries, the Organization for Economic Co-
operation and Development reported that 70-80% of women underwent mammography within the past 5
years [12]. However, the participation rate among Japanese women targeted for breast cancer screening
continues to be low. In 2016, only 44.9% of the target women had undergone mammography screening
within the past 2 years [13], falling short of the targeted participation rate of 50% set in the Basic Plan to
Promote Cancer Control Programs [14]. Effective strategies are therefore urgently needed to increase the
number of target women who undergo breast cancer screening in Japan. Previous studies have revealed that breast cancer screening participation is associated with health-
related lifestyle and behavioral factors, such as knowledge or health literacy about breast cancer [15-17],
smoking [18, 19], alcohol consumption [19, 20], physical activity level [21], and self-rated health (SRH) [20,
22]. Economic factors are also barriers to breast cancer screening participation [23, 24]. Background According to Japan’s
Census on Cancer Control [25], one of the reasons for women not attending cancer screening was
“economic burden” (12.6%). Several other demographic factors are also associated with breast cancer
screening participation, including higher level of education completed, lower occupational class [26, 27],
regular visits to doctors [28, 29], living with a partner [30], having children [31], and older age [24, 28, 29]. Determinants of preventive health behaviors are receiving increased attention in the field of behavioral
economics [32, 33]. Preventive health behaviors and the use of preventive medical care to treat fear of
disease and death are likely affected by tendencies or preferences in behavior or decision-making under
conditions of uncertainty [34, 35]. People often decide to use disease prevention services in consideration
of intertemporal tradeoff—whether they prefer receiving an immediate small reward or a delayed larger
reward—as explained by time preference theory [34, 36]. For example, the decision to participate in cancer
screening is influenced by psychological value, that is, time preference as to whether to place emphasis
on the present or the future. Another tendency in behavior or decision-making under conditions of uncertainty is risk aversion. This
concerns the attitude toward risk that people have when deciding to use disease prevention services to
decrease the probability of disease or death [34, 35]. Even a highly satisfactory behavior may be viewed
as harmful to health and thus avoided. Therefore, participation in cancer screening may be affected by
an individual’s attitude toward confronting or avoiding health problems caused by cancer [34, 35]. Those
who have a higher time preference tend to place value on the current situation without considering the
future and therefore prefer not to engage in preventive health behaviors. Those who are likely to avoid risk
tend to actively engage in preventive health behaviors [34, 35, 37]. Although decision-making tendencies Page 3/20 Page 3/20 under conditions of uncertainty are a crucial factor that could predict preventive health behavior, there are
few empirical research studies addressing this topic. under conditions of uncertainty are a crucial factor that could predict preventive health behavior, there are
few empirical research studies addressing this topic. Communities in Japan are struggling with improving breast cancer screening rates. Given that women
who attend breast cancer screening tend to have healthy habits, strategies have usually been proposed
for people with poorer health behaviors or who are not interested in health behaviors. Background However, such
strategies would not be fully effective considering the low rate of breast cancer screening participation in
Japan. More effective tactics are needed both to encourage women to participate in breast cancer
screening in order to decrease their risk of breast cancer-related health problems in the future and to
improve screening participation rates. Against this background, in this study we examined the relationship between decision-making tendencies
under conditions of uncertainty and breast cancer screening participation among Japanese women. We
also analyzed the relationship of health behaviors and demographics with breast cancer screening to
explore more effective strategies for increasing the breast cancer participation rate in Japanese women. Variables The primary outcome was breast cancer screening participation in the past year (participated or did not
participate). Explanatory variables were variables related to risk aversion, time preference, health behaviors, and
demography. Risk aversion was assessed using a question about deciding whether to take an umbrella
out depending on the percentage likelihood of rain in a weather forecast. Time preference was measured
using a question about the relative value placed on smaller immediate rewards or larger later rewards
[40]: “How satisfied are you with receiving JPY 10,000 after 13 months instead of receiving it after 1
month?” (1 “interest rate = -5%” to 8 “interest rate = 40%”). The KHPS asked about the following health-
related lifestyle variables: alcohol consumption (yes or no), smoking status (smoker or non-smoker),
weekly physical exercise (yes or no), sleep duration (hours per night), and medical treatment received in
the past year (yes or no). SRH (1 “poor” to 5 “excellent”) was also evaluated. The survey also asked about
the following employment characteristics: working hours per week, relative poverty (yearly household
disposable income < JPY 122,000 or not), and type of employment (regular, non-regular, self-employed, or
unemployed). Demographic attributes obtained included gender (male or female), year of birth, marital
status (married or single), children (none or ≥ 1), and level of education completed (junior or senior high
school, junior college or vocational school, or university or graduate school). Residential area was also
recorded. Ratios of time preference and risk aversion were obtained as percentages, with a lower time preference
ratio indicating lower time preference and a higher risk aversion ratio indicating higher risk aversion. To
account for relative poverty, yearly household disposable income was calculated by dividing household
income by the square root of household size and then dividing participants into two groups: “living below
the poverty line” (yearly household disposable income less than JPY 122,000; this is the median yearly
household disposable income in Japan and is defined as the poverty line) and “living above the poverty
line” (yearly household disposable income equal to or more than JPY 122,000). Age was calculated from
year of birth. Methods Data from the Keio Household Panel Survey (KHPS) were used in this preliminary cohort study. The KHPS
survey, conducted by Keio University, provides representative data from panel surveys of Japanese
households [39]. Briefly, the KHPS was approved by the Ministry of Education, Culture, Sports, Science and Technology in
Japan and has been conducted annually since 2004, surveying a total of 4,005 households nationwide. A
stratified two-stage sampling method is used for the survey. KHPS respondents in 2004 were men and
women born between 1935 and 1984. The demographic characteristics of the respondents are
representative of Japanese households nationwide. The KHPS questionnaire includes items on place of
residence, basic demographic data (e.g., year of birth, level of education completed, and gender),
employment status, health status, health-related behaviors, and household economic status. All participants in the KHPS were informed about the purpose of the research, potential use of their data,
the survey methodology, data anonymity, strict protection of individual data, and secondary use of data. The KHPS data are collected by mail, Internet survey, and visit survey by researchers. Applications for
secondary use of these data must be submitted to and approved by Keio University, with assurance
provided that ethical considerations will be met. Before we started this study, Keio University approved
our use of the KHPS data in accordance with our application and assurance that all ethical
considerations will be met. Accordingly, Keio University then provided us with the KHPS data sets. The empirical analysis in this study primarily used the 2017 wave of the KHPS and partially relies on the
2004 wave for basic demographic data. The sample analyzed included women aged 40 years or older,
which corresponds with the recommended age for breast cancer screening in Japan. Page 4/20 Page 4/20 The aim of the study was to explore the relationships between decision-making tendencies under
conditions of uncertainty, health behaviors, demographics, and likelihood of breast cancer screening
participation in Japanese women. Data analysis Frequency (percentage) and descriptive statistics (mean and standard deviation [SD]) were first
confirmed for all variables. Chi-squared tests were then performed and adjusted standardized residuals
(ASRs) were calculated in a preliminary analysis to compare those who participated in breast cancer
screening with those who did not according to age, child status, marital status, level of education
completed, type of employment, relative poverty, medical treatment received in the past year, smoking
status, alcohol consumption, and weekly physical exercise. Furthermore, t-tests were performed as a Page 5/20 Page 5/20 preliminary analysis to compare differences in risk aversion, time preference, SRH, and sleep duration
between the groups. Subsequently, logistic regression analysis was performed and odds ratios were
calculated to identify the factors associated with breast cancer screening participation. Variables that
were significant in the univariate analysis were entered into the model as exploratory variables. preliminary analysis to compare differences in risk aversion, time preference, SRH, and sleep duration
between the groups. Subsequently, logistic regression analysis was performed and odds ratios were
calculated to identify the factors associated with breast cancer screening participation. Variables that
were significant in the univariate analysis were entered into the model as exploratory variables. All statistical analyses were conducted using SPSS Statistics 25.0 for Mac (IBM Corp., Armonk, NY, USA). Statistical significance was set at p < 0.05 (two-tailed). Results Demographic characteristics Demographic characteristics Conversely, breast cancer screening
participation was less likely among those who were self-employed (ASR = 5.5) and those who were living
below the poverty line (ASR = 3.1). Those who had participated in breast cancer screening during the past year had a significantly shorter
sleep duration (p = 0.005), higher SRH (p = 0.025), and greater risk aversion (p = 0.022) than those who
did not. No significant difference was observed in time preference between those who did and did not
attend for breast cancer screening. Demographic characteristics In 2017, the KHPS questionnaire was distributed to 2,945 households, of which 2,729 responded
(response rate: 92.7%). A total of 708 questionnaires had complete responses and met the inclusion
criterion (answered by women aged ≥ 40 years). These responses were analyzed in the course of the
present study (valid response rate: 24.0%). Table 1 shows the descriptive statistics of the sample. Page 6/20 Of the respondents, almost a third underwent breast cancer screening in the past year. Those in the age
ranges of 60-69 years and 50-59 years comprised just over half of all respondents, and those aged 40-49
years comprised almost a quarter. Most lived with families; approximately three-quarters were married
and over half had children. Most had not completed university or graduate school. Relatively few were
regular employees whereas more than a third were unemployed. Analysis of health behavior variables
revealed that most were non-smokers, more than two-thirds did not consume alcohol, almost a quarter Page 7/20 Page 7/20 performed weekly exercise, and almost half had received medical treatment in the past year. Mean time
preference was 0.17 ± 0.15 and mean risk aversion was 0.60 ± 023. performed weekly exercise, and almost half had received medical treatment in the past year. Mean time
preference was 0.17 ± 0.15 and mean risk aversion was 0.60 ± 023. Univariate analysis of explanatory variables and breast cancer screening participation Univariate analysis of explanatory variables and breast cancer screening participation Chi-squared tests and t-tests were performed to examine the differences in explanatory variables
according to breast cancer screening participation (Tables 2 and 3). Chi-squared tests and t-tests were performed to examine the differences in explanatory variables
according to breast cancer screening participation (Tables 2 and 3). Chi-squared tests showed significant differences in breast cancer screening participation according to
family type (χ2 = 3.91, p < 0.048), child status (χ2 = 13.37, p < 0.000), level of education completed (χ2 =
11.18, p < 0.004), type of employment (χ2 = 48.75, p < 0.000), relative poverty (χ2 = 9.61, p < 0.002),
smoking status (χ2 = 32.06, p < 0.000), and alcohol consumption (χ2 = 16.77, p < 0.000). Demographic characteristics Breast cancer
screening participation during the past year was more likely among those who lived with family (ASR =
2.0), had children (ASR = 3.7), had completed junior college or vocational school (ASR = 3.2), had
precarious employment (ASR = 5.4), had received medical treatment in the past year (ASR = 2.3), were a
non-smoker (ASR = 5.7), and did not consume alcohol (ASR = 4.1). Conversely, breast cancer screening
participation was less likely among those who were self-employed (ASR = 5.5) and those who were living
below the poverty line (ASR = 3.1). Chi-squared tests showed significant differences in breast cancer screening participation according to
family type (χ2 = 3.91, p < 0.048), child status (χ2 = 13.37, p < 0.000), level of education completed (χ2 =
11.18, p < 0.004), type of employment (χ2 = 48.75, p < 0.000), relative poverty (χ2 = 9.61, p < 0.002),
smoking status (χ2 = 32.06, p < 0.000), and alcohol consumption (χ2 = 16.77, p < 0.000). Breast cancer
screening participation during the past year was more likely among those who lived with family (ASR =
2.0), had children (ASR = 3.7), had completed junior college or vocational school (ASR = 3.2), had
precarious employment (ASR = 5.4), had received medical treatment in the past year (ASR = 2.3), were a
non-smoker (ASR = 5.7), and did not consume alcohol (ASR = 4.1). Conversely, breast cancer screening
participation was less likely among those who were self-employed (ASR = 5.5) and those who were living
below the poverty line (ASR = 3.1). Chi-squared tests showed significant differences in breast cancer screening participation according to
family type (χ2 = 3.91, p < 0.048), child status (χ2 = 13.37, p < 0.000), level of education completed (χ2 =
11.18, p < 0.004), type of employment (χ2 = 48.75, p < 0.000), relative poverty (χ2 = 9.61, p < 0.002),
smoking status (χ2 = 32.06, p < 0.000), and alcohol consumption (χ2 = 16.77, p < 0.000). Breast cancer
screening participation during the past year was more likely among those who lived with family (ASR =
2.0), had children (ASR = 3.7), had completed junior college or vocational school (ASR = 3.2), had
precarious employment (ASR = 5.4), had received medical treatment in the past year (ASR = 2.3), were a
non-smoker (ASR = 5.7), and did not consume alcohol (ASR = 4.1). Discussion This study investigated the relationships between decision-making tendencies under conditions of
uncertainty, health behaviors, demographics, and breast cancer screening attendance to explore
strategies that would be more effective than those currently implemented for increasing the participation
rate of Japanese women in breast cancer screening. The results showed that those with higher risk
aversion tended to participate in screening. However, time preference was not observed to significantly
affect participation. Women who underwent breast cancer screening were found to have healthy
preventive behaviors, such as not smoking, not drinking alcohol, and having received medical treatment
in the past year. Higher SRH was also associated with breast cancer screening participation. Analysis of
demographic characteristics revealed that the following factors were barriers to breast cancer screening:
being self-employed, being unemployed, and living in relative poverty. Furthermore, having children was
positively associated with participation. This study investigated the relationships between decision
uncertainty, health behaviors, demographics, and breast c
strategies that would be more effective than those current
rate of Japanese women in breast cancer screening. The r
aversion tended to participate in screening. However, time
affect participation. Women who underwent breast cancer
preventive behaviors, such as not smoking, not drinking al
in the past year. Higher SRH was also associated with bre
demographic characteristics revealed that the following fa
being self-employed, being unemployed, and living in relat
positively associated with participation. Association of explanatory variables with breast cancer screening participation The variables that were significantly associated with breast cancer screening participation in the
preliminary analysis (chi-squared tests and t-tests) were subjected to logistic regression analysis, with
age and region of residence used as control variables (Table 4). Page 9/20 Page 10/20
Factors found to significantly contribute to breast cancer screening participation in the past year included
being more risk averse (odds ratio [OR), 2.34; 95% confidence interval [CI] = 1.03-5.32; p = 0.043),
receiving medical treatment in the past year (OR, 1.56; 95% CI = 1.06-2.30; p = 0.026), having a higher
SRH (OR, 1.47; 95% CI = 1.18-1.83; p = 0.001), living above the poverty line (OR, 2.31; 95% CI = 1.13-4.72; p
= 0.022), and having children (OR, 1.57; 95% CI = 1.02-2.42; p = 0.042). Conversely, factors found not to
contribute significantly to breast cancer screening participation were being a smoker (OR, 0.20; 95% CI =
0.10-0.42; p < 0.000), alcohol consumption (OR, 0.56; 95% CI = 0.37-0.86; p = 0.007), being self-employed Page 10/20 Page 10/20 (OR, 0.22; 95% CI = 0.10-0.46; p < 0.000), and being unemployed (OR, 0.48; 95% CI = 0.26-0.90; p = 0.022). Time preference was not found to be significantly associated with breast cancer screening participation. (OR, 0.22; 95% CI = 0.10-0.46; p < 0.000), and being unemployed (OR, 0.48; 95% CI = 0.26-0.90; p = 0.022). Time preference was not found to be significantly associated with breast cancer screening participation. Characteristics of the sample in this study The rate of participation in breast cancer screening in the past year was low (28.8%) in our sample
compared with the rate of participation within the past 2 years obtained in the Comprehensive Survey of
Living Conditions in 2016 (44.9%) [13], even though there was just a 1-year difference in the survey
periods. Analysis of descriptive statistics indicated that most participants in the present study were non-
smokers and did not drink alcohol. Also, their average sleep duration was the same as that reported in the
National Health and Nutrition Survey 2016 [41]. Thus, we can view these women as likely to have
healthier behavior. On the other hand, the women in this study were less likely to have healthy behavior in
relation to weekly physical exercise compared with data from a census on physical fitness [42] showing
that 37.8-71.5% of women engaged in physical exercise on more than 1 day a week. Relationships between decision-making tendencies under conditions of uncertainty and breast cancer
screening participation Page 11/20
Our results indicate that women with higher risk aversion might actively participate in breast cancer
screening to decrease the risk of delayed cancer detection. There is increasing awareness that early
detection of breast cancer leads to more effective treatment and thus to a better prognosis. Therefore,
women with an accurate perception of breast cancer might undergo breast cancer screening to mitigate
their breast cancer risk. A meta-analytic review demonstrated that perceived risk is a predictor of the
likelihood of attending breast cancer screening [43]. Women who recognize the actual risk of developing
breast cancer or are anxious about developing breast cancer tend to participate in cancer screening [43,
44]. Therefore, the breast cancer screening participation rate could be increased by messages that
emphasize the benefits of attending screening, the necessity of screening for improving quality of life
even for women with breast cancer, and the risk of breast cancer being overlooked. Also, to increase participation, it might be effective to provide appropriate health education information about the
accuracy of breast cancer screening, advances in breast cancer treatment, and improvement of prognosis
after treatment. However, the association between risk aversion and breast cancer screening participation
has not yet been fully examined and results are not consistent across studies. Characteristics of the sample in this study Some studies have
reported that women with higher risk aversion do not participate in breast cancer screening to avoid the
risk of a diagnosis of breast cancer, which provokes anxiety or psychological stress [34, 45]. Moreover,
Sasaki and Ohtake suggest that individuals perceive the consequences of decision-making related to
breast cancer screening differently depending on whether they take a gain-framing (safety concerns) or
loss-framing (risk concerns) perspective [35]. For example, on the one hand, individuals who are risk
averse are unlikely to participate in breast cancer screening when considering the risky condition of
“having breast cancer but its treatment might not be successful”. On the other hand, individuals who are
risk-seeking are unlikely to participate in breast cancer screening because they are more tolerant of the
uncertain condition of “not having breast cancer, but it might be detected.” Their survey measured risk
aversion from only the perspective of loss-framing, but future studies should measure it from both the
loss-framing and gain-framing perspectives to examine more effective strategies for improving the breast
cancer screening participation rate. The results of the present study also showed that time preference did not significantly affect the
likelihood of breast cancer screening participation, unlike the findings of past studies [34, 35, 46]. However, a meta-analysis suggested that time preference is associated with addictive health behavior
(e.g., smoking, alcohol consumption, or drug use) and not with preventive health behaviors such as
attending cancer screenings or medical checkups [36]. Some studies have shown that the relationship of
time preference with breast cancer screening participation is weaker than that with other factors [45, 47]. Time preference may be reflected in impulsive, addictive, or emotional behaviors rather than considered
behaviors such as vaccination, medication compliance, or screening [36]. The effect of time preference
might depend on mood or thoughtfulness in decision-making or decision-taking behaviors [35, 36] but
remains incompletely understood. Further research is needed to clarify this effect. Relationship between health behaviors and breast cancer screening participation Smoking and alcohol consumption are risk factors for various cancers, including breast cancer [48]. Past
research has shown a positive association between high breast cancer knowledge and breast cancer
screening participation, and suggested individuals might be more likely to undergo breast cancer
screening if they are knowledgeable about breast cancer, including its risk factors, the usefulness of
mammography, and causes [17, 49, 50]. In addition, individuals who are interested in preventing health
problems or who actively engage in preventive health behaviors would be likely to engage more in healthy
behaviors that can reduce the risk of disease or death. In contrast, individuals who engage in unhealthy
behaviors such as smoking, alcohol consumption, or not attending medical checkups are less likely to
engage in preventive activities [51, 52]. In particular, women who usually engage in preventive health
behaviors would likely participate in breast cancer screening as a part of their preventive health behaviors
[53, 54]. The results of the present study might reflect this behavioral characteristic. The survey that Page 12/20 Page 12/20 provided the data analyzed in this study did not ask about knowledge of breast cancer; further research is
needed to clarify the associations of attending breast cancer screenings with health-related behaviors like
smoking and alcohol consumption and breast cancer knowledge. provided the data analyzed in this study did not ask about knowledge of breast cancer; further research is
needed to clarify the associations of attending breast cancer screenings with health-related behaviors like
smoking and alcohol consumption and breast cancer knowledge. Several studies have demonstrated that frequent visits to medical doctors could increase the
opportunities to recommend breast cancer screening and promote awareness of cancer control [20, 28,
29], whereby women who received medical treatment tended to undergo screening. The association
between higher SRH and participation in breast cancer screening supports the findings of previous
research [55, 56]. Individuals perceived as being in poorer physical health are less likely to attend health
checkups or screenings to avoid knowing the cause of their physical health status [57]. In the present
study, participants with lower SRH had high risk aversion, which might have led to their non-participation
in breast cancer screening. Relationships between demographic factors and breast cancer screening participation Regular employees have been shown to have the highest breast cancer screening participation rate [26,
58, 59], whereas unemployed and self-employed individuals are less likely to participate [58, 60, 61]. Employees of organizations are more likely to be exposed to recommendations for breast cancer
screening or attend organized breast cancer screenings, further promoting their participatory behavior. Self-employed or unemployed women likely have limited cancer control service benefits, so they miss
opportunities for screening. Self-employed women might also find it difficult to take time off to attend
screenings [62, 63]. This might explain the differences in breast cancer screening participation according
to employment status. Exposure to breast cancer screening opportunities or frequent recommendations
for breast cancer screening is likely dependent on living environment and socioeconomic status;
inequalities in either or both could lead to health disparities, and breast cancer is no exception. As such,
carefully designed recommendation strategies for breast cancer screening should be jointly implemented
by the government, medical facilities, and organizations. Social marketing communications could also be
effective, such as combining mass media, social network services, and individual mailings. Our analysis suggests that relative poverty should not be overlooked as a barrier to breast cancer
screening. Income and medical insurance have been shown to affect cancer screening participation [64]. Poverty tends to delay detection of breast cancer worldwide [65], and low income tends to lead to lower
breast cancer screening rates [23, 24, 66]. Free or discount vouchers for mammography have been shown
to significantly improve the breast cancer screening rate in Japan [67-70]. However, there are costs
involved in screening, so the provision of free or low-cost screening to targeted women should be
carefully considered. Individual screening recommendations and free vouchers would likely be effective
in encouraging breast cancer screening participation [71]. Strategies and educational programs should be
prepared that will help women recognize breast cancer risk and the significance of attending screenings
so that they can proactively participate in the screenings regardless of cost. However, as discussed
above, support is urgently needed for women with low income and limited education. Unfortunately,
Japan has no nationwide program for breast cancer screening, which is particularly disadvantageous for Page 13/20 Page 13/20 women in the lower socioeconomic groups and those with limited education. This study indicates that
employment status and income might be triggers for non-participation in breast cancer screening. Conclusions This study used representative data from a panel survey of Japanese households and showed that
women who recognize the actual risk of developing breast cancer and women with a high level of
awareness of the need to prevent breast cancer are likely to attend for breast cancer screening. Healthy
preventive behaviors were also associated with participation in breast cancer screening. However, being
self-employed, being unemployed, and living in relative poverty would be barriers to participation in
screening. Relationships between demographic factors and breast cancer screening participation Women with these risk factors need to be identified so that they are not left out of breast cancer
prevention programs. Empirical studies of the associations of breast cancer screening participation with
economic status and education level are needed so that effective measures can be implemented. Moreover, the findings of this study suggest that lower income might delay participation in breast cancer
screening. Further research is needed to understand the confounders of the association between income
and participation in breast cancer screening, such as educational level, accuracy of knowledge about
breast cancer, accessibility of information about breast cancer screening, the difficulties in taking time off
work for cancer screening and the cost burden of screening, after which effective strategies have to be
built. For example, an education program about breast cancer could be implemented in primary care to
ensure that no woman is without accurate knowledge of breast cancer screening. Alternatively, a public
awareness campaign could be developed to ensure that all women know that screening can help prevent
breast cancer. Finally, this study found that having children is associated with participating in breast cancer screening. Women with children have childcare responsibilities, so it is likely that their health awareness is already
high and that they undertake preventive health behaviors. This study has several limitations. First, it was preliminary in nature and used cross-sectional data to
investigate the associations of breast cancer screening participation with decision-making tendencies
under conditions of uncertainty and preventive health behaviors. As such, causality cannot be inferred. Second, the survey data were all self-reported, so the findings may have been affected by response bias
[72, 73]. Further investigations are needed using objective data. Third, the study participants were from
various regions of Japan, and more factors related to participants’ health status should be considered by
region. Fourth, risk aversion and time preference can be measured from various perspectives, but the
survey used only one questionnaire to address each of these variables. Further examination of the
relationship between these tendencies and breast cancer screening participation should use several types
of questionnaires to explore risk aversion and time preference. Acknowledgments We are grateful to the Keio University Panel Data Research Center for providing the KHPS data. Abbreviations Page 14/20 ASR: adjusted standardized residual; CI: confidence interval; KHPS: Keio Household Panel Survey; OR:
odds ratio; SD: standard deviation; SRH: self-rated health ASR: adjusted standardized residual; CI: confidence interval; KHPS: Keio Household Panel Survey; OR:
odds ratio; SD: standard deviation; SRH: self-rated health Declarations Ethics approval and consent to participate Consent for publication Not applicable. Availability of data and materials Ethics approval and consent to participate We received approval from the Keio University Panel Data Research Center to use the KHPS data for our
research (Approval No. 1322: Data ID 156-JHPS/KHPS2004-2018). The center also provided the data. According to Japan’s Ethical Guidelines for Epidemiological Research, this study did not require ethical
approval. The study complied with Keio University’s data use policy and Japan’s Ethical Guidelines for
Epidemiological Research. Author contributions MS conceptualized and carried out the study and wrote the manuscript. NS provided important scientific
comments on the study design, data analysis, and manuscript content. All authors contributed to and
have approved the final manuscript. Competing interests Competing interests The authors declare that they have no competing interests. The authors declare no significant financial support for this work that could have influenced its outcome. Author contributions Availability of data and materials The data are available from the corresponding author upon reasonable request. The data set supporting
the conclusions of this article is available from the Panel Data Research Center at Keio University with the
approval of this organization (Approval No. 1322: Data ID 156-JHPS/KHPS2004-2018). References Page 15/20
1. Siegel R, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018; doi:
10.3322/caac.21442. Page 15/20 Page 15/20 2. Ministry of Health, Labour and Welfare. Vital statistics. 2017. https://www.mhlw.go.jp/toukei/saikin/hw/jinkou/kakutei17/dl/00_all.pdf. Accessed 17 Mar 2020. (in Japanese). 3. Njor SH, Schwartz W, Blichert-Toft M, Lynge E. Decline in breast cancer mortality: how much is
attributable to screening? J Med Screen. 2015;22:20-7. 4. American Cancer Society. Cancer treatment & survivorship facts & figures. 2019-2021. 2019. https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/cancer-
treatment-and-survivorship-facts-and-figures/cancer-treatment-and-survivorship-facts-and-figures-
2019-2021.pdf. Accessed 10 Apr 2020. 4. American Cancer Society. Cancer treatment & survivorship facts & figures. 2019-2021. 2019. 5. Cancer Research UK. Breast cancer survival statistics. 2019. https://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-
type/breast-cancer/survival#ref-. Accessed 10 Apr 2020. 6. National Cancer Institute. Surveillance, Epidemiology, and End Results Program. 2019. https://seer cancer gov/statfacts/html/breast html Accessed 10 Apr 2020 6. National Cancer Institute. Surveillance, Epidemiology, and End Results Program. 2019. https://seer.cancer.gov/statfacts/html/breast.html. Accessed 10 Apr 2020. 7. Hamashima C, Hattori M, Honjo S, Kasahara Y, Katayama T, Nakai M, et al. The Japanese guidelines
for breast cancer screening. Jpn J Clin Oncol. 2016;46:482-92. (In Japanese). 8. Tonelli M, Connor GS, Joffres M, Dickinson J, Singh H, Lewin G, et al. Recommendations on
screening for breast cancer in average-risk women aged 40–74 years, CMAJ. 2011;183:1991-2001. 8. Tonelli M, Connor GS, Joffres M, Dickinson J, Singh H, Lewin G, et al. Recommendations on
screening for breast cancer in average-risk women aged 40–74 years, CMAJ. 2011;183:1991-2001. 9. Yuan Y, Vu K, Shen Y, Dickinson J, Winget M. Importance of quality in breast cancer screening
practice - a natural experiment in Alberta, Canada, BMJ Open. 2020;10:1-9. 9. Yuan Y, Vu K, Shen Y, Dickinson J, Winget M. Importance of quality in breast cancer screening
practice - a natural experiment in Alberta, Canada, BMJ Open. 2020;10:1-9. 10. Ministry of Health, Labour and Welfare. The measure for education in cancer prevention and cancer
screening. 2004. https://www.mhlw.go.jp/file/06-Seisakujouhou-10900000- 10. Ministry of Health, Labour and Welfare. The measure for education in cancer prevention and cancer
screening. 2004. https://www.mhlw.go.jp/file/06-Seisakujouhou-10900000-
Kenkoukyoku/0000111662.pdf. Accessed 10 Apr 2020. (In Japanese). 10. Ministry of Health, Labour and Welfare. The measure for education in cancer prevention and cancer
screening. 2004. https://www.mhlw.go.jp/file/06-Seisakujouhou-10900000-
Kenkoukyoku/0000111662.pdf. Accessed 10 Apr 2020. (In Japanese). 11. National Cancer Institute. Cancer trends progress report. 2016. https://progressreport.cancer.gov/. Accessed 10 Apr 2020. 11. National Cancer Institute. Cancer trends progress report. 2016. https://progressreport.cancer.gov/. Accessed 10 Apr 2020. 12. OECD Stat. Health status. 2019. https://stats.oecd.org/Index.aspx?ThemeTreeId=9. Accessed 10 Apr
2020. 12. OECD Stat. Health status. 2019. https://stats.oecd.org/Index.aspx?ThemeTreeId=9. Accessed 10 Apr
2020. 13. Ministry of Health, Labour and Welfare. Page 15/20 Comprehensive survey of living conditions. 2016. https://www.mhlw.go.jp/toukei/saikin/hw/k-tyosa/k-tyosa16/dl/16.pdf. Accessed 10 Apr 2020. (In
Japanese). 13. Ministry of Health, Labour and Welfare. Comprehensive survey of living conditions. 2016. https://www.mhlw.go.jp/toukei/saikin/hw/k-tyosa/k-tyosa16/dl/16.pdf. Accessed 10 Apr 2020. (In
Japanese). https://www.mhlw.go.jp/toukei/saikin/hw/k-tyosa/k-tyosa16/dl/16.pdf. Accessed 10 Apr 2020. (In
Japanese). 14. Ministry of Health, Labour and Welfare. The basic plan to promote cancer control programs. 2018. https://www.mhlw.go.jp/file/06-Seisakujouhou-10900000-Kenkoukyoku/0000196975.pdf. Accessed
10 Apr (In Japanese). 15. Guilford K, Mckinley E, Turner L. Breast cancer knowledge, beliefs, and screening behaviors of college
women: application of the health belief model. Am J Health Educ. 2017;48:256-263. 16. Hirai K, Sasaki S, Ohtake F. The relationship between breast cancer screening and health literacy in
breast cancer. Journal of Behavioral Economics and Finance. 2017;10:20-5. (In Japanese). Page 16/20 Page 16/20 17. Rakhshkhorshid M, Navaee M, Nouri N, Safarzaii F. The association of health literacy with breast
cancer knowledge, perception and screening behavior. Eur J Breast Health. 2018; doi:
10.5152/ejbh.2018.3757. 18. Bryan L, Westmaas L, Alcaraz K, Jemal A. Cigarette smoking and cancer screening underutilization
by state: BRFSS 2010. Nicotine Tob Res. 2014;16:1183-9. 19. Lagerlund M, Drake I, Wirfalt E, Sontrop JM, Zackrisson S. Health-related lifestyle factors and
mammography screening attendance in a Swedish cohort. Eur J Cancer Prev. 2015;24:44-50. 20. Eichholzer M, Richard A, Rohrmann S, Schmid SM, Leo C, Huang DJ, et al. Breast cancer screening
attendance in two Swiss regions dominated by opportunistic or organized screening. BMC Health
Serv Res. 2016; doi: 10.1186/s12913-016-1760-4. 21. Martin-Lopez R, Hernandez-Barrera V, De Andres AL, Garrido PC, De Miguel AG, Garcia RJ. Breast and
cervical cancer screening in Spain and predictors of adherence. Eur J Cancer Prev. 2010;19:239-45. 22. Schoofs J, Krijger K, Vandevoorde J, Devroey D. Health‑related factors associated with adherence to
breast cancer screening, J Midlife Health. 2017;8:63-9. 22. Schoofs J, Krijger K, Vandevoorde J, Devroey D. Health‑related factors associated with adherence to
breast cancer screening, J Midlife Health. 2017;8:63-9. 23. Gathirua-Mwangi W, Cohee A, Tarver WL, Marley A, Biederman E, Stump T, et al. Factors associated
with adherence to mammography screening among insured women differ by income levels. Women’s
Health Issues. 2018;28:462-9. 23. Gathirua-Mwangi W, Cohee A, Tarver WL, Marley A, Biederman E, Stump T, et al. Factors associated
with adherence to mammography screening among insured women differ by income levels. Women’s
Health Issues. 2018;28:462-9. 24. Henry KA, McDonald K, Sherman R, Kinney AY, Stroup AM. Page 15/20 Association between individual and
geographic factors and nonadherence to mammography screening guidelines. J Womens Health. 2014;23:664-74. 24. Henry KA, McDonald K, Sherman R, Kinney AY, Stroup AM. Association between individual and
geographic factors and nonadherence to mammography screening guidelines. J Womens Health. 2014;23:664-74. 25. Cabinet Office. Census on cancer control. 2016. https://survey.gov-online.go.jp/h28/h28-
gantaisaku/index.html. Accessed 10 Apr 2020. (In Japanese). 25. Cabinet Office. Census on cancer control. 2016. https://survey.gov-online.go.jp/h28/h28-
gantaisaku/index.html. Accessed 10 Apr 2020. (In Japanese). 26. Damiani G, Federico B, Basso D, Ronconi A, Bianchi CB, Anzellotti GM, et al. Socioeconomic
disparities in the uptake of breast and cervical cancer screening in Italy: a cross sectional study. BMC
Public Health. 2012; doi: 10.1186/1471-2458-12-99. 26. Damiani G, Federico B, Basso D, Ronconi A, Bianchi CB, Anzellotti GM, et al. Socioeconomic
disparities in the uptake of breast and cervical cancer screening in Italy: a cross sectional study. BMC
Public Health. 2012; doi: 10.1186/1471-2458-12-99. 27. Willems B, Bracke P. The education gradient in cancer screening participation: a consistent
phenomenon across Europe? Int J Public Health. 2018;63:93-103. 27. Willems B, Bracke P. The education gradient in cancer screening participation: a consistent
phenomenon across Europe? Int J Public Health. 2018;63:93-103. 28. Kempe KL, Larson RS, Shetterley S, Wilkinson A. Breast cancer screening in an insured population:
whom are we missing? Perm J. 2013;17:38-44. 28. Kempe KL, Larson RS, Shetterley S, Wilkinson A. Breast cancer screening in an insured population:
whom are we missing? Perm J. 2013;17:38-44. 29. Vyas A, Madhavan S, Kelly K, Metzger A, Schreiman J, Remick S. Predictors of self-reported
adherence to mammography screening guidelines in West Virginia women visiting a stationary
facility. W V Med J. 2014;110:20-6. 29. Vyas A, Madhavan S, Kelly K, Metzger A, Schreiman J, Remick S. Predictors of self-reported
adherence to mammography screening guidelines in West Virginia women visiting a stationary
facility. W V Med J. 2014;110:20-6. 30. Martin-Lopez R, Jimenez-Garcia R, Lopez-de-Andres A, Hernandez-Barrera V, Jimenez-Trujillo I, Gil-de-
Miguel A, et al. Inequalities in uptake of breast cancer screening in Spain: analysis of a cross-
sectional national survey. Public Health. 2013;127:822-7. 30. Martin-Lopez R, Jimenez-Garcia R, Lopez-de-Andres A, Hernandez-Barrera V, Jimenez-Trujillo I, Gil-de-
Miguel A, et al. Inequalities in uptake of breast cancer screening in Spain: analysis of a cross-
sectional national survey. Public Health. 2013;127:822-7. Page 17/20
31. Vetter L, Keller M, Bruckner T, Golatta M, Eismann S, Evers C, et al. Page 15/20 Adherence to the breast cancer
surveillance program for women at risk for familial breast and ovarian cancer versus overscreening:
a monocenter study in Germany. Breast Cancer Res Treat 2016;156:289-99. Page 17/20 32. Roberto CA, Kawachi I. Use of psychology and behavioral economics to promote healthy eating. Am
J Prev Med. 2014;47:832-7. 33. Thorgeirsson T, Kawachi I. Behavioral economics: merging psychology and economics for lifestyle
interventions. Am J Prev Med. 2013;44:185-9. 33. Thorgeirsson T, Kawachi I. Behavioral economics: merging psychology and economics for lifestyle
interventions. Am J Prev Med. 2013;44:185-9. 34. Goldzahl L. Contributions of risk preference, time orientation and perceptions to breast cancer
screening regularity. Soc Sci Med. 2017;185:147-57. 35. Sasaki S, Ohtake F. Behavioral economics in health and medical fields: decisions, biases, and
nudges. Journal of Behavioral Economics and Finance. 2018;11:110-20. (In Japanese). 35. Sasaki S, Ohtake F. Behavioral economics in health and medical fields: decisions, biases, and
nudges. Journal of Behavioral Economics and Finance. 2018;11:110-20. (In Japanese). 36. Chapman G. Short-term cost for long-term benefit: time preference and cancer control. Health
Psychology. 2005;4:41-8. 36. Chapman G. Short-term cost for long-term benefit: time preference and cancer control. Health
Psychology. 2005;4:41-8. 37. Anderson LR, Mellor JM. Predicting health behaviors with an experimental measure of risk
preference. J Health Econ. 2008;27:1260-74. 37. Anderson LR, Mellor JM. Predicting health behaviors with an experimental measure of risk
preference. J Health Econ. 2008;27:1260-74. 38. Ministry of Health, Labour and Welfare. Japan’s ethical guidelines for epidemiological research. 2017. https://www.mhlw.go.jp/file/06-Seisakujouhou-10600000-
Daijinkanboukouseikagakuka/0000153339.pdf. Accessed 10 Apr 2020. (In Japanese). 38. Ministry of Health, Labour and Welfare. Japan’s ethical guidelines for epidemiological research. 2017. https://www.mhlw.go.jp/file/06-Seisakujouhou-10600000-
Daijinkanboukouseikagakuka/0000153339.pdf. Accessed 10 Apr 2020. (In Japanese). kouseikagakuka/0000153339.pdf. Accessed 10 Apr 2020. (In Japanese). 39. Panel Data Research Center at Keio University. 2020. Japan Household Panel Survey. https://www.pdrc.keio.ac.jp/en/paneldata/datasets/jhpskhps/. Accessed 10 Apr 2020. 39. Panel Data Research Center at Keio University. 2020. Japan Household Panel Survey. https://www.pdrc.keio.ac.jp/en/paneldata/datasets/jhpskhps/. Accessed 10 Apr 2020. 40. Frederick S, Loewenstein G, O’Donoghue T. Time discounting and time preference: a critical review. J
Economic Literature. 2002;40:351-401. 40. Frederick S, Loewenstein G, O’Donoghue T. Time discounting and time preference: a critical review. J
Economic Literature. 2002;40:351-401. 41. Ministry of Health, Labour and Welfare. National health and nutrition survey 2016. 2017. https://www.mhlw.go.jp/content/10904750/000351576.pdf. Accessed 10 Apr 2020. (In Japanese). 41. Ministry of Health, Labour and Welfare. National health and nutrition survey 2016. 2017. https://www.mhlw.go.jp/content/10904750/000351576.pdf. Accessed 10 Apr 2020. (In Japanese). 42. doi: 10.1371/journal.pone.0194409. doi: 10.1371/journal.pone.0194409. 49. Wu T, Ronis D. Correlates of recent and regular mammography screening among Asian-American
women. J Adv Nurs. 2009;65:2434-46. 50. Guilford K, McKinley E, Turner L. Breast cancer knowledge, beliefs, and screening behaviors of
college women: application of the health belief model. Am J Health Educ. 2017; doi:
10.1080/19325037.2017.1316694. 51. Rakowski W, Clark MA, Ehrich B. Smoking and cancer screening for women ages 42–75:
associations in the 1990–1994 National Health Interview Surveys. Prev Med. 1999;29:487–95. 52. Fredman L. Cigarette smoking, alcohol consumption, and screening mammography among women
ages 50 and older. Prev. Med. 1999;28:407–17. 52. Fredman L. Cigarette smoking, alcohol consumption, and screening mammography among women
ages 50 and older. Prev. Med. 1999;28:407–17. 53. Kanetou C, Toyokawa S, Miyoshi Y, Suzuki T, Suyama Y, Kobayashi Y. The factors relate to cervical
cancer screening attendance among Japanese female wokers.Journal of Health and Welfare
Statistics. 2010;57:1-7.(In Japanese). 53. Kanetou C, Toyokawa S, Miyoshi Y, Suzuki T, Suyama Y, Kobayashi Y. The factors relate to cervical
cancer screening attendance among Japanese female wokers.Journal of Health and Welfare
Statistics. 2010;57:1-7.(In Japanese). 54. Elewonibi B, Miranda PY. Using mammograms to predict preventive health services behavior and
mortality in women. Prev Med Rep. 2017;5:27-32. 54. Elewonibi B, Miranda PY. Using mammograms to predict preventive health services behavior and
mortality in women. Prev Med Rep. 2017;5:27-32. 55. Lagerlund M, Drake I, Wirfalt E, Sontrop JM, Zackrisson S. Health-related lifestyle factors and
mammography screening attendance in a Swedish cohort study. Eur J Cancer Prev. 2015;24:44-50. 55. Lagerlund M, Drake I, Wirfalt E, Sontrop JM, Zackrisson S. Health-related lifestyle factors and
mammography screening attendance in a Swedish cohort study. Eur J Cancer Prev. 2015;24:44-50. 56. Gandhi PK, Gentry WM, Kibert Ⅱ JL, Lee EY, Jordan W, Bottorff MB, et al. The relationship between
four health-related quality-of-life indicators and use of mammography and Pap test screening in US
women. Qual Life Res. 2015;24:2113-28. 56. Gandhi PK, Gentry WM, Kibert Ⅱ JL, Lee EY, Jordan W, Bottorff MB, et al. The relationship between
four health-related quality-of-life indicators and use of mammography and Pap test screening in US
women. Qual Life Res. 2015;24:2113-28. 57. Wada T. Significance and utility of Ningen Dock from viewpoint of subjective health feeling. Official
Journal of Japan Society of Ningen Dock. 2019;33:675-82. (In Japanese). 57. Wada T. Significance and utility of Ningen Dock from viewpoint of subjective health feeling. Page 15/20 Ministry of Education, Culture, Sports, Science and Technology. Census about physical fitness 2016. 2017. https://www.mext.go.jp/sports/b_menu/toukei/chousa04/sports/1402343.htm. Accessed 10
Apr 2020. (In Japanese). 42. Ministry of Education, Culture, Sports, Science and Technology. Census about physical fitness 2016. 2017. https://www.mext.go.jp/sports/b_menu/toukei/chousa04/sports/1402343.htm. Accessed 10
Apr 2020. (In Japanese). 43. Katapodi MC, Lee KA, Facione NC, Dodd MJ. Predictors of perceived breast cancer risk and the
relation between perceived risk and breast cancer screening: a meta-analytic review. Prev Med. 2004;38:388-402. 44. Carman KG, Kooreman P. Probability perceptions and preventive health care. J Risk Uncertain. 2014;
49:43-71. 45. Picone G, Sloan F, Taylor D. Effects of risk and time preference and expected longevity on demand
for medical tests. J Risk Uncertain. 2004;28:39-53. 46. Fang H, Wang Y. Estimating dynamic discrete choice models with hyperbolic discounting, with an
appli-cation to mammography decisions. Int Econ Rev. 2015;56:565-96. 47. Levy AG, Micco E, Putt M, Armstrong K. Value for the future and breast cancer–preventive health
behavior. Cancer Epidemiol Biomakers Prev. 2006;15:955-60. 48. Katz D, Tengekyon AJ, Kahan NR, Calderon-Margalit R. Patient and physician characteristics affect
adherence to screening mammography: a population-based cohort study. PLoS One. 2018; Page 18/20 Page 18/20 doi: 10.1371/journal.pone.0194409. Official
Journal of Japan Society of Ningen Dock. 2019;33:675-82. (In Japanese). 58. Ministry of Health, Labour and Welfare. Comprehensive survey of living conditions 2016. 2017. 58. Ministry of Health, Labour and Welfare. Comprehensive survey of living conditions 2016. 2017. http://www.gender.go.jp/research/kenkyu/pdf/ishiki_3.pdf. Accessed 10 Apr (In Japanese). 58. Ministry of Health, Labour and Welfare. Comprehensive survey of living conditions 2016. 2017. http://www.gender.go.jp/research/kenkyu/pdf/ishiki_3.pdf. Accessed 10 Apr (In Japanese). 58. Ministry of Health, Labour and Welfare. Comprehensive survey of living conditions 2016. 2017. http://www.gender.go.jp/research/kenkyu/pdf/ishiki_3.pdf. Accessed 10 Apr (In Japanese). 59. Gray N, Picone G. Evidence of large-scale social interactions in mammography in the United States. Atl Econ J, 2018;46:441-57. 59. Gray N, Picone G. Evidence of large-scale social interactions in mammography in the United States. Atl Econ J, 2018;46:441-57. 60. Ohara K, Saeki K, Nezu S, Obayashi K, Tomioka K, Okamoto N, et al. The predictive factors of cancer
screening behaviors. Journal of Health and Welfare Statistics. 2014;61:13-20. (In Japanese). 60. Ohara K, Saeki K, Nezu S, Obayashi K, Tomioka K, Okamoto N, et al. The predictive factors of cancer
screening behaviors. Journal of Health and Welfare Statistics. 2014;61:13-20. (In Japanese). 61. Kim SJ, Song JH, Oh YM, Park SM. Disparities in the utilisation of preventive health services by the
employment status: an analysis of 2007-2012 South Korean national survey. PloS one. 2018; doi:
10.1371/journal.pone.0207737. 61. Kim SJ, Song JH, Oh YM, Park SM. Disparities in the utilisation of preventive health services by the
employment status: an analysis of 2007-2012 South Korean national survey. PloS one. 2018; doi:
10.1371/journal.pone.0207737. 62. Shimada T, Kato K, Inomata Y, Kikuchi R, Shibuya D. Evaluation of recall letters and application
methods in view of increasing gastric cancer screening rates.Journal of Gastrointestinal Cancer
Screening. 2010;48:647-54.(In Japanese). 62. Shimada T, Kato K, Inomata Y, Kikuchi R, Shibuya D. Evaluation of recall letters and application
methods in view of increasing gastric cancer screening rates.Journal of Gastrointestinal Cancer
Screening. 2010;48:647-54.(In Japanese). 63. Shim HY, Jun JK, Shin JY. Employment conditions and use of gastric cancer screening services in
Korea: a nationwide survey. BMC Public Health. 2019; doi: 10.1186/s12889-019-6841-y. 63. Shim HY, Jun JK, Shin JY. Employment conditions and use of gastric cancer screening services in
Korea: a nationwide survey. BMC Public Health. 2019; doi: 10.1186/s12889-019-6841-y. Page 19/20 64. Watanabe R. An analysis of participation in cancer screening in Japan. Health Care and Society. 2003;13:113-32. (In Japanese). 65. Coughlin SS. doi: 10.1371/journal.pone.0194409. Social determinants of breast cancer risk, stage, and survival. Breast Cancer Res Treat. 2019; doi: 10.1007/s10549-019-05340-7. 66. Ferdous M, Goopy S, Yang H, Rumana N, Abedin T, Turin TC. Barriers to breast cancer screening
among immigrant populations in Canada. J Immigr Minor Health. 2020; doi: 10.1007/s10903-019-
00916-3. 67. Ban K, Watanabe S, Ono Y. The efficacy of issuing a free coupon for breast cancer screening. Japan
Association of Breast Cancer Screening. 2011;20:127-134. (In Japanese). 67. Ban K, Watanabe S, Ono Y. The efficacy of issuing a free coupon for breast cancer screening. Japan
Association of Breast Cancer Screening. 2011;20:127-134. (In Japanese). 68. Sugawara S, Matsuda T. Measuring the effects of cancer screening on non-participants of the
generation who works using a questionnaire. Japan Journal of Public Health. 2013;60:396-402. (In
Japanese). 69. Konishi H. Trial of free coupon for screening mammography. Japan Association of Breast Cancer
Screening. 2013;22:37-9. (In Japanese). 69. Konishi H. Trial of free coupon for screening mammography. Japan Association of Breast Cancer
Screening. 2013;22:37-9. (In Japanese). 70. Nakajima H, Kageyama N, Sawai K, Fujiwara I, Mizuta N, Sakaguchi K, et al. A project for increasing
the rate of participation in mammographic breast cancer screening in Kyoto prefecture to 50%. Japan Association of Breast Cancer Screening. 2007;16:38-43. (In Japanese). 71. Komoto S, Takeda K. Influence of a free coupon and an educational booklet on breast cancer
screening in Mie prefecture, Japan. Japan Association of Breast Cancer Screening. 2010;19:179-183. (In Japanese). 72. Paulhus DL. Socially desirable responding: the evolution of a construct. In: Braun HI, Jackson DN,
Wiley DE, editors. The role of constructs in psychological and educational measurement. Mahwah
NJ: Erlbaum; 2002. p. 49-69. 72. Paulhus DL. Socially desirable responding: the evolution of a construct. In: Braun HI, Jackson DN,
Wiley DE, editors. The role of constructs in psychological and educational measurement. Mahwah
NJ: Erlbaum; 2002. p. 49-69. 73. Howard M, Agarwal G, Lytwyn A. Accuracy of self-reports of Pap and mammography screening
compared to medical record: a meta-analysis. Cancer Causes Control. 2009;20:1-13. 73. Howard M, Agarwal G, Lytwyn A. Accuracy of self-reports of Pap and mammography screening
compared to medical record: a meta-analysis. Cancer Causes Control. 2009;20:1-13. Page 20/20
|
https://openalex.org/W2605942370
|
https://repository.rothamsted.ac.uk/download/f490af430ff5bbc95728ef592b127d7c16c757127f52058877a5307097de652d/3898952/Burman-2018-Participatory-evaluation-guides-the.pdf
|
English
| null |
Participatory evaluation guides the development and selection of farmers’ preferred rice varieties for salt- and flood-affected coastal deltas of South and Southeast Asia
|
Field crops research
| 2,018
|
cc-by
| 13,800
|
Rothamsted Research is a Company Limited by Guarantee
Registered Office: as above. Registered in England No. 2393175.
Registered Charity No. 802038. VAT No. 197 4201 51. g
y
Founded in 1843 by John Bennet Lawes. Patron: Her Majesty The Queen
Rothamsted Research
Harpenden, Herts, AL5 2JQ
Telephone: +44 (0)1582 763133
Web: http://www.rothamsted.ac.uk/
Rothamsted Repository Download
A - Papers appearing in refereed journals
Burman, D., Maji, B., Singh, Sudhanshu, Mandal, Subhasis, Sarangi,
Sukanta K., Bandyopadhyay, B. K., Bal, A. R., Sharma, D. K.,
Krishnamurthy, S. L., Singh, H. N., delosReyes, A. S., Villanueva, D.,
Paris, T., Singh, U. S., Haefele, S. M. and Ismail, Abdelbagi M. 2018.
Participatory evaluation guides the development and selection of farmers’
preferred rice varieties for salt- and flood-affected coastal deltas of South
and Southeast Asia. Field Crops Research. 220, pp. 67-77. Rothamsted Research
Harpenden, Herts, AL5 2JQ
Telephone: +44 (0)1582 763133
Web: http://www.rothamsted.ac.uk/ Rothamsted Research
Harpenden, Herts, AL5 2JQ Patron: Her Majesty The Queen Harpenden, Herts, AL5 2JQ
Telephone: +44 (0)1582 763133
Web: http://www.rothamsted.ac.uk/
Rothamsted Repository Download
A - Papers appearing in refereed journals
Burman, D., Maji, B., Singh, Sudhanshu, Mandal, Subhasis, Sarangi,
Sukanta K., Bandyopadhyay, B. K., Bal, A. R., Sharma, D. K.,
Krishnamurthy, S. L., Singh, H. N., delosReyes, A. S., Villanueva, D.,
Paris, T., Singh, U. S., Haefele, S. M. and Ismail, Abdelbagi M. 2018. Participatory evaluation guides the development and selection of farmers’
preferred rice varieties for salt- and flood-affected coastal deltas of South
and Southeast Asia. Field Crops Research. 220, pp. 67-77. The publisher's version can be accessed at:
• https://dx.doi.org/10.1016/j.fcr.2017.03.009
The output can be accessed at:
https://repository.rothamsted.ac.uk/item/8v655/participatory-evaluation-guides-the-
development-and-selection-of-farmers-preferred-rice-varieties-for-salt-and-flood-
affected-coastal-deltas-of-south-and-southeast-asia. © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license
http://creativecommons.org/licenses/by-nc-nd/4.0/
14/01/2020 09:23
repository.rothamsted.ac.uk
library@rothamsted.ac.uk g
g
g
Registered Charity No. 802038. VAT No. 197 4201 51. Rothamsted Repository Download A - Papers appearing in refereed journals Burman, D., Maji, B., Singh, Sudhanshu, Mandal, Subhasis, Sarangi,
Sukanta K., Bandyopadhyay, B. K., Bal, A. R., Sharma, D. K.,
Krishnamurthy, S. L., Singh, H. N., delosReyes, A. S., Villanueva, D.,
Paris, T., Singh, U. S., Haefele, S. M. and Ismail, Abdelbagi M. 2018. Participatory evaluation guides the development and selection of farmers’
preferred rice varieties for salt- and flood-affected coastal deltas of South
and Southeast Asia. Field Crops Research. 220, pp. 67-77. A B S T R A C T Keywords:
Farmers’ preference
Participatory varietal selection
Salinity tolerance
Sundarbans
Waterlogging Rice is the staple food and provides livelihood for smallholder farmers in the coastal delta regions of South and
Southeast Asia. However, its productivity is often low because of several abiotic stresses including high soil
salinity and waterlogging during the wet (monsoon) season and high soil and water salinity during the dry
season. Development and dissemination of suitable rice varieties tolerant of these multiple stresses encountered
in coastal zones are of prime importance for increasing and stabilizing rice productivity, however adoption of
new varieties has been slow in this region. Here we implemented participatory varietal selection (PVS) processes
to identify and understand smallholder farmers’ criteria for selection and adoption of new rice varieties in coastal
zones. New breeding lines together with released rice varieties were evaluated in on-station and on-farm trials
(researcher-managed) during the wet and dry seasons of 2008–2014 in the Indian Sundarbans region. Significant
correlations between preferences of male and female farmers in most trials indicated that both groups have
similar criteria for selection of rice varieties. However, farmers’ preference criteria were different from
researchers’ criteria. Grain yield was important, but not the sole reason for variety selection by farmers. Several other factors also governed preferences and were strikingly different when compared across wet and dry
seasons. For the wet season, farmers preferred tall (140–170 cm), long duration (160–170 d), lodging resistant
and high yielding rice varieties because these traits are required in lowlands where water stagnates in the field
for about four months (July to October). For the dry season, farmers’ preferences were for high yielding, salt
tolerant, early maturing (115–130 d) varieties with long slender grains and good quality for better market value. Pest and disease resistance was important in both seasons but did not rank high. When farmers ranked the two
most preferred varieties, the ranking order was sometimes variable between locations and years, but when the
top four varieties that consistently ranked high were considered, the variability was low. This indicates that at
least 3–4 of the best-performing entries should be considered in succeeding multi-location and multi-year trials,
thereby increasing the chances that the most stable varieties are selected. These findings will help improve
breeding programs by providing information on critical traits. Participatory evaluation guides the development and selection of farmers’
preferred rice varieties for salt- and flood-affected coastal deltas of South
and Southeast Asia D. Burmana,1, B. Majia,1, Sudhanshu Singhb,1, Subhasis Mandala,1, Sukanta K. Sarangia,1,
B.K. Bandyopadhyaya, A.R. Bala, D.K. Sharmac, S.L. Krishnamurthyc, H.N. Singhd,
A.S. delosReyese, D. Villanuevae, T. Parise, U.S. Singhb, S.M. Haefelef, Abdelbagi M. Ismaile,⁎
a Indian Council of Agricultural Research-Central Soil Salinity Research Institute, Regional Research Station, Canning Town, India
b International Rice Research Institute, Delhi Office, India
c Indian Council of Agricultural Research-Central Soil Salinity Research Institute, Karnal, India
d Govind Ballabh Pant University of Agriculture and Technology, Uttarakhand, India
e International Rice Research Institute, Los Baños, Philippines
f University of Adelaide, Adelaide, Australia ⁎ Corresponding author at: Genetics and Biotechnology Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines.
1 Authors contributed equally to manuscript writing
E-mail addresses: burman.d@gmail.com (D. Burman), b.maji57@gmail.com (B. Maji), sud.singh@irri.org (S. Singh), subhasis2006@gmail.com (S. Mandal),
sksarangicanning@gmail.com (S.K. Sarangi), bimalbkb@gmail.com (B.K. Bandyopadhyay), asitbal@yahoo.com (A.R. Bal), DK.Sharma@icar.gov.in (D.K. Sharma),
krishnagene@gmail.com (S.L. Krishnamurthy), hns_eco@rediffmail.com (H.N. Singh), a.cueno@irri.org (A.S. delosReyes), d.villanueva@irri.org (D. Villanueva),
t.paris@irri.org (T. Paris), u.singh@irri.org (U.S. Singh), stephan.haefele@acpfg.com.au (S.M. Haefele), a.ismail@irri.org (A.M. Ismail). The publisher's version can be accessed at: The publisher's version can be accessed at: The publisher's version can be accessed at:
• https://dx.doi.org/10.1016/j.fcr.2017.03.009
The output can be accessed at:
https://repository.rothamsted.ac.uk/item/8v655/participatory-evaluation-guides-the-
development-and-selection-of-farmers-preferred-rice-varieties-for-salt-and-flood-
affected-coastal-deltas-of-south-and-southeast-asia. © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license
http://creativecommons.org/licenses/by-nc-nd/4.0/
14/01/2020 09:23
repository.rothamsted.ac.uk
library@rothamsted.ac.uk The publisher's version can be accessed at:
• https://dx.doi.org/10.1016/j.fcr.2017.03.009
The output can be accessed at:
https://repository.rothamsted.ac.uk/item/8v655/participatory-evaluation-guides-the-
development-and-selection-of-farmers-preferred-rice-varieties-for-salt-and-flood-
affected-coastal-deltas-of-south-and-southeast-asia. © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license
http://creativecommons.org/licenses/by-nc-nd/4.0/
14/01/2020 09:23
repository.rothamsted.ac.uk
library@rothamsted.ac.uk • https://dx.doi.org/10.1016/j.fcr.2017.03.009 Field Crops Research 220 (2018) 67–77 Contents lists available at ScienceDirect http://dx.doi.org/10.1016/j.fcr.2017.03.009
Received 1 August 2016; Received in revised form 7 March 2017; Accepted 23 March 2017
Available online 23 April 2017
0378-4290/ ©
2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecom
m
ons.org/licenses/BY/4.0/). 1. Introduction Rice is the staple food for about half of the world’s population, and
about 90% of the world’s rice is produced and consumed in Asia
(Mackill et al., 2012). Rice is a major crop in tropical coastal deltas in
South and Southeast Asia. However, several climatic adversities
including abiotic stresses like soil and water salinity in both wet
(Kharif) and dry (Rabi) seasons, waterlogging or flash floods, coastal
storms and cyclones during the wet season, often affects its productivity
(Burman et al., 2013; Islam and Gregorio, 2013; Ismail and Tuong,
2009; Ismail et al., 2010; Singh et al., 2010). Globally, about 230
million ha of coastal areas are saline (Li et al., 2014) of which about 27
million ha are in coastal zones of South and Southeast Asia (Ismail
et al., 2010). In India alone, 3.1 million ha are affected by salinity in
coastal regions (Yadav et al., 1983). g
g
y
y
Rice is grown in about 98% of the cultivated area as a rainfed crop
during the wet season. Growing other crops during this season is
difficult due to excessive wetting and waterlogging of low-lying fields
(Sarangi et al., 2015, 2016). In the dry (winter) season, rice is grown on
a limited area (20%), and most of the remaining fields are left fallow
due to lack of good quality water for irrigation and high soil salinity. Cultivation of rice during the dry season in coastal saline soils is
challenging and requires careful choices of suitable rice varieties and
good management practices (Sarangi et al., 2014). In both seasons, the
productivity of rice is low due to high soil and water salinity, water-
logging, and submergence, besides other problems. Mostly traditional
rice varieties are grown during the wet season with little adoption of
modern varieties. Evaluation of breeding material in actual field
conditions in such diverse ecosystem, and with input from farmers,
will ensure selection of proper types and subsequent uptake and
adoption. Rice farming is the primary source of livelihood for millions of poor
and smallholder farmers in these areas, despite being sensitive to salt
stress, with an upper threshold limit of 3 dS m−1 (Maas and Hoffmann,
1977). Above this threshold, rice yield decreases by 12% for every
additional unit increase in salinity (ECe, dS m−1) (Maas and Grattan,
1999). 2.1. Characterization of the experimental sites It comprises 102 islands of which 54 are inhabited, spreading
across 19 blocks of the two southern most districts of West Bengal,
North 24 Parganas (6 blocks) and South 24 Parganas (13 blocks). While
the Sundarbans region is one of the richest ecosystems in the world, the
inhabitants often face severe poverty, which both contributes to and
arises from the vulnerability of the population to a range of natural The PVS approach has been successfully implemented in marginal
lands before, considering the social context of end users during
evaluation, validation, and promotion of new varieties (Joshi et al.,
2007, 2012; Manzanilla et al., 2011; Morris and Bellon, 2004; Ortiz-
Ferrara et al., 2007; Paris et al., 2001, 2002; Singh et al., 2010, 2014,
2016). It is designed to include viewpoints of resource-limited farmers,
identify varieties suitable for different stress conditions and incorporate
a wider range of traits that match specific client preferences (Dorward
et al., 2007; Morris and Bellon, 2004; Sperling et al., 2001). Participa-
tory research approaches involving farmers in decision making pro-
cesses help improve the effectiveness of technology development,
increase the speed of adoption and payoffs to agricultural research
(Atlin et al., 2002; Ceccarelli and Grando, 2007; Ceccarelli et al., 2000;
Freeman, 2001; Gregorio et al., 2004; Joshi et al., 2005; Knox and Lilza,
2004; Tiwari et al., 2009; Trouche et al., 2011; Witcombe et al., 1996). The climate at the experimental sites is characterized by high
monsoon rainfall in a hot and humid summer and a dry, mild winter. Data on weather conditions, hydrology, and salinity were collected
from the ICAR-CSSRI-RRS station. The seasonal variability in mean
rainfall, surface soil salinity and depth of field water (stagnant water)
during 2008–2014, along with the timing of rice cultural operations are
presented in Fig. 2. The monthly depth of field water during the wet
season and monthly topsoil salinity (0–15 cm depth) were recorded
from the experimental fields. Most rainfall is received during May to
October. An average of 1630 mm (range of 1140–2160 mm) rainfall The Sundarbans represent typical salt-affected areas of the coastal
delta regions of South Asia, located between 21° 32′ and 21° 40′ N, and
88° 05′ and 89° 00′ E, in the Ganges delta of eastern coastal part of
India. A B S T R A C T Selected varieties through PVS are also more
likely to be adopted by farmers and will ensure higher and more stable productivity in the salt- and flood-
affected coastal deltas of South and Southeast Asia. D. Burman et al. Field Crops Research 220 (2018) 67–77 hazards (World Bank, 2014). The percentage of households below the
poverty line in the 19 Sundarbans blocks of South and North 24 Pargans
districts is 43.5%, compared with about 23% in the remaining non-
Sundarbans blocks of these two districts (CSE, 2012). Farmers in the
region are mostly smallholders, resource poor and dominated by
scheduled castes and tribes (Mandal et al., 2011). About 90% of the
farmers are marginal (< 1 ha landholding) and small (1–2 ha land-
holding). High monsoon rainfall, poor soil, and water quality and
natural adversaries like coastal storms and cyclones make agriculture in
this region highly non-remunerative, complex and risky. 1. Introduction This sensitivity to salt stress is one of the reasons why the
average productivity of rice in coastal delta regions is far below the
national average in several countries. For example, in India, the average
productivity of rice in this area is less than 1.5 t ha−1, which is
considerably below the 2.8 t ha−1 of the country’s average rice yield
(Singh, 2006). High-yielding and stress-tolerant rice varieties are
expected to provide a yield increase of about 2 t ha−1 in these areas
(Ponnamperuma, 1994). However, limited progress has been made in
developing and disseminating suitable varieties that can enhance and
sustain the productivity of this ecosystem to exploit its considerable
potential for food supply. In this study, we conducted PVS trials on a research station and on-
farm locations over seven years in these coastal saline regions. The
objectives were to identify stress-tolerant rice varieties suitable for the
target areas for use during the wet and dry seasons and to understand
smallholder farmers’ preferences and bases of their cultivars’ selection. We also conducted critical analyses to understand the effectiveness of
the PVS process, to guide necessary adjustments to ensure selection of
appropriate varieties for the spatially and temporally variable produc-
tion conditions encountered in these regions. There is a need to develop stress tolerant varieties adapted to local
conditions and also meet the preferences of local farming communities
to ensure adoption. Conventionally, breeders evaluate breeding lines
and make decisions on release for commercial use, a process that
ignores farmers’ preferences. This approach has probably contributed to
the slow adoption of new rice varieties, as breeders mostly prefer traits
that do not always match the needs of local farmers, such as agronomic
and eating quality traits. The participatory varietal selection (PVS;
Witcombe et al., 1996) process, where farmers and other stakeholders
are involved in the selection of desired breeding lines before their
formal commercialization may address this issue to a great extent. 2.1. Characterization of the experimental sites The
experiments
were
conducted
at
the
Indian
Council
of
Agricultural
Research-Central
Soil
Salinity
Research
Institute,
Regional Research Station (ICAR-CSSRI-RRS), Canning Town, located
in the South 24 Parganas district in the coastal Sundarbans region, and
also in numerous farmers’ fields at locations spreading across both
South 24 Parganas and North 24 Parganas districts (Fig. 1). Trials were
conducted
in
the
wet
(Kharif)
and
dry
(Rabi)
seasons
during
2008–2014. Soil characteristics were initially determined using stan-
dard methods before the start of the experiments. Soil samples were
analyzed for organic carbon (Walkley and Black, 1934), available N
(Subbiah and Asija, 1956), available P (Olsen et al., 1954), and
available K (Hanway and Heidel, 1952). The soil at the experimental
sites was mostly heavy textured, varying from silty clay to clay. The top
(0–15 cm depth) soil pH ranged from 6.85 to 7.68, and average organic
carbon was 0.62%. Available N, P, and K concentrations in the topsoil
were 243 kg ha−1, 10.5 kg ha−1 and 482 kg ha−1, respectively. y
g
The PVS approach has been successfully implemented in marginal
lands before, considering the social context of end users during
evaluation, validation, and promotion of new varieties (Joshi et al.,
2007, 2012; Manzanilla et al., 2011; Morris and Bellon, 2004; Ortiz-
Ferrara et al., 2007; Paris et al., 2001, 2002; Singh et al., 2010, 2014,
2016). It is designed to include viewpoints of resource-limited farmers,
identify varieties suitable for different stress conditions and incorporate
a wider range of traits that match specific client preferences (Dorward
et al., 2007; Morris and Bellon, 2004; Sperling et al., 2001). Participa-
tory research approaches involving farmers in decision making pro-
cesses help improve the effectiveness of technology development,
increase the speed of adoption and payoffs to agricultural research
(Atlin et al., 2002; Ceccarelli and Grando, 2007; Ceccarelli et al., 2000;
Freeman, 2001; Gregorio et al., 2004; Joshi et al., 2005; Knox and Lilza,
2004; Tiwari et al., 2009; Trouche et al., 2011; Witcombe et al., 1996). The Sundarbans represent typical salt-affected areas of the coastal
delta regions of South Asia, located between 21° 32′ and 21° 40′ N, and
88° 05′ and 89° 00′ E, in the Ganges delta of eastern coastal part of
India. 2.1. Characterization of the experimental sites It comprises 102 islands of which 54 are inhabited, spreading
across 19 blocks of the two southern most districts of West Bengal,
North 24 Parganas (6 blocks) and South 24 Parganas (13 blocks). While
the Sundarbans region is one of the richest ecosystems in the world, the
inhabitants often face severe poverty, which both contributes to and
arises from the vulnerability of the population to a range of natural 68 D. Burman et al. Field Crops Research 220 (2018) 67–77 Fig. 1. Experimental sites in different locations in North and South 24 Parganas districts in Sundarbans region, West Bengal, India. Fig. 1. Experimental sites in different locations in North and South 24 Parganas districts in Sundarbans region, West Bengal, India. Fig. 2. Seasonal variability in soil salinity, depth of floodwater in the field and rainfall during 2008–2014, averaged across sites and months. Arrows indicate the time of different rice
cultural operations during the wet and dry seasons. Fig. 2. Seasonal variability in soil salinity, depth of floodwater in the field and rainfall during 2008–2014, averaged across sites and months. Arrows indicate the time of different rice
cultural operations during the wet and dry seasons. 69 D. Burman et al. Field Crops Research 220 (2018) 67–77 was 15 cm x 20 cm in all trials. Recommended management practices
were used as described in all on-station and on-farm trials. was received during the experimental years. Of this, around 85% (range
of 80–89%) was during the wet season; and only 6% (1–12%) occurred
during the dry season. The mean monthly maximum temperature
varied from 24.6 °C in January to 35.4 °C in April and mean monthly
minimum temperature was lowest in January (12.9 °C) and highest
(26.8 °C) in June. Field waterlogging was deepest in August and
September, reaching 40–50 cm at the trials sites. The lowest salinity
in the topsoil was about 2 dS m−1 reached in October, and the highest
was 9 dS m−1 in May. Nurseries were established at the start of each season and 40 and
30 days old seedlings were transplanted during the wet and dry seasons,
respectively, with 1–2 seedlings per hill. Fertilizers were applied at
rates of 120-20-0 and 50-20–10 kg of N-P2O5-K2O ha−1 during the wet
and dry seasons, respectively. Table 1 Table 1
Overview of rice genotypes included in various researcher-managed trials in different
years during the wet and dry seasons of 2008–2014. No. Wet season
Dry season
Variety/breeding line
No. of
trials
Variety/breeding line
No. of
trials
1
Sabita*
17
Lal Minikit (WGL
20471)
13
2
Amal-Mana [CSRC(S) 7-
1-4]
17
Khitish
6
3
SR 26B
13
Lalat*
13
4
Geetanjali
15
Satabdi (IET 4786)
13
5
CST 7-1
4
Gontra Bidhan-2 (IET
19571)
13
6
Sumati
2
Boby
13
7
Bhutnath
2
Canning 7
10
8
Utpala
2
CSR 4
7
9
Pankaj
2
CSR 36
3
10
Dinesh
6
CSR 22
6
11
Patnai 23
2
CSR38
3
12
Manasswarabar
5
Annada
13
13
Swarna-Sub1
2
Rasi (IET 1444)
3
14
BINA 8
2
Sankarsaru
3
15
CSRC(S) 21-2-5-B-1-1**
17
Super Minikit
3
16
CSRC(D) 7-0-4**
9
N Sankar
3
17
CSRC(D) 2-17-5**
4
Super Sankar
3
18
CSRC(D) 13-16-9**
4
Parijat
3
19
CSRC(D) 12-8-12**
4
BRRIdhan47
3
20
CN 1039-9**
8
BINA 8
3
21
CN 1233-39-9**
4
IR 64 Saltol**
3
22
CN 12133-3-9**
4
IR 72593-B-18-2-2-2**
3
23
CSRC(S) 47-7-B-B**
1
IR 72593-B-3-2-3-3**
3
24
NC 678
5
IR 76346-B-B-10-1-1-
1**
3
25
CR 2006-71-2**
1
IR 76393-2B-7-1-1-3-
1**
3
26
CR 2095-181-1**
1
27
CR 2070-52-2**
1
28
CR 2094-46-3**
1
29
IR 76393-28-7**
1
30
IR 206-29-2-1-1**
1 Table 1
Overview of rice genotypes included in various researcher-managed trials in different
years during the wet and dry seasons of 2008–2014. 2.2. On-station and on-farm participatory varietal selection trials Promising rice varieties and new breeding lines were evaluated
following the PVS approach in on-station and on-farm trials, both
managed by researchers (Paris et al., 2011), and in both wet and dry
seasons. A total of 17 wet season trials (4 on-station and 13 on-farm)
and 16 dry season trials (3 on-station and 13 on-farm) were conducted,
each using a randomized complete block design (RCBD) with three
replications. The entries included released varieties and new breeding
lines recommended from different breeding programs and networks,
with 8–13 entries evaluated in each trial. Within a year, the set of
entries were the same for all on-station and on-farm trials but were
different between years. The entries selected as the best in a preceding
year were included in subsequent years along with newly added entries. The entries and the total number of times they have been included in
the trials in different years and seasons are presented in Table 1. Plot
size varied from 30 m2 (5 m x 6 m) to 40 m2 (5 m x 8 m), and spacing 2.1. Characterization of the experimental sites All P and K were incorporated before
transplanting, whereas N in the form of urea was applied in three equal
splits at seven days after transplanting (DAT), maximum tillering (45
DAT) and at the initiation of flowering (75 DAT) during the wet season. During the dry season, all of P, K and 25% N were applied before land
leveling. Half of N was broadcasted 21 DAT, and the remaining 25%
was broadcasted at 60 DAT. Hand weeding was accomplished twice (30
and 60 DAT) during both seasons. Chloropyriphos at 2 ml l−1 water and
tricyclazole at 0.6 g l−1 water were used to control insects and diseases,
respectively. 2.3. Preference analysis A Preference analysis (PA) through casting votes was conducted
during a pre-harvest period when most varieties reached around 80%
maturity (Paris et al., 2011). At each PA, a group of male and female
farmers and researchers were invited to vote for two most preferred
(positive vote) and two least preferred (negative votes) entries, using
paper ballots and envelopes placed at the head of each plot. Names of
entries were kept anonymous with codes used for each entry through-
out the voting process. Votes were then used to identify the most and
least preferred rice genotypes and farmers interviewed to understand
the reasons behind their choices. This resulted in two types of data; (i)
the quantitative preference score (PS) for each variety generated by
expressing the number of positive votes minus the negative votes
divided by the total number of votes, and (ii) a list of characteristics
considered by farmers as the basis for selecting the most and least
preferred entries. *, ** represent ‘check’ and ‘breeding line’, respectively. 2.4. Data collection and analysis Ten hills were randomly selected at harvest from each plot, and
plant height was measured from stem base to the tip of the longest leaf
or panicle, whichever was longer. The depth of water in the field was
monitored using a meter stick installed in each plot. Soil salinity was
measured using an electrical conductivity meter in 1:2 (soil: water) and
converted to ECe by multiplying with an appropriate conversion factor. At maturity, plants were harvested, then sun dried and weighed to
determine grain yield, which was then adjusted to 14% grain moisture
content. The Pearson correlation coefficient (r) was used for correlation
analysis between male and female farmers’ PS, between all farmers’ and
researchers’ PS and between yield and all farmers’ PS. The ‘r’ value was
used for comparing the extent of agreement or disparity in preference
choices between any two groups. Growth parameters and yield data
were collected from the inner rows, leaving 50 cm border rows at both
ends of the plot. Descriptive statistical methods were then used for
analyzing the effects of waterlogging and salt stress on rice grain yield. To rank the various preference criteria, the Rank Based Quotient
(RBQ) analysis was employed (Mandal et al., 2013). The criteria used
by farmers for their selection of the most preferred lines through PVS
trials were listed first, and then they were asked to rank them according
to their individual priority on a scale of 1–5. The most preferred criteria
were ranked as 1, the least preferred as 5. The analysis allowed ranking
farmers’ preferences based on RBQ. A total of 60 farmers (30% females)
from 2 locations were interviewed for this preference analysis during
the wet season. A similar methodology was used in the dry season. Farmers’ choice of a particular rice variety was influenced by several
attributes like salinity tolerance, capacity to withstand waterlogging
(plant height), resistance to pests and diseases, grain and straw quality, The Pearson correlation coefficient (r) was used for correlation
analysis between male and female farmers’ PS, between all farmers’ and
researchers’ PS and between yield and all farmers’ PS. The ‘r’ value was
used for comparing the extent of agreement or disparity in preference
choices between any two groups. Growth parameters and yield data
were collected from the inner rows, leaving 50 cm border rows at both
ends of the plot. 2.4. Data collection and analysis Descriptive statistical methods were then used for
analyzing the effects of waterlogging and salt stress on rice grain yield. To rank the various preference criteria, the Rank Based Quotient
(RBQ) analysis was employed (Mandal et al., 2013). The criteria used
by farmers for their selection of the most preferred lines through PVS
trials were listed first, and then they were asked to rank them according
to their individual priority on a scale of 1–5. The most preferred criteria
were ranked as 1, the least preferred as 5. The analysis allowed ranking
farmers’ preferences based on RBQ. A total of 60 farmers (30% females)
from 2 locations were interviewed for this preference analysis during
the wet season. A similar methodology was used in the dry season. Farmers’ choice of a particular rice variety was influenced by several
attributes like salinity tolerance, capacity to withstand waterlogging
(plant height), resistance to pests and diseases, grain and straw quality, 70 Field Crops Research 220 (2018) 67–77 D. Burman et al. resistance to lodging and duration of the crop. These attributes were
identified from the feedback of farmers during multi-location PVS trials
across both seasons and over the years (2008–2014). These attributes
were ranked through RBQ analysis during the 2013-14 season. RBQ is a
problem identification technique, mathematically presented as follows: preference for farmers in the wet season included tall plants, long
panicles with more grains, less infestation with pest and diseases,
numerous tillers, good grain type, overall good crop performance,
enough straw for fodder, thatching and fuel, apparent suitability for
parboiling, optimum maturity period, suitability for specific field
situations (low/medium land), good lodging resistance, and high grain
yield. RBQ
fi n
i
N n
= Σ
(
+ 1 −)*100
*
j
n
=1 The analyses reflected significant correlation at 5% and 1% levels
between preference scores of male and female farmers in 59% of the
sites. However, for other sites, a positive but non-significant correlation
was observed. Correlations between the preference scores of all farmers
versus researchers were strong and significant at 5% and 1% levels in
47% of the trial sites, which is 12% lower than that of male and female
farmers. Significant correlations between preference scores of all farm-
ers and yield were observed in only 41% of the sites. 3.1. Preferences of varieties and breeding lines in the wet season 3.1. Preferences of varieties and breeding lines in the wet season Waterlogging was a major limiting factor for rice growth and
productivity at the experimental sites in the monsoon season (Fig. 2
and Table 2). Due to poor or non-existence of drainage facilities, flat
topography, low-lying lands and heavy rainfall, the experimental fields
remained waterlogged with a depth of more than 30 cm from July to
October (Fig. 2). Soil salinity was high (> 4.0 dS m−1) during early
seedling stages (Table 2) when rice is highly sensitive; but lower during
reproductive stage, which is also highly sensitive to salt stress. The
performance of different rice varieties and breeding lines tested in 17
on-station and on-farm trials during the wet seasons between 2008 and
2013 is summarized in Table 3. The entries were of medium to long
duration (140–170 d) and are medium to tall (90–175 cm). Grain yield
varied between 2.58–4.39 t ha−1. Chi-square test was applied to assess associations between the
preference criteria and ranks assigned by respondents. For the wet
season, the calculated chi-square was 40.925, with P value = 0.055. With P > 0.05, we accepted the null hypothesis and concluded that
there was no association between preference criteria and ranks assigned
by the respondents (Table 5). Hence preference criteria and ranks
assigned by the respondents are independent for the wet season. Cyclones with strong winds are common in these coastal areas and
can cause considerable damage to standing rice crop. Farmers also
selected some genotypes with medium height (100–115 cm) like
Sumati, CSRC(S) 21-2-5-B-1-1, and Swarna-Sub1, as in their opinion,
they are suitable for medium lands (water depth of 20–30 cm for most
of the wet season with no flooding problem). The duration of the crop
was another important consideration by farmers. They preferred long
duration varieties (160–170 d) like CSRC(D) 12-8-12, Geetanjali, Amal-
Mana, CSRC(D) 7-0-4, CSRC(D) 13-16-9, Sabita, SR 26B, and Patnai 23
for lowlands to avoid harvesting in standing water. Medium duration
(140–150 d) rice varieties like Sumati, CSRC(S) 21-2-5-B-1-1, and
Swarna-Sub1 were targeted for medium-lands. Farmers did not prefer
early maturing varieties because they are not suitable for local land
conditions and are often damaged by rats and rodents as was evidenced
in the trials. 2.4. Data collection and analysis This variation
indicates more frequent mis-matches between characteristics of highest
yielding rice varieties and breeding lines selected by breeders versus
farmers’ preferences. Thus, grain yield was not the most preferred trait
for farmers in the wet season; other traits seem equally important for
the selection of best rice varieties for this region. Where N = total number of farmers, n = total number of ranks (there
are five ranks, n = 5), i = the rank for which the RBQ is calculated (for
a problem), and f = number of farmers reporting the rank i (for the
problem). Since RBQ scores are index values, tests of significance are not
applicable. The data consists of numbers of persons assigning the
different ranks, so is considered discrete data, and Chi-square test
was used for testing associations between the preference criteria and
ranks assigned by the respondents. The null hypothesis “there is no
association between preference criteria and ranks assigned by the
respondents,” was tested against the alternative hypothesis, i.e. “there
exists an association between preference criteria and ranks assigned by
the respondents”. Farmers’ preference traits were ranked using the RBQ analysis
(Table 5) and the results revealed that the taller genotypes were most
preferred, followed by high grain yield. The quality and quantity of
straw constituted the third most important trait. Farmers prefer tall
varieties (140–170 cm; Table 4) because they are more suited for the
lowland situations in coastal areas where land remains waterlogged
(> 30 cm depth) for about 4 months (July to October). Taller varieties
also provide sufficient straw for fodder, for roof thatching and for use as
fuel. Resistance to lodging is another important trait preferred by
farmers, as they perceive that lodging of tall varieties might cause yield
losses. 3.1. Preferences of varieties and breeding lines in the wet season Criteria such as pest and disease resistance, grain quality The participants in the PA for rice variety selection (681 persons
during 2008–2013) were 44% male farmers, 36% female farmers, and
20% researchers. Correlation analysis was performed between the
preference scores of male and female farmers, between all farmers
and researchers, and all farmers versus grain yield, using Pearson’s
correlation (Table 4). Based on farmers’ preferences scores, the four top
ranking varieties or breeding lines in each year from the PA are also
presented in Table 4. Out of a total of 30 entries, 11 entries were ranked
at least once among the top 4, in the 17 on-station and on-farm trials. However the most preferred entries differed across locations and years,
and the most preferred entries in a preceding year were not always the
most preferred in subsequent years. The most preferred varieties and
breeding lines during the six wet seasons (2008–2013) included
Geetanjali, Amal-Mana, CSRC(S)21-2-5-1-1, and Sabita. The traits of Table 3 Table 3
Agronomic characteristics of rice varieties and breeding lines evaluated in multi-location trials during the wet and dry seasons of 2008–2014. Grain yield represents mean values across
the number of trials in which particular entry was used ± SE. Table 3 Grain yield represents mean values across
the number of trials in which particular entry was used ± SE. *, **, *** significant at P < 0.05, 0.01 and 0.001, respectively. Table 3 Wet Season
Dry season
Varieties/breeding lines
Plant height
(cm)
Duration
(d)
Grain yield ± SE
Varieties/breeding lines
Plant height
(cm)
Duration
(d)
Grain yield
± SE
CSRC(D) 12-8-12
145–165
165–170
4.39 ± 0.08
Gontra Bidhan-2
105–110
120–125
4.77 ± 0.09
Geetanjali
150–170
165–170
4.12 ± 0.12
BINA 8
90–100
115–120
4.62 ± 0.11
Amal-Mana
150–162
160–170
4.11 ± 0.12
Boby
100–110
125–130
4.34 ± 0.14
Swarna-Sub1
105–110
145–150
4.02 ± 0.33
Annada
95–105
120–125
4.28 ± 0.07
CSRC(D) 7-0-4
140–155
165–170
3.99 ± 0.14
N Sankar
95–105
120–125
4.25 ± 0.06
CSRC(D) 2-17-5
145–155
165–170
3.85 ± 0.06
BRRI dhan47
105–110
120–125
4.25 ± 0.06
CSRC(S) 21-2-5-B-1-1
115–125
140–145
3.84 ± 0.09
IR 76393-2B-7-1-1-3-1
85–95
120–125
4.18 ± 0.23
CSRC(D) 13-16-9
155–165
165–170
3.82 ± 0.07
Lal Minikit
90–105
120–125
4.15 ± 0.15
Manasswarabar
140–145
160–165
3.80 ± 0.32
Parijat
85–95
115–120
4.15 ± 0.04
Sabita
150–165
165–170
3.78 ± 0.14
Super Sankar
90–100
115–120
4.08 ± 0.07
CN 12133-3-9
105–115
3.76 ± 0.10
Rasi
95–105
115–120
4.03 ± 0.08
SR 26B
140–160
165–170
3.71 ± 0.18
Canning 7
95–105
125–130
3.98 ± 0.14
Patnai 23
140–155
160–165
3.63 ± 0.11
Satabdi
85–95
120–125
3.96 ± 0.09
NC 678
155–165
165–170
3.53 ± 0.032
IR 76346-B-B-10-1-1-1
95–1005
120–125
3.92 ± 0.022
BINA 8
90–110
145–150
3.52 ± 0.04
IR 64 Saltol
95–105
120–125
3.91 ± 0.05
CST 7-1
110–120
140–150
3.49 ± 0.13
CSR 4
95–100
125–130
3.82 ± 0.06
CN 1039-9
105–115
3.42 ± 0.02
CSR38
95–100
125–130
3.70 ± 0.12
Sumati
100–105
140–145
3.33 ± 0.17
CSR 22
95–105
125–130
3.65 ± 0.12
CSRC(S) 47-7-B-B
90–100
145–150
3.27
Lalat
100–105
125–130
3.58 ± 0.26
Utpala
105–115
140–145
3.25 ± 0.20
Super Minikit
90–100
115–120
3.54 ± 0.15
CN 1233-39-9
125–135
3.17 ± 0.35
IR 72593-B-3-2-3-3
120–125
3.45 ± 0.23
CR 2094-46-3
150–160
160–165
3.17
Sankarsaru
95–105
110–115
3.36 ± 0.34
CR 2095-181-1
150–160
160–165
3.13
Khitish
85–95
115–120
3.34 ± 0.26
Bhutnath
95–105
130–135
3.13 ± 0.16
IR 72593-B-18-2-2-2
120–125
3.21 ± 0.25
Pankaj
130–150
140–145
3.10 ± 0.14
CSR 36
95–105
125–130
2.19 ± 0.16
IR 206-29-2-1-1
105–115
145–150
3.10
Dinesh
145–175
165–170
3.09 ± 0.38
CR 2006-71-2
150–165
2.98
CR 2070-52-2
145–155
160–165
2.87
IR 76393-28-7
90–110
145–150
2.58 Table 3
Agronomic characteristics of rice varieties and breeding lines evaluated in multi-location trials during the wet and dry seasons of 2008–2014. a Numbers in parenthesis in the second column are on-farm trials. Table 2 Year
Trials
Sites
Ranking of most preferred varieties/breeding lines
Correlations between preferences
1st
2nd
3rd
4th
Male vs. Female
Farmers vs. Researchers
Farmers vs. yield
2008
On-station
Site 1
Amal-Mana
Sabita
SR 26 B
Sumati
0.61**
0.84***
0.35
On-farm
Site 1
SR 26B
Amal-Mana
CSRC(S) 21-2-5-B-1-1
Sumati
0.75**
0.92***
0.15
2009
On-station
Site 1
CSRC(S) 21-2-5-
1-1
Geetanjali
Amal-Mana
Sabita
0.85***
0.90***
0.86***
On-farm
(3)a
Site 1
CSRC(S) 21-2-5-
1-1
Geetanjali
Amal-Mana
SR 26 B/ Sabita
0.10
0.48
0.96***
Site 2
CSRC(S) 21-2-5-
1-1
Geetanjali
Amal-Mana
SR 26 B
0.62
0.91***
0.19
Site 3
CSRC(S) 21-2-5-
1-1
Geetanjali
Amal-Mana
Sabita
0.85**
0.90***
0.73**
2010
On-station
Site 1
Geetanjali
Amal-Mana
Sabita
CSRC(S) 21-2-5-1-1/
SR 26B
0.91***
0.94***
0.72*
On-farm
(3)
Site 1
Geetanjali
Amal-Mana
CSRC(D) 7-0-4/ CSRC(S)
21-2-5-1-1
–
0.94***
0.91***
0.50
Site 2
Amal-Mana
Geetanjali
CSRC(S) 21-2-5-1-1/
Sabita
–
0.85**
0.81**
0.74*
Site 3
Geetanjali
Amal-Mana
CSRC(S) 21-2-5-1-1
CSRC(D) 7-0-4
0.93***
0.78*
0.74
2011
On-station
Site 1
CSRC(D) 7-0-4
SR 26 B
Amal-Mana/Sabita
–
0.65*
0.48
0.79**
2012
On-farm
(3)
Site 1
Sabita
CSRC(S) 21-2-5-
1-1
CSRC(D) 7-0-4
CSRC(D) 13-16-9
0.73*
0.71*
0.51
Site 2
Sabita
CSRC(D) 12-8-12
Amal-Mana
Geetanjali
0.37
0.44
0.67
Site 3
CSRC(D) 12-8-12
Sabita
Geetanjali
Amal-Mana
0.74*
0.57
0.49
Site 4
CSRC(D) 12-8-12
Sabita
Amal-Mana
CSRC(D) 13-16-9
0.81*
0.72*
0.88**
2013
On-farm
Site 1
CSRC(S) 21-2-5-
1-1
Swarna-Sub1
Patnai-23
Geetanjali
0.81***
0.56*
0.66**
Sabita
Amal-Mana
Swarna-Sub1
CSRC (S) 21-2-5-1-1
0.75**
0.15
0.87***
*, **, *** significant at P < 0.05, 0.01 and 0.001, respectively. a Numbers in parenthesis in the second column are on-farm trials. D. Burman et al. Field Crops Research 220 (2018) 67–77 Field Crops Research 220 (2018) 67–77 D. Burman et al. D. Burman et al. Table 2 Table 2
The depth of water in the field and top soil salinity (0–15 cm depth) at the two salt sensitive growth stages of rice (early seedling and reproductive stages) during the wet and dry seasons. Values are averages across seasons and years during 2008–2014. Sensitive growth stages of rice
Wet season (Kharif)
Dry season (Rabi)
Depth of field water
(cm)
Soil salinity
(ECe, dS m−1)
Soil salinity
(ECe, dS m−1)
Range
Mean ± SE
Range
Mean ± SE
Range
Mean ± SE
Early seedling
26.0–37.0
31.4 ± 0.60
4.10–5.80
4.58 ± 0.11
3.80–5.60
4.45 ± 0.11
Reproductive
27.0–42.0
33.2 ± 1.00
1.80–2.80
2.05 ± 0.06
4.90–6.90
5.73 ± 0.11 The depth of water in the field and top soil salinity (0–15 cm depth) at the two salt sensitive growth stages of rice (early seedling and reproduct
Values are averages across seasons and years during 2008–2014. 71 Table 3
Agronomic characteristics of rice varieties and breeding lines evaluated in multi-location trials during the wet and dry seasons of 2008–2014. Grain yield represents mean values across
the number of trials in which particular entry was used ± SE. Table 2 Wet Season
Dry season
Varieties/breeding lines
Plant height
(cm)
Duration
(d)
Grain yield ± SE
Varieties/breeding lines
Plant height
(cm)
Duration
(d)
Grain yield
± SE
CSRC(D) 12-8-12
145–165
165–170
4.39 ± 0.08
Gontra Bidhan-2
105–110
120–125
4.77 ± 0.09
Geetanjali
150–170
165–170
4.12 ± 0.12
BINA 8
90–100
115–120
4.62 ± 0.11
Amal-Mana
150–162
160–170
4.11 ± 0.12
Boby
100–110
125–130
4.34 ± 0.14
Swarna-Sub1
105–110
145–150
4.02 ± 0.33
Annada
95–105
120–125
4.28 ± 0.07
CSRC(D) 7-0-4
140–155
165–170
3.99 ± 0.14
N Sankar
95–105
120–125
4.25 ± 0.06
CSRC(D) 2-17-5
145–155
165–170
3.85 ± 0.06
BRRI dhan47
105–110
120–125
4.25 ± 0.06
CSRC(S) 21-2-5-B-1-1
115–125
140–145
3.84 ± 0.09
IR 76393-2B-7-1-1-3-1
85–95
120–125
4.18 ± 0.23
CSRC(D) 13-16-9
155–165
165–170
3.82 ± 0.07
Lal Minikit
90–105
120–125
4.15 ± 0.15
Manasswarabar
140–145
160–165
3.80 ± 0.32
Parijat
85–95
115–120
4.15 ± 0.04
Sabita
150–165
165–170
3.78 ± 0.14
Super Sankar
90–100
115–120
4.08 ± 0.07
CN 12133-3-9
105–115
3.76 ± 0.10
Rasi
95–105
115–120
4.03 ± 0.08
SR 26B
140–160
165–170
3.71 ± 0.18
Canning 7
95–105
125–130
3.98 ± 0.14
Patnai 23
140–155
160–165
3.63 ± 0.11
Satabdi
85–95
120–125
3.96 ± 0.09
NC 678
155–165
165–170
3.53 ± 0.032
IR 76346-B-B-10-1-1-1
95–1005
120–125
3.92 ± 0.022
BINA 8
90–110
145–150
3.52 ± 0.04
IR 64 Saltol
95–105
120–125
3.91 ± 0.05
CST 7-1
110–120
140–150
3.49 ± 0.13
CSR 4
95–100
125–130
3.82 ± 0.06
CN 1039-9
105–115
3.42 ± 0.02
CSR38
95–100
125–130
3.70 ± 0.12
Sumati
100–105
140–145
3.33 ± 0.17
CSR 22
95–105
125–130
3.65 ± 0.12
CSRC(S) 47-7-B-B
90–100
145–150
3.27
Lalat
100–105
125–130
3.58 ± 0.26
Utpala
105–115
140–145
3.25 ± 0.20
Super Minikit
90–100
115–120
3.54 ± 0.15
CN 1233-39-9
125–135
3.17 ± 0.35
IR 72593-B-3-2-3-3
120–125
3.45 ± 0.23
CR 2094-46-3
150–160
160–165
3.17
Sankarsaru
95–105
110–115
3.36 ± 0.34
CR 2095-181-1
150–160
160–165
3.13
Khitish
85–95
115–120
3.34 ± 0.26
Bhutnath
95–105
130–135
3.13 ± 0.16
IR 72593-B-18-2-2-2
120–125
3.21 ± 0.25
Pankaj
130–150
140–145
3.10 ± 0.14
CSR 36
95–105
125–130
2.19 ± 0.16
IR 206-29-2-1-1
105–115
145–150
3.10
Dinesh
145–175
165–170
3.09 ± 0.38
CR 2006-71-2
150–165
2.98
CR 2070-52-2
145–155
160–165
2.87
IR 76393-28-7
90–110
145–150
2.58
Table 4
Results of the preference analyses conducted during the wet seasons of 2008–2013. Table 5 Table 5
Ranking of farmers’ preference criteria for selection of rice entries in the wet and dry seasons. Preference traits were established based on farmers’ feedback during 2008–2013, and
ranking was done in 2014 wet and dry seasons, each involving 60 farmers. selection of rice entries in the wet and dry seasons. Preference traits were established based on farmers’ feedback during 2008–2013, and
asons, each involving 60 farmers. Table 5
Ranking of farmers’ preference criteria for selection of rice entries in the wet and dry seasons. Preference traits were established based on farmers’ feedback during 2008–2013, and
ranking was done in 2014 wet and dry seasons, each involving 60 farmers. Wet season
Dry season
Preference Criteria
Ranks as assigned by respondents (5 years)
RBQa Score
Rank
Ranks as assigned by respondents (5 years)
RBQ Score
Rank
1
2
3
4
5
1
2
3
4
5
Yield
18
12
10
6
6
62.00
2
24
12
12
10
10
78.00
1
Tolerant to salinity
2
0
2
4
8
10.67
8
22
14
8
6
8
70.00
2
Capacity to withstand waterlogging
(height)
10
14
14
14
12
62.67
1
0
0
4
8
8
12.00
8
Pest and disease resistant
4
6
4
10
6
27.33
6
2
8
8
6
4
27.33
5
Quality of straw for thatching/fodder/
fuel
10
10
10
8
6
47.33
3
2
2
4
6
8
16.67
7
Resistance to lodging
12
8
8
6
6
44.67
4
0
6
4
8
6
19.33
6
Grain quality for better market price
2
2
4
6
8
16.67
7
2
8
8
6
8
28.67
4
Duration of crop
2
8
8
6
8
28.67
5
8
10
12
10
8
48.00
3
Chi Square Statistic
40.925
76.082***
RBQa, Rank Based Quotient. *** P < 0.001. of all farmers and grain yields were recorded in 69% of all trials, which
was much higher than in the wet season (41%). This variation indicates
that yield was the most preferred criteria for farmers in the dry season. The RBQ analysis (Table 5) also reflected that ranking of preference
criteria was different compared to that of the wet season. The yield was
the most critical trait for choosing suitable rice varieties and breeding
lines in the dry season. 3.2. Preferences of varieties and breeding lines in the dry season During the monsoon season, excess salt is mostly washed away with
drainage water and pushed down the soil profile. Therefore, salinity
stress was lowest in September-October (Fig. 2). However, in the dry
season, rice is grown using irrigation, and soil salinity is the major
abiotic stress. Salinity built up at the study sites due to gradual drying
of the soil after the monsoon season and capillary rise of salinity from
the subsoil. Starting in December, topsoil salinity increased progres-
sively and reached a maximum in April-May (Fig. 2). Therefore, soil
salinity was high (> 4.0 dS m−1) during both sensitive growth stages of
rice at the experimental sites (Table 2). The plant height, duration and
grain yield of different rice varieties and breeding lines evaluated in
sixteen on-station and on-farm trials during the dry seasons of 2008–09
to 2013-14 are presented in Table 3. Entries were mostly of short
duration (115–130 d), short to medium height (85–110 cm) and with
grain yield of 2.19–4.77 t ha−1. High grain quality for better market value was identified as another
critical trait for selection. Farmers grow rice in the dry season mostly
for marketing because the productivity is higher compared with the wet
season. Farmers have better control over crop management, especially
water and nutrients. During field days, participating farmers indicated
that about 90% of their rice production in the dry season was sold. Consequently, farmers in the study areas preferred rice with long
slender grains (Tables 4 and 6). Farmers in general do not keep seeds of
the dry season rice varieties for the succeeding dry season because good
storage facilities are required to maintain optimum humidity, especially
during the hot and humid monsoon months. However, this is less of an
issue for storage for the wet season’s rice seed because of the dry
conditions after harvest. To select suitable rice genotypes, 384 farmers participated in the
evaluation process from 2008 to 2014. Out of these, 45% were male
farmers, 40% were females, and the rest were researchers. A total of 25
entries were included in these trials, 13 of them were favored by
farmers and ranked from 1st to 4th (Table 6). The most frequently
selected genotypes were Gontra Bidhan-2, Boby, Lal Minikit, and
Annada. Pest and disease resistance and resistance to lodging were also
considered important for selection of rice lines for the dry season. Table 5 After yield, the tolerance to salinity was 2nd
because salinity is a major constraint for farming in coastal saline areas
in the dry season. Crop growth duration was another important
criterion for selection. Farmers in the region preferred short duration
(115–130 d) varieties, because of freshwater scarcity, and together with
high soil salinity, most of the land (about 80%) remains fallow during
the dry season. Where possible, farmers irrigate their crops from
shallow tube wells. Farmers are mostly smallholders with limited
resources to invest in purchasing water from neighboring farmers
who own shallow tube wells. Soil salinity also builds up gradually as
the dry season progresses. Short duration rice varieties will have fewer
irrigation requirements and will mature before the build up of high soil
salinity towards the end of the season. for better market price, and tolerance to salinity ranked least according
to the PA. Farmers in this region are mostly small and marginal,
growing rice during the wet season for their consumption. They keep
some seed for the next wet season planting and sell the only remaining
surplus. Farmers preferred bold grain types as they feel that bold types
satiate longer after cooking and are also more suitable for parboiling. Because salinity is not a major stress except at early seedling stage,
salinity tolerance was not considered important for farmers during the
wet season. Table 4 Table 4
Results of the preference analyses conducted during the wet seasons of 2008–2013. Year
Trials
Sites
Ranking of most preferred varieties/breeding lines
Correlations between preferences
1st
2nd
3rd
4th
Male vs. Female
Farmers vs. Researchers
Farmers vs. yield
2008
On-station
Site 1
Amal-Mana
Sabita
SR 26 B
Sumati
0.61**
0.84***
0.35
On-farm
Site 1
SR 26B
Amal-Mana
CSRC(S) 21-2-5-B-1-1
Sumati
0.75**
0.92***
0.15
2009
On-station
Site 1
CSRC(S) 21-2-5-
1-1
Geetanjali
Amal-Mana
Sabita
0.85***
0.90***
0.86***
On-farm
(3)a
Site 1
CSRC(S) 21-2-5-
1-1
Geetanjali
Amal-Mana
SR 26 B/ Sabita
0.10
0.48
0.96***
Site 2
CSRC(S) 21-2-5-
1-1
Geetanjali
Amal-Mana
SR 26 B
0.62
0.91***
0.19
Site 3
CSRC(S) 21-2-5-
1-1
Geetanjali
Amal-Mana
Sabita
0.85**
0.90***
0.73**
2010
On-station
Site 1
Geetanjali
Amal-Mana
Sabita
CSRC(S) 21-2-5-1-1/
SR 26B
0.91***
0.94***
0.72*
On-farm
(3)
Site 1
Geetanjali
Amal-Mana
CSRC(D) 7-0-4/ CSRC(S)
21-2-5-1-1
–
0.94***
0.91***
0.50
Site 2
Amal-Mana
Geetanjali
CSRC(S) 21-2-5-1-1/
Sabita
–
0.85**
0.81**
0.74*
Site 3
Geetanjali
Amal-Mana
CSRC(S) 21-2-5-1-1
CSRC(D) 7-0-4
0.93***
0.78*
0.74
2011
On-station
Site 1
CSRC(D) 7-0-4
SR 26 B
Amal-Mana/Sabita
–
0.65*
0.48
0.79**
2012
On-farm
(3)
Site 1
Sabita
CSRC(S) 21-2-5-
1-1
CSRC(D) 7-0-4
CSRC(D) 13-16-9
0.73*
0.71*
0.51
Site 2
Sabita
CSRC(D) 12-8-12
Amal-Mana
Geetanjali
0.37
0.44
0.67
Site 3
CSRC(D) 12-8-12
Sabita
Geetanjali
Amal-Mana
0.74*
0.57
0.49
Site 4
CSRC(D) 12-8-12
Sabita
Amal-Mana
CSRC(D) 13-16-9
0.81*
0.72*
0.88**
2013
On-farm
Site 1
CSRC(S) 21-2-5-
1-1
Swarna-Sub1
Patnai-23
Geetanjali
0.81***
0.56*
0.66**
Sabita
Amal-Mana
Swarna-Sub1
CSRC (S) 21-2-5-1-1
0.75**
0.15
0.87***
* ** *** significant at P < 0 05 0 01 and 0 001 respectively f the preference analyses conducted during the wet seasons of 2008–2 72 Field Crops Research 220 (2018) 67–77 D. Burman et al. D. Burman et al. 3.3. Selection of best varieties based on farmers’ preferences During the PA, farmers were asked to cast their votes for the two
most preferred and two least preferred entries, which allowed ranking
of all entries based on cumulative farmers’ preference scores. The
ranking of most preferred entries changed from year to year in both
seasons (Tables 4 and 6), and even between sites within same years. For
example, Amal-Mana, which was included in all 17 wet season trials,
had quite different rankings between trial locations and years (Table 4). Similarly, in the dry season, Annada, which was included in 13 out of
the 16 trials, had different rankings in different locations and years
(Table 6). Based on farmers’ preference scores across locations and
years, eight entries in both seasons were ranked 1st or 2nd, while 11
and 13 entries were ranked among the top 4 in the wet and dry seasons,
respectively (Table 7). 3.2. Preferences of varieties and breeding lines in the dry season Farmers preferred varieties with sturdy culms to avoid lodging and
yield losses because of occasional rain and storms that occur during the
dry season. Quality of straw and height were ranked as least important
compared
to
other
criteria. Farmers
preferred
medium
height
(85–100 cm) because they believe it is associated with resistance to
lodging. Similar to the wet season, a ranking of the most preferred entries in
the dry season varied across locations and years. Preferred entries were
selected based on a range of traits includingd tolerance of soil salinity,
long panicles with more grains, no or minimum infestation by pest and
diseases, more tillers, good grain type, overall good crop performance,
sufficient straw for fodder, thatching and for use as fuel, suitability for
parboiling, optimum maturity period, and high yield. Correlations
between preferences of male and female farmers were significant in
75% of the trials (Table 6). Significant correlations were also observed
between preference scores of all farmers versus researchers in 56% of
the trials. Similarly, significant correlations between preference scores Chi-square tests were applied to assess the associations between
preference criteria and ranks assigned by the respondents. Based on
preference ranking under dry season, the calculated Chi-square was
76.082 (P < 0.001). This indicated a stronger association between
preference criteria and ranks assigned by the respondents in the dry 73 Field Crops Research 220 (2018) 67–77 D. Burman et al. season than during the wet season. season than during the wet season. Year
Trials
Sites
No. of entries
Ranking of most preferred varieties/lines
Correlation between preferences
1st
2nd
3rd
4th
Male vs. Female
Farmers vs. Researchers
Farmers vs. 3.2. Preferences of varieties and breeding lines in the dry season yield
2008−09
On-station
Site 1
12
Gontra Bidhan-2
Canning 7
Lal Minikit
Satabdi
0.81**
0.85**
0.86*
On-farm
(2)a
Site 1
12
Gontra Bidhan-2
Canning 7
Satabdi
Lalat
0.73**
0.86**
0.69*
Site 2
12
Gontra Bidhan-2
Canning 7
Satabdi
Khitish
0.93**
0.79**
0.66*
2009−10
On-station
Site 1
8
Gontra Bidhan-2
Annada
Rasi
Boby
0.81**
0.85***
0.86*
On-farm
(2)
Site 1
8
Gontra Bidhan-2
Boby
Rasi
Canning 7
0.93**
0.08
0.72**
Site 2
8
Canning 7
Annada
Rasi
Boby
0.97**
0.86**
0.16
2011−12
On-station
Site 1
8
Boby
Bidhan-2
Lal Minikit
Annada
0.91*
0.82*
0.89*
On-farm
(3)
Site 1
8
Lal Minikit
Annada
Gontra Bidhan-2
Boby
0.83
0.51
0.66
Site 2
8
Gontra Bidhan-2
Boby
Annada
Lal Minikit
0.83
0.64
0.70
Site 3
8
Gontra Bidhan-2
Annada
Lal Minikit
Satabdi
0.93*
0.82
0.73
2012−13
On-farm
(3)
Site 1
10
Gontra Bidhan-2
Boby
Annada
Parijat/Super Sankar
0.46
0.39
0.80**
Site 2
10
Super Sankar
Gontra Bidhan-2
Boby
N Sankar
0.78**
0.52
0.84**
Site 3
10
Boby
Gontra Bidhan-2
Super Sankar
Annada
0.94**
0.90***
0.32
2013−14
On-farm
(3)
Site 1
10
Lal Minikit
Lalat
Satabdi
Boby
0.83**
0.95**
0.65*
Site 2
10
Lal Minikit
Lalat
Annada/IR 64 Saltol
–
0.20
0.95**
0.68*
Site 3
10
Lal Minikit
Lalat/IR 64 Saltol
Boby
–
0.83**
0.92**
0.65* 4. Discussion Most rural people living in coastal tropical deltas of south and
southeast Asia are dependent on agriculture for their livelihood. The
majority of them are smallholders, facing severe poverty and regular
hunger periods because of low land productivity and limited alter-
natives. Rice productivity in the coastal region is low and unstable
because of the persistence of several abiotic stresses like waterlogging
in the wet season and soil and water salinity in both wet and dry
seasons (Sarangi et al., 2014; Sarangi et al., 2016). The assumption in
this study is that the rice productivity in these areas can be enhanced by
developing and disseminating suitable stress tolerant varieties presently
not accessible to farmers. Participation of local smallholder farmers in
the process of selecting suitable rice varieties is crucial to understand
their preferences and to select material adapted to these difficult
conditions for subsequent use (Gyawali et al., 2007). The need for
participatory approaches to identify such material is highlighted by the
fact that conventional breeding has not brought significant crop
improvement to smallholder farmers in these marginal environments
(Kerr and Kolavalli, 1999; Lipton and Longhurst, 1989; Tiwari et al.,
2009). The PVS trial approach is a simple and cost-effective method to
understand farmer’s selection process and to identify their most
preferred varieties for these less favorable areas. This approach was
found successful especially in remote and marginal areas where farmers
had limited resources and opportunities (Belay et al., 2005; Joshi and
Witcombe, 2002; van Asten et al., 2009; Witcombe et al., 1996). Consequently, germplasm evaluation and selection and knowledge of
varietal preferences of farmers were the primary reasons behind this
PVS study. Farmers are the key stakeholders for the adoption of new
technologies (Antle and Crissman, 1990). The feedback will help
rationalize breeding strategies by targeting necessary traits and will
also contribute to optimizing resource use (Bhuiyan et al., 2004;
Waldman et al., 2014). Table 6
Results of the preference analyses conducted during the dry seasons of 2008–2014. *, ** significant at P < 0.05, and 0.01, respectively. a Numbers in parenthesis in the second column are on-farm trials. *, ** significant at P < 0.05, and 0.01, respectively.
a Numbers in parenthesis in the second column are on-farm trials. a conducted during the wet and dry seasons of 2008 −2014, in the coastal regions of the Indian Sundarbans. Trial details presented in Table 1. b Frequency of preferences indicates the number of times a particular variety was ranked as 1st to 4th. preferred varieties, but also other traits necessary for their local farming
conditions (Joshi et al., 1997; Mandal et al., 2002; Manzanilla et al.,
2011; Singh et al., 2010, 2014, 2016). Site-specific characteristics other
than grain yield are expressly important in the wet season, as reflected
by the significant correlations with yield in fewer trials than in the dry
season (Tables 4 and 6). The ranking of preference criteria following the
RBQ analysis showed that plant height rather than grain yield is the
most critical trait for farmers in the wet season, because of the
predominance of waterlogging (Table 5). importance for various uses, including construction of homes, fodder
for livestock, and fuel for cooking. The fourth-ranked trait was
resistance to lodging, which is of particular importance for taller
varieties to maintain their high yield, thus a consequence of the first
and second preferences. Least important traits for wet season varieties
were market- driven grain quality and salinity tolerance, though the
latter might have been ranked higher if the PA was conducted at the
sensitive seedling stage. importance for various uses, including construction of homes, fodder
for livestock, and fuel for cooking. The fourth-ranked trait was
resistance to lodging, which is of particular importance for taller
varieties to maintain their high yield, thus a consequence of the first
and second preferences. Least important traits for wet season varieties
were market- driven grain quality and salinity tolerance, though the
latter might have been ranked higher if the PA was conducted at the
sensitive seedling stage. In the dry season, there is no flooding risk, but salinity increases as
the season progresses, and most of the crop is sold rather than used for
household consumption. Consequently, farmers prefer high yielding
varieties with at least some salinity tolerance, short duration, and good
grain quality for high market value. Least important were plant height
and straw characteristics. Farmers' overall germplasm choices as well as
their preference rankings were, therefore seamlessly rational and
emulated the constraints and opportunities of their production envir-
onment. *, ** significant at P < 0.05, and 0.01, respectively.
a Numbers in parenthesis in the second column are on-farm trials. p
gg
g (
)
Another outcome of these PVS trials is that farmers’ preferences are
seldom uniform across different locations or years. In this process,
farmers select and rank varieties based on visual traits of the standing
crop during a field day, usually organized between flowering and
maturity (at about 80% maturity in the present study). If for some
reasons, any variety does not perform well in a given year or at a
specific site, farmers will not select it even if it was ranked high in
previous seasons. Misiko (2013) pointed out this problem as a funda-
mental dilemma for PVS. The farmers who participate in PVS and the
final selection mostly see these entries only once during the field day
without prior knowledge of their performance throughout the season or
in previous years. They also have no information about the environ-
mental conditions at the specific site, e.g., presence/absence of diseases
or abiotic stresses like floods or salinity. Farmers might also select
entries because of some attractive traits like high yield or profuse
tillering, neglecting other important traits. This ‘impulse buying’
(Misiko, 2013) can adversely affect the proper selection of germplasm. Misiko (2013) proposed involvement of participating farmers at
different crop development stages to give them a better grasp of the
characteristics and performance of the germplasm being evaluated,
which then would enable farmers to more accurately select appropriate
genotypes for their local conditions. Other studies (Bellon and Reeves,
2002; Paris et al., 2011; Sperling et al., 1993), pointed that selection of
rice varieties by smallholder farmers is also influenced by other factors,
such as farmers’ socio-economic conditions, available resources, spe-
cific needs, and preferences. In-depth discussions with farmers during
PA field days at various locations, seasons and years provided similar
evidence. Although the exact ranking of entries was not always
consistent between sites and years (Tables 4 and 6), several entries
were repeatedly among the top four at different sites and in different
years. And farmers’ preference criteria (Table 5) did not indicate much
‘impulse buying’ but rather a good understanding of their environment
without the necessity for a particular stress occurring at the trial site. *, ** significant at P < 0.05, and 0.01, respectively.
a Numbers in parenthesis in the second column are on-farm trials. Our study showed that characteristics of suitable rice varieties for
smallholder farmers in this coastal salt and waterlogging troubled
region are variable, as farmers need to adjust to the complex production
systems and stresses typical of their local conditions. Preferential traits
were obviously different for the wet versus the dry season (Table 5). Sarangi et al. (2016) observed that preference for new varieties by
farmers during the wet season largely depends on traits necessary for
survival and higher yields in flood-affected areas of the Sundarbans
region of West Bengal. In the wet season, uncontrolled waterlogging
and poor drainage are the dominant risks that require taller varieties or
varieties capable of elongation with rising water (Singh et al., 2011;
Kato et al., 2014); therefore, plant height at maturity was the most
preferred trait. Because the wet season crop is the main source of food
supply for most farmers, grain yield ranked second. The third pre-
ference rank is quality and quantity of straw, demonstrating its 74 74 Field Crops Research 220 (2018) 67–77 D. Burman et al. D. Burman et al. Table 7
Farmers’ most preferred varieties and breeding lines in trialsa conducted during the wet and dry seasons of 2008 −2014, in the coastal regions of the Indian Sundarbans. Most preferred varieties/lines
Wet season
Most preferred varieties/lines
Dry season
Frequency of preferences (no.)b
Frequency of preferences (no.)
1st rank
2nd rank
3rd rank
4th rank
1st rank
2nd rank
3rd rank
4th rank
Amal-Mana
2
5
7
1
Gontra Bidhan-2
8
3
1
–
SR 26B
1
1
1
2
Boby
2
3
2
4
CSRC(S) 21-2-5-1-1
5
1
3
2
Lal Minikit
4
3
1
Geetanjali
3
5
1
2
Super Sankar
1
1
1
CSRC(D) 7-0-4
1
2
1
Canning 7
1
3
Sabita
3
3
3
3
Annada
–
4
3
2
CSRC(D) 12-8-12
2
1
Lalat
–
3
1
Swarna-Sub1
–
1
1
–
IR64 Saltol
–
1
1
Patnai-23
–
–
1
–
Satabdi
–
–
3
2
Sumati
–
–
–
2
Rasi
–
–
3
–
CSRC(D) 13-16-9
–
–
–
2
Khitish
–
–
–
1
Parijat
–
–
–
1
N Sankar
–
–
–
1
a Trial details presented in Table 1. b Frequency of preferences indicates the number of times a particular variety was ranked as 1st to 4th. References Antle, J.M., Crissman, C.C., 1990. Risk, efficiency, and the adoption of modern crop
varieties: evidence from the Philippines. Econ. Dev. Cult. Chang. 38, 517–537. A standard second step in PVS involves further evaluation of one or
two top preferred lines in farmer-managed trials in subsequent season
(s). However, when only a few top-ranked entries are selected for these
trials, there are chances of losing some promising lines early in the
process. As was observed in our study, the 2 top ranked entries were not
selected as most preferred varieties every year (Table 7). Therefore we
would propose that more than two entries (e.g., 4), should be tested in
follow-up multi-year researcher and farmer managed trials, to increase
the chances of selecting the best and stable varieties that are most
preferred by farmers, as some preferences might be site- or condition-
specific. Our PVS results indicated that choices of farmers were not
always consistent because the preference criteria (also the performance
of a particular variety) vary across groups. Increasing the number of
most preferred rice varieties while providing more options for the
farmers will also contribute to increasing farmers’ acceptance. Addi-
tional entries will also increase varietal diversity and provide more
stability in these unfavorable areas, as one of the benefits of PVS trial
system (Tshewang and Ghimiray, 2010; Subedi et al., 2011; Witcombe
et al., 2001). Atlin, G., Paris, T., Courtois, B., 2002. Sources of variation in participatory varietal
selection trials with rainfed rice: implications for the design of mother-baby trial
networks. In: Bellon, M.R., Reeves, J. (Eds.), Quantitative Analysis of Data From
Participatory Methods in Plant Breeding. Centro Internacional de Mejoramiento de
Maíz y Trigo, Mexico, D.F, pp. 36–43. Belay, G., Tefera, H., Tadesse, B., Metaferia, G., Jarra, D., Tadesse, T., 2005. Participatory
variety selection in the Ethiopian cereal. Tef. Exp. Agric. 42, 91–101. Bellon, M.R., Reeves, J. (Eds.), 2002. Quantitative Analysis of Data From Participatory
Methods in Plant Breeding. International Maize and Wheat Improvement Center,
Mexico, DF (143 p). Rice Research and Development in the Flood-Prone Ecosystem. In: Bhuiyan, S.I., Abedin,
M.Z., Singh, V.P., Hardy, B. (Eds.), Proceedings of the International Workshop on
Flood-Prone Rice Systems Held in Gazipur, Bangladesh. 9–11 January 2001. International Rice Research Institute, Los Baños, Philippines. pp. 283. Burman, D., Bandyopadhyay, B.K., Mandal, S., Mandal, U.K., Mahanta, K.K., Sarangi,
S.K., Maji, B., Rout, S., Bal, A.R., Gupta, S.K., Sharma, D.K., 2013. 5. Conclusions I l
M R G
i
G B
2013 P
f
li i
l
i
i
d
l
i Islam, M.R., Gregorio, G.B., 2013. Progress of salinity tolerant rice variety development in
Bangladesh. SABRAO J. Breed. Genet. 45, 21–30. Ismail, A.M., Tuong, T.P., 2009. Brackish water coastal zones of the monsoon tropics:
challenges and opportunities. In: Haefele, S.M., Ismail, A.M. (Eds.), Natural Resource
Management for Poverty Reduction and Environmental Sustainability in Fragile Rice-
based Systems. Proceedings No. 15. International Rice Research Institute, Los Baños,
Laguna, Philippines, pp. 113–121. Ismail, A.M., Thomson, M.J., Vergara, G.V., Rahman, M.A., Singh, R.K., Gregorio, G.B.,
Mackill, D.J., 2010. Designing resilient rice varieties for coastal deltas using modern
tools. In: Hoanh, C.T., Szuster, B.W., Suan-Pheng, K., Ismail, A.M., Nobel, A.D. (Eds.),
Tropical Deltas and Coastal Zones: Food Production, Communities and Environment
at the Land-Water Interface. CAB International, Nosworthy Way,
Wallingford,Oxfordeshire, UK, pp. 166–182. Joshi, K.D., Witcombe, J.R., 2002. Participatory varietal selection in rice in Nepal in
favorable agricultural environments—a comparison of two methods assessed by
varietal adoption. Euphytica 127, 445–458. Joshi, K., Subedi, M., Rana, R., Kadayat, K., Sthapit, B., 1997. Enhancing on farm varietal
diversity through participatory varietal selection: a case study for Chaiterice in Nepal. Exp. Agric. 33, 335–344. Joshi, K.D., Biggs, S., Gauchan, D., Devkota, K.P., Devkota, C.K., Shrestha, P.K., Sthapit,
B.R., 2005. Institutional innovations in the Nepal rice improvement system. Asp. Appl. Biol. 75, 93–97. Joshi, K.D., Musa, A.M., Johansen, C., Gyawali, S., Harris, D., Witcombe, J.R., 2007. Highly client-oriented breeding: using local preferences and selection produces
widely adapted rice varieties. Field Crops Res. 100, 107–116. References Land Shaping – a
Unique Technology for Improving Productivity of Coastal Land. CSSRI/Canning
Town/Bulletin/2013/02. ICAR-Central Soil Salinity Research Institute, Regional
Research Station, Canning Town, West Bengal, India (38 p). Town/Bulletin/2013/02. ICAR-Central Soil Salinity Research Institute, Regional
Research Station, Canning Town, West Bengal, India (38 p). CSE, 2012. In: Chaudhuri, J. (Ed.), Living with Changing Climate: Impact, Vulnerability
and Adaptation Challenges in Indian Sundarbans. Centre for Science and
Environment Centre for Science and Environment, New Delhi, India (108 p). Ceccarelli, S., Grando, S., 2007. Decentralized-participatory plant breeding: an example
of demand driven research. Euphytica 155, 349–360. Ceccarelli, S., Grando, S., Tutwiler, R., Baha, J., Martini, A.M., Salahieh, H., Goodchild,
A., Michael, M., 2000. A methodological study on participatory barley breeding I. Selection phase. Euphytica 111, 91–104. 5. Conclusions y
Chi, T.T.N., Liem, P.V., Paris, T.R., 2007. Farmers’ participation in rice variety selection. Omonrice 15, 159–163. The study assessed smallholder farmers’ criteria when selecting new
rice varieties suitable for salt-affected coastal delta regions of tropical
Asia. The data showed that farmers have different preference criteria
for rice varieties for the wet and dry seasons. Farmers’ assessment was
multivariate and involves multiple traits, including agronomic char-
acteristics, tolerance of prevailing abiotic stresses, and socio-economic
conditions. Farmers have a clear understanding of their rice environ-
ment, and the major traits that the new varieties must possess. In
addition to grain yield, conventional breeding programs should
obviously consider these important and sometimes site and season
specific traits such as tolerance to waterlogging, quality and quantity of
straw, and lodging resistance for wet season rice varieties. Besides grain
yield in the dry season, preferred traits include salinity tolerance,
medium to short duration, and good grain quality for better market
value. Comparative analyses of the order of ranking of top-preferred
entries in these complex ecosystems suggested the need for testing
entries over multiple locations and years to select the best performing
and stable genotypes. Furthermore, the study suggests preferably more
(e.g. four rather than two) better-performing varieties should be
retained over the years and promoted through farmers’ managed PVS
trials, thereby increasing the chances of the best entries being selected. These findings highlighted the importance and effectiveness of PVS
process in these less favorable, marginal, and complex environments in
providing useful feedback to inform breeding programs and facilitate
the development of adapted varieties that meet farmers’ expectations in
coastal regions of tropical South and Southeast Asia. Dorward, P., Craufurd, P., Marfo, K., Dogbe, W., Bam, R., 2007. Improving participatory
varietal selection processes: Participatory varietal selection and the role of informal
seed diffusion mechanisms for upland rice in Ghana. Euphytica 155, 315–327. Freeman, H.A., 2001. Comparison of Farmer-participatory Research Methodologies: Case
Studies in Malawi and Zimbabwe. Working Paper Series No. 10, Socio-economics and
Policy Program. International Crops Research Institute for the Semi-Arid Tropics,
Nairobi, Kenya (28 p.). Gregorio, G.B., Salam, M.A., Karim, N.H., Seraj, Z.I., 2004. Evaluation Report of Sub-
project on Development of High-yielding Varieties for Coastal Wetlands of
Bangladesh. International Rice Research Institute, Dhaka, Bangladesh. Gyawali, S., Sunwar, S., Subedi, M., Tripathi, M., Joshi, K.D., Witcombe, J.R., 2007. Collaborative breeding with farmers can be effective. Field Crops Res. 101, 88–95. *, ** significant at P < 0.05, and 0.01, respectively.
a Numbers in parenthesis in the second column are on-farm trials. Moreover, our results imply that PVS should be conducted over
multiple years and locations to be able to judge consistency in farmers’ Correlation between preference scores of male and female farmers,
an indicative of consensus, reflected a strong to very strong agreement
in most of the trials across locations and years, especially in the dry
season (Tables 4 and 6). This agreement showed that male and female
smallholder farmers in the target areas had similar requirements in new
varieties. Women and men often work together for a range of different
agricultural operations in the Sundarbans region (Mandal et al., 2011). And because of the common cases of male labor migration in this
region, women become the major working force for rice production
(Mondal, 2013), reflecting that they have full knowledge of the rice
production system and needs. Nevertheless, there remain some dis-
crepancies between male and female preferences. In addition to
agronomic characters of new varieties, the PVS process can also be
extended to include post-harvest characteristics, where obviously
women will have some additional preference criteria such as good
eating quality, softness after cooking, and high market value (Chi et al.,
2007; Paris et al., 2011). The relatively low correlation in preference criteria between farm-
ers and researchers during both wet and dry seasons indicated that
farmers often had different priorities than the researchers, as also
reported before (Singh et al., 2010). Farmers’ preferences and their
reasoning are important criteria for breeders to consider when devel-
oping new varieties to ensure they will be acceptable by farmers and
adapted to local conditions. This concern is particularly highlighted by
the weak correlation between the preference scores of all farmers and
grain yield, with the latter being a central criterion in most breeding
programs. Apparently, farmers consider yield when selecting their most 75 D. Burman et al. Field Crops Research 220 (2018) 67–77 preferences. Similarly, Thapa et al. (2009) suggested multi-year trials
while identifying superior wheat cultivars on resource-poor farms
through PVS. References 130–139. Participatory plant breeding research: opportunities and
challenges for the international crop improvement system. Euphytica 136, 21–35. Subedi, S., Rosyara, U.R., Adhikari, B.B., Ojha, B.R., Ghimire, D.P., Dhakal, D.D., Gurung,
H.B., Pandey, S., 2011. Participatory crop improvement: effect of farmers’ selection
criteria on diversity of rice grown in uplands of Nepal. J. Agric. Res. 49, 83–96. Olsen, S.R., Cole, C.V., Watanabe, F.S., Dean, L.A., 1954. Estimation of available
phosphorus in soils by extraction with sodium bicarbonate. USDA Circular. U.S. Gov. Print. Office, Washington, DC, 939, pp. 1–19. Thapa, D.B., Sharma, R.C., Mudwari, A., Ortiz-Ferrara, G., Sharma, S., Basnet, R.K.,
Witcombe, J.R., Virk, D.S., Joshi, K.D., 2009. Identifying superior wheat cultivars in
participatory research on resource poor farms. Field Crops Res. 112, 124–130. Ti
i T P
Vi k D S
Si
l i
F L
2009 R
id
i
i
i ld
d
d
i
f Ortiz-Ferrara, G., Joshi, A.K., Chand, R., Bhatta, M.R., Mudwari, A., Thapa, D.B., Sufian,
M.A., Saikia, T.P., Chatrath, R., Witcombe, J.R., Virk, D.S., Sharma, R.C., 2007. Partnering with farmers to accelerate adoption of new technologies in South Asia to
improve wheat productivity. Euphytica 157, 399–407. Tiwari, T.P., Virk, D.S., Sinclair, F.L., 2009. Rapid gains in yield and adoption of new
maize varieties for complex hillside environments through farmer participation. I. Improving options through participatory varietal selection (PVS). Field Crops Res. 111, 137–143. Paris, T.R., Singh, A., Luis, J., 2001. Listening to male and female farmers in rice varietal
selection: a case in eastern India. In: An Exchange of Experiences from South and
Southeast Asia. In: Proceedings of the International Symposium on Participatory
Plant Breeding and Participatory Plant Genetic Resource Enhancement. Pokhara,
Nepal, 1–5 May 2000. CIAT, Cali, Colombia. pp. 459. Trouche, G., Acuna, S.A., Briones, B.C., Palacios, N.G., Lancon, J., 2011. Comparing
decentralized participatory breeding with on-station conventional sorghum breeding
in Nicaragua: I. Agronomic performance. Field Crops Res. 121, 19–28. Tshewang, S., Ghimiray, M., 2010. Participatory variety selection: increasing rice varietal
diversity. J. Renew. Resour. Bhutan 6, 1–10. Paris, T.R., Singh, R.K., Atlin, G., Sarkarung, S., McLaren, G., Courtois, B., McAllister, K.,
Piggin, C., Pandey, S., Singh, A., Singh, H.N., Singh, O.N., Singh, S., Singh, R.K.,
Mandal, N.P., Prasad, K., Sahu, R.K., Sahu, V.N., Sharma, M.L., Singh, R.K.P., Thakur,
R., Singh, N.K., Chaudhary, D., Ram, S., 2002. Breeding rainfed rice for drought-
prone environments: integrating conventional and participatory plant breeding in
South and Southeast Asia. 130–139. Lipton, M., Longhurst, R., 1989. New Seeds and Poor People. Unwin Hyman, London
(473 p). p
g
j
Singh, R.K., Redona, E., Gregorio, G.B., Salam, M.A., Islam, M.R., Singh, D.P., Sen, P.,
Saha, S., Mahata, K.R., Sharma, S.G., Pandey, M.P., Sajise, A.G., Mendoza, R.D.,
Toledo, M.C., Dante, A., Ismail, A.M., Paris, T.R., Haefele, S.M., Thomson, D.K.,
Zolvinski, S., Singh, Y.P., Nayak, A.K., Singh, R.B., Mishra, V.K., Sharma, D.K.,
Gautam, R.K., Ram, P.C., Singh, P.N., Verma, O.P., Singh, A., Lang, N.T., 2010. The
right rice in the right place: Systematic exchange and farmer-based evaluation of rice
germplasm for salt affected areas. In: Hoanh, C.T., Szuster, B.W., Suan-Pheng, K.,
Ismail, A.M., Nobel, A.D. (Eds.), Tropical Deltas and Coastal Zones: Food Production,
Communities and Environment at the Land-Water Interface. CAB International,
Nosworthy Way, Walliningford, Oxfordeshire, UK, pp. 155–165. g
j
ingh, R.K., Redona, E., Gregorio, G.B., Salam, M.A., Islam, M.R., Singh, D.P., Sen, P.,
Saha, S., Mahata, K.R., Sharma, S.G., Pandey, M.P., Sajise, A.G., Mendoza, R.D., Maas, E.V., Grattan, S.R., 1999. Crop yields as affected by salinity. In: Skaggs, R.W., van
Schilfgaarde, J. (Eds.), Agricultural Drainage, Agron. Monograph 38. ASA, CSSA,
SSA, Madison, USA, pp. 55–108. Toledo, M.C., Dante, A., Ismail, A.M., Paris, T.R., Haefele, S.M., Thomson, D.K.,
Zolvinski, S., Singh, Y.P., Nayak, A.K., Singh, R.B., Mishra, V.K., Sharma, D.K., Maas, E.V., Hoffmann, G.J., 1977. Crop salt tolerance: current assessment. J. Irrig. Drain. Engineering ASCE 103, 115–134. Gautam, R.K., Ram, P.C., Singh, P.N., Verma, O.P., Singh, A., Lang, N.T., 2010. The
right rice in the right place: Systematic exchange and farmer-based evaluation of rice
germplasm for salt affected areas. In: Hoanh, C.T., Szuster, B.W., Suan-Pheng, K., Mackill, D.J., Ismail, A.M., Singh, U.S., Labios, R.V., Paris, T.R., 2012. Development and
rapid adoption of submergence-tolerant (Sub1) rice varieties. Adv. Agron. 115,
299–352. Mandal, N.P., Sinha, P.K., Singh, R.K., Variar, M., Singh, P.K., Atlin, G.N., 2002. Farmers’
participatory breeding for upland rice in eastern India. In: Witcombe, J.R., Parr, L.B.,
Atlin, G.N. (Eds.), Breeding Rainfed Rice for Drought-prone Environments:
Integrating Conventional and Participatory Plant Breeding in South and Southeast
Asia. Proceedings of a DFID Plant Sciences Research Program. IRRI Conference 12–15
March 2002. International Rice Research Institute, Los Baños, Philippines. (104 p). Singh, S., Mackill, D.J., Ismail, A.M., 2011. Tolerance of longer-term partial stagnant
flooding is independent of the SUB1 locus in rice. Field Crops Res. 121, 311–323. 130–139. In: Witcombe, J.R., Parr, L.B., Atlin, G.N. (Eds.),
Proceedings of a DFID Plant Sciences Research Program. IRRI Conference 12–15
March 2002. International Rice Research Institute, Los Baños, Philippines. (104 p). van Asten, P.J.A., Kaaria, S., Fermont, A.K., Delve, R.J., 2009. Challenges and lessons
when using farmer knowledge in agricultural research and development projects in
Africa. Exp. Agric. 45, 1–14. Waldman, K.B., Kerr, J.M., Isaacs, K.B., 2014. Combining participatory crop trials and
experimental auctions to estimate farmer preferences for improved common bean in
Rwanda. Food Policy 46, 183–192. Walkley, A.J., Black, I.A., 1934. Estimation of soil organic carbon by the chromic acid
titration method. Soil Sci. 37, 29–38. Paris, T.R., Manzanilla, D., Tatlonghari, G., Labios, R., Cueno, A., Villanueva, D., 2011. Guide to Participatory Varietal Selection for Submergence-tolerant Rice. International Rice Research Institute, Los Baños, Philippines (111p). P
F N
1994 E
l
i
d i
f l
d f
l
d i Witcombe, J.R., Joshi, A., Joshi, K.D., Sthapit, B.R., 1996. Farmer participatory crop
improvement. I. varietal selection and breeding methods and their impact on
biodiversity. Exp. Agric. 32, 445–460. http://dx.doi.org/10.1017/
S0014479700001526. Ponnamperuma, F.N., 1994. Evaluation and improvement of lands for wetland rice
production. In: Senadhira, D. (Ed.), Rice and Problem Soils in South and Southeast
Asia. IRRI Discussion Paper Series No. 4. International Rice Research Institute,
Manila, Philippines, pp. 3–19. Witcombe, J.R., Joshi, K.D., Rana, R.B., Virk, D.S., 2001. Increasing genetic diversity by
participatory varietal selection in high potential production systems in Nepal and
India. Euphytica 122, 575–588. Sarangi, S.K., Maji, B., Singh, S., Sharma, D.K., Burman, D., Mandal, S., Ismail, A.M.,
Haefele, S.M., 2014. Crop establishment and nutrient management for dry season
(boro) rice in coastal areas. Agron. J. 106, 2013–2023. http://dx.doi.org/10.2134/
agronj14.0182. World Bank, 2014. Building resilience for sustainable development of the Sundarbans
through estuary management, poverty reduction, and biodiversity conservation. In:
Sánchez-Triana, E., Paul, T., Ortolano, L., Ruitenebeek, J. (Eds.), Strategy Report No. 88061. South Asia Region, Sustainable Development Department, Environment and
Water Resources Management Unit, The International Bank for Reconstruction and
Development/The World Bank (273 p). Sarangi, S.K., Maji, B., Singh, S., Burman, D., Mandal, S., Sharma, D.K., Singh, U.S.,
Ismail, A.M., Haefele, S.M., 2015. Improved nursery management further enhances
the productivity of stress-tolerant rice varieties in coastal rainfed lowlands. Field
Crops Res. 174, 61–70. http://dx.doi.org/10.1016/j.fcr.2015.01.011. Yadav, J.S.P., Bandyopadhyay, A.K., Bandyopadhyay, B.K., 1983. The extent of coastal
saline soils of India. J. Indian Soc. Coast. 130–139. Si
h Y P
N
k A K
Sh
D K
G
R K
Si
h R K
Si
h R
Mi h
V K Singh, Y.P., Nayak, A.K., Sharma, D.K., Gautam, R.K., Singh, R.K., Singh, R., Mishra, V.K.,
Paris, T., Ismail, A.M., 2014. Farmers’ participatory varietal selection: a sustainable
crop improvement approach for the 21st Century. Agroecol. Sustain. Food Syst. 38,
427–444. Mandal, S., Bandyopadhyay, B.K., Burman, D., Sarangi, S.K., Mahanta, K.K., 2011. Baseline Report of the NAIP Project on ‘Strategies for Sustainable Management of
Degraded Coastal Land and Water for Enhancing Livelihood Security of Farming
Communities”. ICAR-Central Soil Salinity Research Institute, Regional Research
Station, Canning Town, West Bengal, India (74p). Singh, Y.P., Mishra, V.K., Singh, Sudhanshu, Sharma, D.K., Singh, D., Singh, U.S., Singh,
R.K., Haefele, S.M., Ismail, A.M., 2016. Productivity of sodic soils can be enhanced
through the use of salt tolerant rice varieties and proper agronomic practices. Field
Crop Res. 190, 82–90. http://dx.doi.org/10.1016/j.fcr.2016.02.007. Mandal, S., Sarangi, S.K., Burman, D., Bandyopadhyay, B.K., Maji, B., Mandal, U.K.,
Sharma, D.K., 2013. Land shaping models for enhancing agricultural productivity in
salt-affected coastal areas of West Bengal –An economic analysis. Indian J. Agric. Econ. 68, 389–401. op Res. 190, 82–90. http://dx.doi.org/10.1016/j.fcr.2016.02.007. Singh, A.K., 2006. Mantra for the new millennium. The Hindu Survey of Indian
Agriculture (39 p). Sperling, L., Loevinsohn, M.E., Ntabomvuras, B., 1993. Rethinking the farmer’s role in
plant breeding: local bean experts and on station selection in Rwanda. Exp. Agric. 29,
509–519. Manzanilla, D.O., Paris, T.R., Vergara, G.V., Ismail, A.M., Pandey, S., Labios, R.V.,
Tatlonghari, G.T., Acda, R.D., Chi, T.T.N., Duoangsila, K., Siliphouthone, I.,
Manikmas, M.O.A., Mackill, D.J., 2011. Submergence risks and farmers’ preferences:
implications for breeding Sub1 rice in Southeast Asia. Agric. Syst. 104, 335–347. Sperling, L., Ashby, J., Smith, M., Weltzien, E., McGuire, S., 2001. A framework for
analyzing participatory plant breeding approaches and results. Euphytica 122,
439–450. Misiko, M., 2013. Dilemma in participatory selection of varieties. Agric. Syst. 119, 35–42. Mondal, M., 2013. The role of rural women in agriculture sector of Sagar Island West
Bengal. India.The Int. J. Eng. Sci. 2, 81–86. Subbiah, B.V., Asija, G.L., 1956. A rapid procedure for the determination of available
nitrogen in soils. Curr. Sci. 25, 259–260. Mondal, M., 2013. The role of rural women in agriculture sector of Sagar Island West
Bengal. India.The Int. J. Eng. Sci. 2, 81–86. Morris, M.L., Bellon, M.R., 2004. Yadav, J.S.P., Bandyopadhyay, A.K., Bandyopadhyay, B.K., 1983. The extent of coastal
saline soils of India. J. Indian Soc. Coast. Agric. Res. 1, 1–6. Acknowledgements We acknowledge the assistance of farmers, scientists, extension
workers and NGOs who participated in the PVS activities for the
duration of this study. We are grateful to the Bill and Melinda Gates
Foundation (BMGF) for providing funding through STRASA (Stress
tolerant rice for Africa and South Asia) project. The administrative and
logistic support of the International Rice Research Institute Offices in
the Philippines and India, and ICAR-Central Soil Salinity Research
Institute, Karnal, Haryana, India are greatly acknowledged. The authors
appreciate the assistance of Dr. M. R. Verma, ICAR- Indian Veterinary
Research Institute, Izatnagar, India for statistical analysis and Mr. D. B. Nayak,
ICAR-
Central
Soil
Salinity
Research
Institute,
Regional
Research Station, Canning Town, India in preparing the figures. Joshi, K.D., Devkotab, K.P., Harrisc, D., Khanald, N.P., Paudyal, B., Sapkotad, A.,
Witcombec, J.R., 2012. Participatory research approaches rapidly improve household
food security in Nepal and identify policy changes required for institutionalization. Field Crops Res. 131, 40–48. Kato, Y., Collard, B.C.Y., Septiningsih, E.M., Ismail, A.M., 2014. Physiological analyses of
traits associated with tolerance of long-term partial submergence in rice. AoB
PLANTS 6, plu058. http://dx.doi.org/10.1093/aobpla/plu058. Kerr, J., Kolavalli, S., 1999. Impact of Agricultural Research in Poverty Alleviation:
Conceptual Framework with Illustrations from Literature. EPTD Discussion Paper. IFPRI, Washington, DC (195p). Knox, A., Lilza, N., 2004. Farmer research and extension. In: Meinzen-Dick, R.S.,
Gregorio, M.D. (Eds.), Collective Action and Property Rights for Sustainable
Development: Focus 11, Brief 14. IFPRI, Washington, DC, USA, pp. 29–30. Li, J., Pu, L., Zhu, M., Zhang, J., Li, P., Dai, X., Xu, Y., Liu, L., 2014. Evolution of soil
properties following reclamation in coastal areas: a review. Geoderma 226-227, 76 D. Burman et al. Field Crops Research 220 (2018) 67–77 Ismail, A.M., Haefele, S.M., 2016. Using improved variety and management enhances
rice productivity in stagnant flood-affected coastal zones. Field Crops Res. 190,
70–81. http://dx.doi.org/10.1016/j.fcr.2015.10.024. 130–139. Agric. Res. 1, 1–6. Sarangi, S.K., Maji, B., Singh, S., Sharma, D.K., Burman, D., Mandal, S., Singh, U.S., 77
|
https://openalex.org/W4372354569
|
https://www.nature.com/articles/s41598-023-34542-9.pdf
|
English
| null |
Innovative solid desiccant dehumidification using distributed microwaves
|
Scientific reports
| 2,023
|
cc-by
| 9,318
|
Innovative solid desiccant
dehumidification using distributed
microwaves
Doskhan Ybyraiymkul 1*, Qian Chen 2, Muhammad Burhan 1, Faheem Hassan Akhtar 3,
Raid AlRowais4, Muhammad Wakil Shahzad5, M. Kum Ja1 & Kim Choon Ng1
OPEN Doskhan Ybyraiymkul 1*, Qian Chen 2, Muhammad Burhan 1, Faheem Hassan Akhtar 3,
Raid AlRowais 4, Muhammad Wakil Shahzad 5, M. Kum Ja 1 & Kim Choon Ng 1 Dehumidification is one of the key challenges facing the air conditioning (AC) industry in the
treatment of moist air. Over many decades, the dual role of heat exchangers of AC chillers for the
sensible and latent cooling of space has hindered the thermal-lift reduction in the refrigeration cycle
due to the requirements of water vapor removal at dew-point and heat rejection to the ambient
air. These practical constraints of AC chillers have resulted in the leveling of energy efficiency of
mechanical vapor compressors (MVC) for many decades. One promising approach to energy efficiency
improvement is the decoupling of dehumidification from sensible processes so that innovative but
separate processes can be applied. In this paper, an advanced microwave dehumidification method
is investigated in the laboratory, where the microwave (2.45 GHz) energy can be irradiated onto the
dipole structure of water vapor molecules, desorbing rapidly from the pores of adsorbent. Results
show a significant improvement in performance for microwave dehumidification, up to fourfold, as
compared to data available in the literature. Dehumidification is water vapor removal from the air to maintain human comfort and a healthy environment
(relative humidity (RH) at 40%-60%)1–4. Presently, dehumidification is provided by cooling the airstream to its
dew-point to condense water vapor using a dual-role AC chiller5; and air-cooled AC chillers have reached their
asymptotic performance limit, 0.7–0.85 kW/Rton (equivalent to a coefficient of performance (COP)6 of 4–5)7. Much literature on chiller manufacturers cites low kW/Rton is attributed to acceptance test conditions ignoring
electricity consumption incurred by long chilled water piping losses5. One of the solutions to improve the
performance of AC is to decouple dehumidification from sensible cooling, thus permitting the incorporation of
new dehumidification methods. It is well known that microwaves can desorb water molecules from adsorbent or
sorbent. Therefore, the mechanism is used in microwave dehumidification, which is an emerging environmental-
friendly method. In microwave dehumidification, the air becomes dehumidified due to the attraction of water
molecules onto a solid adsorbent (desiccant) pore surface by physio-sorption (physical adsorption)8–11, a
characteristic of weak Van der Walls forces12–15. When adsorbent pores are saturated with water, microwave-
assisted desorption (removal of water) initiates, and highly humid air purges out. The process is schematically
presented in Fig. www.nature.com/scientificreports www.nature.com/scientificreports 1BESE Division, Water Desalination and Reuse Center, King Abdullah University of Science and Technology,
Thuwal 23955, Saudi Arabia. 2Institute for Ocean Engineering, Shenzhen International Graduate School,
Tsinghua University, Tsinghua Campus, University Town, Shenzhen 518055, China. 3Department of Chemistry
and Chemical Engineering, Lahore University of Management Sciences, Lahore 54792, Pakistan. 4Department
of Civil Engineering, College of Engineering, Jouf University, Sakakah 72388, Saudi Arabia. 5Mechanical and
Construction Engineering Department, Northumbria University, Newcastle Upon Tyne NE1 8ST, UK. *email:
doskhan.ybyraiymkul@kaust.edu.sa Figure 1. Schematically representation of microwave dehumidification. (a) Dehumidification of airstream by
adsorption of water vapor from the moist air onto the pore surface of the adsorbent due to Van der Walls forces. Nitrogen and oxygen molecules in the air are very little attracted at ambient pressure and temperatures; (b)
Desorption of water vapor from pores of adsorbent due to irradiation by microwave energy, where microwave
energy (energized by oscillating) is directly delivered to polar adsorbed water molecules. Desorbed water
molecules and air molecules are almost not adsorbing microwave energy as they can freely move in a gas state. Microwave desorption is needed to regain the adsorbent’s ability to attract water molecules. Figure 1. Schematically representation of microwave dehumidification. (a) Dehumidification of airstream by
adsorption of water vapor from the moist air onto the pore surface of the adsorbent due to Van der Walls forces. Nitrogen and oxygen molecules in the air are very little attracted at ambient pressure and temperatures; (b)
Desorption of water vapor from pores of adsorbent due to irradiation by microwave energy, where microwave
energy (energized by oscillating) is directly delivered to polar adsorbed water molecules. Desorbed water
molecules and air molecules are almost not adsorbing microwave energy as they can freely move in a gas state. Microwave desorption is needed to regain the adsorbent’s ability to attract water molecules. Figure 1. Schematically representation of microwave dehumidification. (a) Dehumidification of airstream by
adsorption of water vapor from the moist air onto the pore surface of the adsorbent due to Van der Walls forces. Nitrogen and oxygen molecules in the air are very little attracted at ambient pressure and temperatures; (b)
Desorption of water vapor from pores of adsorbent due to irradiation by microwave energy, where microwave
energy (energized by oscillating) is directly delivered to polar adsorbed water molecules. Desorbed water
molecules and air molecules are almost not adsorbing microwave energy as they can freely move in a gas state. Microwave desorption is needed to regain the adsorbent’s ability to attract water molecules. systems. MCOP can be calculated using microwave power, microwave exposure duration, and water desorbed
amount. The calculated values of MCOP for other authors were extremely low, as summarized in Table 1. The
system’s performance depends on the uniform propagation of the electric field25, the geometry of the microwave
chamber, microwave irradiation time, the type of irradiation (continuous, pulsed), and the reflected power
amount. Innovative solid desiccant
dehumidification using distributed
microwaves
Doskhan Ybyraiymkul 1*, Qian Chen 2, Muhammad Burhan 1, Faheem Hassan Akhtar 3,
Raid AlRowais4, Muhammad Wakil Shahzad5, M. Kum Ja1 & Kim Choon Ng1
OPEN 1a,b.ii p
g
From the available literature, Roussy and Chenot demonstrated the first microwave dehumidification process
with a single-mode waveguide in 198116. They presented the dependence of desiccant temperature on the electric
field16. Moreover, Roussy et al. proposed a model to represent the fast kinetics of microwave desorption17. Most
of the research within 40 years has been focused on developing the microwave-assisted desorption method with
small volumes16–22. Notably, the investigation was extended with different adsorbents (activated alumina, zeolite,
and silica gel)18. Many advantages of microwave desorption were revealed, such as transferring energy more
efficiently than convection energy transport23 and desorbing at low temperatures due to direct energy transport24. However, a critical parameter such as COP was usually omitted in the literature. In addition, no electrical power
values were provided; instead, microwave power was shown. Therefore, the microwave coefficient of performance
(MCOP) was introduced, which can be the platform for comparing different microwave dehumidification 1BESE Division, Water Desalination and Reuse Center, King Abdullah University of Science and Technology,
Thuwal 23955, Saudi Arabia. 2Institute for Ocean Engineering, Shenzhen International Graduate School,
Tsinghua University, Tsinghua Campus, University Town, Shenzhen 518055, China. 3Department of Chemistry
and Chemical Engineering, Lahore University of Management Sciences, Lahore 54792, Pakistan. 4Department
of Civil Engineering, College of Engineering, Jouf University, Sakakah 72388, Saudi Arabia. 5Mechanical and
Construction Engineering Department, Northumbria University, Newcastle Upon Tyne NE1 8ST, UK. *email:
doskhan.ybyraiymkul@kaust.edu.sa 1BESE Division, Water Desalination and Reuse Center, King Abdullah University of Science and Technology,
Thuwal 23955, Saudi Arabia. 2Institute for Ocean Engineering, Shenzhen International Graduate School,
Tsinghua University, Tsinghua Campus, University Town, Shenzhen 518055, China. 3Department of Chemistry
and Chemical Engineering, Lahore University of Management Sciences, Lahore 54792, Pakistan. 4Department
of Civil Engineering, College of Engineering, Jouf University, Sakakah 72388, Saudi Arabia. 5Mechanical and
Construction Engineering Department, Northumbria University, Newcastle Upon Tyne NE1 8ST, UK. *email:
doskhan.ybyraiymkul@kaust.edu.sa | https://doi.org/10.1038/s41598-023-34542-9 | https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 www.nature.com/scientificreports/ Figure 1. Schematically representation of microwave dehumidification. (a) Dehumidification of airstream by
adsorption of water vapor from the moist air onto the pore surface of the adsorbent due to Van der Walls forces. Nitrogen and oxygen molecules in the air are very little attracted at ambient pressure and temperatures; (b)
Desorption of water vapor from pores of adsorbent due to irradiation by microwave energy, where microwave
energy (energized by oscillating) is directly delivered to polar adsorbed water molecules. A multi-mode chamber system similar to a home oven was used for desorption19. Furthermore, the fixed
zeolite-coated desiccant rotor was regenerated using microwave and temperature swing desorption methods,
but the performance was low, with MCOP around 0.2221,22.i p
Despite many decades of studying microwave dehumidification, we have discovered a knowledge gap
that hindered its development. It is associated with narrowing research focus on methods advancement with
small samples over the past half-century. The bounding of research by small single-mode cavities is due to
the uniform electric field distribution, which led to impeding factor neglect, such as low performance. So far,
most experiments have been carried out on scales less than 160 g with a volume of 1 L16–22. Consequently, all
bench-scale studies were not developed to the pilot level to dehumidify the air. Scaling up the bench-scale
system can lead to an uneven distribution of the electric field, heating of narrow sections (close to the surface),
and, consequently, a drop in efficiency25. For achieving a high-performance pilot system, the following critical
limitations had to be solved: (I) Internal entropy generation due to unheated areas (the non-uniformity of the
electric field); (II) Enormous energy wasting (a high reflection of microwaves power); (III) Excess microwave
irradiating time. g
In this paper, we experimentally demonstrated that 97–99.5% of irradiated microwave power could be
efficiently utilized, increasing performance by fourfold at a laboratory-scale pilot for the first time. Optimization
allowed us to reduce a reflected power to 0.5–3% of input microwave power and distribute microwaves
homogeneously. The key novelties are as follows: (a) A rotating reflector with attached honeycomb desiccant
was proposed; it makes a more uniform distribution of the electric field and prevents overheating of desiccant;
(b) a new approach of optimization was proposed to decrease reflected microwave power and unheated areas;
(c) The effects of microwave irradiation time and heat recovery on COP were experimentally evaluated. The
proposed dehumidification system with an optimized structure and operating parameters will help overcome
all of the above limitations and achieve sustainable green dehumidification goals. We hope our work will help Table 1. A comparison of microwave dehumidification systems that are available in the literature. MCOP
refers to the ratio of desorbed enthalpy of water vapor to irradiated microwave energy. MCOP values were
calculated or obtained from the literature. Calculation of MCOP for literature is provided in supplementary
materials. *calculated values. Innovative solid desiccant
dehumidification using distributed
microwaves
Doskhan Ybyraiymkul 1*, Qian Chen 2, Muhammad Burhan 1, Faheem Hassan Akhtar 3,
Raid AlRowais4, Muhammad Wakil Shahzad5, M. Kum Ja1 & Kim Choon Ng1
OPEN Desorbed water
molecules and air molecules are almost not adsorbing microwave energy as they can freely move in a gas state. Microwave desorption is needed to regain the adsorbent’s ability to attract water molecules. Results Microwave dehumidification was developing with negligence of performance hitherto, so numerical optimization
and experiments were highly focused on performance. The RD-type silica gel-coated desiccant wheel with a
honeycomb structure was used as a desiccant wheel. The diameter of the wheel was 0.448 m, the height was
0.4 m, the composite material density was 668 kg/m3, the volume, including voids, was 0.063 m3, and the dry
mass of the desiccant wheel was 11.8 kg. The thickness of honeycomb channel wall’s average thickness (including
coating + cellulose + binder) was obtained from a cross-sectional SEM image (thickness was 208 µm), which
is shown in Fig. 2a. Figure 2b depicts a fractured desiccant coating surface bound with fiber material. These
fractures increase mass diffusion and flow of moisture. The selection of honeycomb structure and RD-type
silica gel was due to its high-water adsorption capacity and high microwave penetration depth. Additionally,
adsorption isotherms of the desiccant wheel, which is a combined (honeycomb cellulose, adsorbent, and binder)
desiccant structure, were obtained, as shown in Fig. 2c. According to the results, the composite structure can
adsorb moisture up to 30% of its dry bone mass at higher humidity. However, it can adsorb around 20% of its
dry bone mass within a moderate humidity region. Besides, Fig. 2c demonstrates a composite desiccant sample
on the crucible of the dynamic vapor sorption analyzer "Aquadyne DVS," which operates on the gravimetric
principle and is fully automated for measuring adsorption isotherms. An Agilent impedance analyzer was used
to measure the complex permittivity of composite desiccant material that was uniformly sampled without voids,
and then effective values were determined according to Eq. (2). Figure 2d illustrates the complex permittivity Figure 2. Characteristics of composite (honeycomb cellulose, adsorbent, and binder) desiccant structure. (a)
SEM image of desiccant cross-section; (b) SEM image of desiccant surface; (c) Isotherms of composite desiccant
structure. A composite desiccant sample on the crucible of dynamic vapor sorption analyzers "Aquadyne DVS";
(d) Dependency of effective complex permittivity and penetration depth of microwaves on adsorption water
uptake at 2.45 GHz. Figure 2. Characteristics of composite (honeycomb cellulose, adsorbent, and binder) desiccant structure. (a)
SEM image of desiccant cross-section; (b) SEM image of desiccant surface; (c) Isotherms of composite desiccant
structure. References
MCOP
Irradiation duration (min)
Desorbed water mass (g)
Microwave energy (kJ)
Roussy et al.17
0.09*
1.66
0.67
17.5
Polaet et al.18
0.018*
130
2.73
390
Kubota et al.22
0.22
–
4
47 References
MCOP
Irradiation duration (min)
Desorbed water mass (g)
Microwave energy (kJ)
Roussy et al.17
0.09*
1.66
0.67
17.5
Polaet et al.18
0.018*
130
2.73
390
Kubota et al.22
0.22
–
4
47 Table 1. A comparison of microwave dehumidification systems that are available in the literature. MCOP
refers to the ratio of desorbed enthalpy of water vapor to irradiated microwave energy. MCOP values were
calculated or obtained from the literature. Calculation of MCOP for literature is provided in supplementary
materials. *calculated values. https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 | www.nature.com/scientificreports/ further model microwave processes and develop emerging technologies. A detailed explanation of the system
is provided in the following sections. further model microwave processes and develop emerging technologies. A detailed explanation of the system
is provided in the following sections. www.nature.com/scientificreports/ (dielectric properties) of a composite desiccant material with different adsorption uptakes. Penetration depth
is the depth at which the electric field is reduced to 37% of its entrance value in the medium. It should be noted
that microwaves can penetrate to the center of the desiccant wheel as the wheel’s radius (0.224 m) is lower
than the penetration depth at the operating range of adsorption uptake (0.05–0.2). As the amount of adsorbed
water reduces, the microwave’s penetration depth is increased with decreasing in the desiccant wheel’s complex
permittivity. Moreover, it reveals that the penetration depth varies little at the operating range of adsorption
uptake (0.05–0.02), which helps in simulation simplification. Chamber design and optimization. Three global optimizations were done for 3 cases (cylindrical,
rectangular, and hopper shape) with random initial control variables, as explained in the section “Methods”
(subsection “Multi-objective optimization of the microwave chamber”). Figure 3a shows the evolution of
reflected power and low electric field ratio for the most optimal 3 cases of global optimization with a random
initial control variable. Moreover, within 3 cases, case-3 was the most optimal. The optimum value of the control
variable (illustrated in Fig. 6b) for case-3 was equal to 0.14 or c = 0.14. The difference in low electric field ratio
was insignificant in 3 cases at optimal values. Results show that microwave emitting waveguide should be
placed at the central part of the chamber than aside to get a more homogeneous (uniform) propagation of
electric field, which corresponds to the lowest electric field ratio. The reflected power for case-3 is 44 W (0.7%
of emitted power), which is the lowest value among other cases. The low reflection cannot be explained by 4
Reports | (2023) 13:7386 |
https://doi.org/10.1038/s41598-023-34542-9
Figure 3. Optimization results of a microwave dehumidification system. (a) Reflected power and low
electric field ratio for the most optimal 3 cases of global optimization with random initial control variable; (b)
Streamline of the “Poynting” vector of microwaves at the cross-section. The desiccant wheel is embedded in
the cylindrical microwave chamber. The wavelength of the microwave is 0.122 m. The radius of the cylinder is
r = 0.5 m. The color scale refers to the normalized value of S. (c) Objective functions space for multi-objective
global optimization with random initial control values. Figure 3. Optimization results of a microwave dehumidification system. Results A composite desiccant sample on the crucible of dynamic vapor sorption analyzers "Aquadyne DVS";
(d) Dependency of effective complex permittivity and penetration depth of microwaves on adsorption water
uptake at 2.45 GHz. Scientific Reports | (2023) 13:7386 | https://doi.org/10.1038/s41598-023-34542-9 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Experiments were carried out in two modes: without heat recovery and with heat
recovery (from the outflow of purged air).hfif Experimental results. Based on the optimization results, the system with an efficient multi-mode chamber
was built by the authors. Experiments were carried out in two modes: without heat recovery and with heat
recovery (from the outflow of purged air).hfif l
The most efficient one (maximum COP) within the experimental results (different microwave irradiation,
air flow rate) were demonstrated in Fig. 4 a,b for each mode. Figure 4a shows temperature and humidity ratio
profiles at the inlet and outlet of the system without heat recovery. Microwave radiation time was equal to 17 min. However, the desorption time was longer than the radiation time due to the residual energy (thermal mass of
the desiccant wheel). Inlet air temperature was constant during the adsorption and desorption cycles, and it was
equivalent to 24 °C. Similarly, the inlet humidity ratio (ω) was equal to 10.3 g/kg throughout the experiment. As
presented in Fig. 4a, the temperature of the desiccant wheel increased sharply at the start of microwave emission. The outlet air temperature increased over time, but its temperature was lower than the wheel temperature. h
p
p
p
It proved that energy was transported by microwaves directly to adsorbed water. Consequently, pressure on
the surface of the adsorbent increased, raising the outlet value of the humidity ratio to 43 g/kg. Nevertheless, the
humidity ratio started declining after 500 s and reached 40 g/kg at the stopping time of microwave irradiation. As
the airflow rate during the desorption was constant and equal to 185 m3/h, decreasing the outlet humidity ratio
evidently reduced the system’s performance. The outlet humidity ratio was meager due to the thermal mass of
adsorbed water and desiccant at the starting time of microwave emission. The thermal mass effect needs more
microwave emission time. However, the decreasing trend of the humidity ratio demonstrates that it cannot be
very long. 2 kg of water was desorbed for the current mode during the desorption cycle, showing that many
water vapors can be captured and turned into potable water or used to run IEC systems. The COP of the system
was 0.55, and MCOP was 0.83. The temperature of the desiccant wheel did not go very high, which proves the
excellent distribution of the electric field obtained by numerical results. www.nature.com/scientificreports/ (a) Reflected power and low
electric field ratio for the most optimal 3 cases of global optimization with random initial control variable; (b)
Streamline of the “Poynting” vector of microwaves at the cross-section. The desiccant wheel is embedded in
the cylindrical microwave chamber. The wavelength of the microwave is 0.122 m. The radius of the cylinder is
r = 0.5 m. The color scale refers to the normalized value of S. (c) Objective functions space for multi-objective
global optimization with random initial control values. Figure 3. Optimization results of a microwave dehumidification system. (a) Reflected power and low
electric field ratio for the most optimal 3 cases of global optimization with random initial control variable; (b)
Streamline of the “Poynting” vector of microwaves at the cross-section. The desiccant wheel is embedded in
the cylindrical microwave chamber. The wavelength of the microwave is 0.122 m. The radius of the cylinder is
r = 0.5 m. The color scale refers to the normalized value of S. (c) Objective functions space for multi-objective
global optimization with random initial control values. Scientific Reports | (2023) 13:7386 | https://doi.org/10.1038/s41598-023-34542-9 www.nature.com/scientificreports/ the position of the waveguide as the reflected power increases during optimization to 914 W (15% of emitted
power). Nevertheless, circular geometry had the lowest microwave power reflection during the optimization. Optimization results demonstrate that the shape of the chamber has a significant impact on performance than
the position of the waveguide. Figure 3b illustrates streamlines of the Poynting vector of microwaves at the cross-
section of the chamber. These streamlines indicate the propagation path of microwaves. The color scale refers
to the normalized value of S (power flow). According to the results, most of the microwave power was adsorbed
during the reflection (bouncing) of the microwaves inside the chamber. Owing to the parabolic form (which can
be seen at the cross-section), the metal cover of the chamber in case-3 prevents returning of microwave rays back
to the magnetron. Figure 3c shows objective function space, where we can also spot the optimum values for 3
cases located near the optimum front line, which is also called the Pareto front. The sum of objective functions
at case-3 (cylindrical shape) reached extremum value, so the global optimum condition belongs to case-3 with
c = 0.14 m. Experimental results. Based on the optimization results, the system with an efficient multi-mode chamber
was built by the authors. www.nature.com/scientificreports/ Hot spots or decreasing performance
of the system were not observed due to the continuous operation of the stirrer at the center of the desiccant
wheel and wheel rotation that made the system safe and sustainable. Moreover, the desiccant temperature did
not exceed 80 °C. Nevertheless, some transported energy is wasted by heating the outlet temperature to 51 °C,
so it is motivated to consider the mode with heat recovering from hot outlet air to inlet air by the heat exchanger. Figure 4b presents temperature and humidity ratio profiles for microwave desorption with a heat recovering
mode. A schematic diagram of the mode is shown in Fig. 7b. Microwaving time was equal to 12 min 20 s, and the
airflow rate was 140 m3/h. Inlet air temperature increased due to heat recovering from the outlet temperature. Besides, the temperature profile was different from the temperature profile without heat recovery; notably,
the outlet air temperature reached 51 °C in a shorter time than the previous mode. As a result, the system has Figure 4. Experimental results of a microwave dehumidification system. (a) Humidity ratio and temperature
for non-heat recovery mode; (b) Humidity ratio and temperature for heat recovery mode. Experimental results
correspond to the maximum COP value of each mode. Figure 4. Experimental results of a microwave dehumidification system. (a) Humidity ratio and temperature
for non-heat recovery mode; (b) Humidity ratio and temperature for heat recovery mode. Experimental results
correspond to the maximum COP value of each mode. Scientific Reports | (2023) 13:7386 | https://doi.org/10.1038/s41598-023-34542-9 www.nature.com/scientificreports/ the highest COP than other modes, and COP is equal to 0.58, and MCOP is equivalent to 0.87. Moreover,
high performance can be seen from the humidity ratio profile that increased with time until microwave
irradiation was stopped. Compared with the non-heat recovering mode, some wasted heat is used efficiently,
thus increasing system performance. Around 1.54 kg of water vapor was desorbed from the desiccant wheel
during the desorption process. Figure 5b demonstrates system performance and amount of desorbed water for
the different duration (3.5–17 min) of microwave emission for both modes. The desorbed amount of water had
almost linear dependence from time. Results show that COP increases with the duration increasing of microwave
irradiation for non-heat recovery modes because of the thermal mass of saturated composite desiccant. www.nature.com/scientificreports/ At the
beginning of microwave radiation, some portion of energy was used for rapid heating of the saturated desiccant
wheel from 24 °C to 48 °C (Fig. 4a), so COP at the short time was low. Running the microwave longer, we can
reduce the effect of thermal mass and increase the COP of the system. However, microwave irradiation was not
more than 17 min as most of the water was desorbed (adsorption uptake was 0.03). www.nature.com/scientificreports/ www.nature.com/scientificreports/ recovery modes was more elevated than for non-heat recovery modes. The performance of microwave desorption
should be considered as the general coefficient of performance (COP) and microwave power-based coefficient
of performance (MCOP) by the following equation: (1)
MCOP = m ∗hfg
Emw
(2)
COP = m ∗hfg
Pelec (1)
MCOP = m ∗hfg
Emw MCOP = m ∗hfg
Emw (1) (2)
COP = m ∗hfg
Pelec (2) where m is desorbed (water) moisture mass in [kg], hfg is a heat of evaporation in [J/kg], Emw is microwave
energy emitted from the magnetron in [J], and Pelec is electrical energy consumed in [J]. Dehumidification by
desiccant always works as a cycle (adsorption and desorption), so the amount of adsorbed and desorbed water
mass are the same. To focus on the performance of microwave dehumidification in the calculation, desorbed
water mass was used. Hence the conversion efficiency η is 0.7, Fig. 5a illustrates the difference between MCOP
and COP in the methodology of estimation. Figure 5c shows a comparison in MCOP for different authors with
microwave desorption. It can be seen that the current system that was developed, designed, and built by authors
at King Abdullah University of Science and Technology (KAUST) has the highest COP and MCOP. It verifies
that numerical optimization of chamber shape and obtaining the best operating conditions can overcome both
limitations. Quantum jump was achieved in microwave dehumidification, with a fourfold increase in the MCOP,
up to 0.87, as compared with available literature. Improving the microwave-based MCOP allowed us to cross-
compare the microwave performances of assorted authors. Another issue was not considering microwaving time
on the performance of the system.h p
y
The system was improved to commercial large system performance. It shows that building a microwave
system needs an understanding of the physics and mechanics of electromagnetic waves, which can help design a
system with high safety standards and high performance. The role of numerical optimization methods is crucial,
as well as experiments. Conclusion A laboratory-scale microwave dehumidifier pilot was successfully tested to achieve a fourfold improvement in
the MCOP up to 0.87 compared with available literature. These dehumidification improvements were attributed
to the better waveguide and chamber design, demonstrating the sensitivity of water molecules desorption
(dehumidification) to microwave energy delivery. However, the parasitic losses of electricity conversion to
microwave, around 30% of electricity input, have resulted in an overall COP of 58%. Hence, there is much room
for the electrical technology of microwave generation research, should a sustainable microwave dehumidifier
COP of 75% is desired. Methods
h
f Theory of microwave dehumidification. Van der Waals force (or energy sites) attract water molecules
onto the surface of sold desiccant material during dehumidification (Fig. 1a). Adsorbed water can be removed
by pressure swing26,27 and thermal swing12,28. It is the most energy-consuming process in dehumidification29. On
the other hand, attraction forces have electrostatic behavior, so oscillating dipole-structured water molecules
with electromagnetic waves (microwaves) could detach from the surface faster and with less energy compared
to the above-mentioned methods. Microwave-assisted desorption is an emerging method (Fig. 1b), where two
desorption mechanisms are applied: the direct microwave effect on molecules (selective energy transport) and
the thermal microwave effect23. It does not require heating of purged air stream to transport energy as in thermal
swing; instead, the energy is transported directly to the water molecule23. Microwaves are electromagnetic
waves ranging from about 1 m to 0.001 m (with frequencies between 0.3 GHz and 300 GHz)30, and like all
electromagnetic waves, it obeys Maxwell’s equation systems. The time-harmonic electromagnetic field can be
represented by the following differential equation that is obtained from Maxwell equation systems by applying
a frequency-domain approach: (3)
∇×
∇× E
−k2
0
εr,eff
E = 0, (3) where ∇ (nabla) is a vector differential operator, −→
E (Ex, Ey, Ez) is the vector field of an electric field in [V/m], k0
is wavenumber in [rad/m]. εr,eff is effective complex permittivity, and it has real and complex components, as
shown by the following equation: where ∇ (nabla) is a vector differential operator, −→
E (Ex, Ey, Ez) is the vector field of an electric field in [V/m], k0
is wavenumber in [rad/m]. εr,eff is effective complex permittivity, and it has real and complex components, as
shown by the following equation: (4)
εr,eff= ε
′
r,eff+ iε
′′
r,eff (4) where ε"
r,eff is the real part of effective complex permittivity (dielectric constant), ε"
r,eff is the imaginary part of
effective complex permittivity (dielectric loss factor). In simulations, averaged microwave power consumed by
the desiccant wheel is calculated according to the Poynting equation: Pmw = Vπf ε0ε"
r,effE2 , where Pmw is the
microwave power in [W], V is the desiccant wheel’s volume in [m3], f is the microwave’s frequency in [Hz], ε0 is
the free space permittivity in [F/m]. Another important parameter is the time-averaged vector field ( −→S ), which
showed the power flow and microwave direction. Discussion Not focusing on the performance of microwave dehumidification was the gap in research hitherto. Since
simulation and experiment were targeted at getting the maximum performance, the most efficient microwave
dehumidification system was developed with COP of 0.58 and MCOP of 0.87. MCOP of 0.87 means most of
the microwave energy was delivered to water molecules directly, with the least internal entropy generation and
low reflected power to the magnetron. Internal entropy generation is attributed to uneven heating within the
volume. It occurs in high values region of “low electric field ratio.” Reflected microwave power was at the lowest
value (0.5–3% of input microwave power), which has a tremendous impact on microwave dehumidification
performance. The microwave generator’s conversion efficiency, which turns electrical energy into microwave
energy, was 0.7, which was accountable for the COP of 0.58 when MCOP was as high as 0.87. However, COP
was the highest within the available literature. The highest COP (0.58) for heat recovery modes corresponds
to the time when the humidity ratio reaches the highest value. Recovering heat can increase performance, but
heat recovery has less effect for a short time or a long time. Meanwhile, the desorbed amount of water for heat Figure 5. Performance of microwave dehumidification system. (a) Methodology for calculation of MCOP
to compare the efficiency of different authors’ microwave dehumidification systems and their difference from
COP calculation concept. (b) COP and desorbed amount of water vs. duration of microwave emission. (c)
Comparison of dehumidification performance with reference authors with current advance, which is 4 times
higher than other authors. Figure 5. Performance of microwave dehumidification system. (a) Methodology for calculation of MCOP
to compare the efficiency of different authors’ microwave dehumidification systems and their difference from
COP calculation concept. (b) COP and desorbed amount of water vs. duration of microwave emission. (c)
Comparison of dehumidification performance with reference authors with current advance, which is 4 times
higher than other authors. https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 | www.nature.com/scientificreports/ cellulose) are almost transparent to microwave radiation; hence, microwave energy is solely focused on ejecting
the water molecules from the pores of the adsorbent. cellulose) are almost transparent to microwave radiation; hence, microwave energy is solely focused on ejecting
the water molecules from the pores of the adsorbent. Effective complex permittivity and penetration depth. The honeycomb-based adsorbent wheel
permits airflow through its channeled voids. For accurate modeling, it is necessary to obtain the effective
complex permittivity of the honeycomb wheel, which is a function of complex permittivity of air and desiccant
materials (silica gel, binder, cellulose), that is31: (5)
εr,eff= fopεr,air + fcdεr,cd = fop
ε
′
r,air
+ fcd
ε
′
r,cd + iε
′′
r,cd (5) where fop is the volume fraction of air in the openings (honeycomb), and fcd = 1-fop is the volume fraction of
composite desiccant. The penetration depth of microwaves is also calculated with effective complex permittivity
by the following formula23: where fop is the volume fraction of air in the openings (honeycomb), and fcd = 1-fop is the volume fraction of
composite desiccant. The penetration depth of microwaves is also calculated with effective complex permittivity
by the following formula23: (6)
Dp =
0
2π
2ε
′
r,eff
1
1 +
ε′′
r,eff
ε′
r,eff
2
−1
(6) where Dp is the penetration depth of microwaves in [m] and 0 is the wavelength of microwaves in [m]. Multi‑objective optimization of the microwave chamber. Optimization was conducted, minimizing
the reflected microwave power and unheated areas to increase heating performance within the honeycomb
wheel. Three multi-objective optimizations with the weighted sum of objectives were carried out as three shapes
were considered, namely, (i) the first is a rectangular block chamber with a pyramidal hopper-shaped side,
(ii) the second is a rectangular block, and (iii) a cylindrical chamber. The control variables were denoted by
a,b,c,d and displayed in Fig. 6a. Global optimum value was obtained with random initial control values within
the constraining range. Moreover, the optimization was constrained by the chamber’s geometry, with wheel
dimensions kept constant at a radius of 0.224 m and height of 0.4 m (Fig. 6b). www.nature.com/scientificreports/ For mathematical modeling,
the following assumptions were used: 1) The complex permittivity and the effective complex permittivity of
honeycomb material are homogeneous and isotropic; 2) The perforated metal sheet was assumed to have the
same reflective characteristics as the non-perforated one due to the much smaller perforation hole diameter
(4 mm) than the wavelength of microwaves (124 mm). For the design of the waveguide and chamber, Eq. (1)
was solved to obtain the electric field (V/m) subjected to the boundary conditions. At the entrance of the
waveguide (from the magnetron), the electric field of the x-direction is designed according to Eq. (7) whilst the
corresponding values in the y and z directions are zero32: (7) (7)
Ex =
Pin/Pmode∗sin
w −y
/w∗pi
/w, Ey = 0, Ez = 0. Equation (7) would satisfy the microwave irradiation classified as the TE10 mode under the standard industrial
waveguide (WR340) at a frequency (f) of 2.45 GHz. The assumption of the perfect electrical conductor was
applied for all walls and perforated sheets, where tangential components of the electric field were set equal to zero: Equation (7) would satisfy the microwave irradiation classified as the TE10 mode under the standard industrial
waveguide (WR340) at a frequency (f) of 2.45 GHz. The assumption of the perfect electrical conductor was
applied for all walls and perforated sheets, where tangential components of the electric field were set equal to zero: (8) (8)
n × E = 0 n × E = 0 The computation region consists of two domains (Fig. 6b) because air (gray) and the desiccant wheel (red)
have different effective complex permittivity. The Nelder-Mead algorithm was used for optimization calculations. Nelder–Mead algorithm is a nonlinear optimization method that uses the simplex concept. At each iteration,
a new vertex is defined by the four operations known as reflection, expansion, contraction, and shrinkage. The
value of the objective function at n + 1 vertex of a simplex is calculated as it is moved toward the minimum
point33. Two objective functions were defined such as reflected power ratio and low electric field ratio: (9)
fp = wc ∗Prefl/Pin and flow =
∫
Vhmpz
ElowdV/Vhmpz (9) eighting coefficient ( wc ) was equal to 5 as usually 20% of microwave power was reflected back. where the weighting coefficient ( wc ) was equal to 5 as usually 20% of microwave power was reflected back. Methods
h
f −→S = −→
E × −→
H∗ , where −→
H∗ is the vector field of the magnetic
field and complex conjugate. Silica gel was chosen among adsorbents and coated on a cellulose-based honeycomb
structured wheel to achieve a high surface area per unit volume of the wheel. These materials (silica gel and where ε"
r,eff is the real part of effective complex permittivity (dielectric constant), ε"
r,eff is the imaginary part of
effective complex permittivity (dielectric loss factor). In simulations, averaged microwave power consumed by
the desiccant wheel is calculated according to the Poynting equation: Pmw = Vπf ε0ε"
r,effE2 , where Pmw is the
microwave power in [W], V is the desiccant wheel’s volume in [m3], f is the microwave’s frequency in [Hz], ε0 is
the free space permittivity in [F/m]. Another important parameter is the time-averaged vector field ( −→S ), which
showed the power flow and microwave direction. −→S = −→
E × −→
H∗ , where −→
H∗ is the vector field of the magnetic
field and complex conjugate. Silica gel was chosen among adsorbents and coated on a cellulose-based honeycomb
structured wheel to achieve a high surface area per unit volume of the wheel. These materials (silica gel and https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ (10)
Elow =
1,
if (Enorm < Ethreshold);
0,
otherwise, (10) where Ethreshold was equal to 3000 V/m, which was discovered from the authors’ waveguide experiment. A low
electric field ratio is needed to maintain uniform heating by microwaves. Control variables and their bounds
for all cases are presented below: where Ethreshold was equal to 3000 V/m, which was discovered from the authors’ waveguide experiment. A low
electric field ratio is needed to maintain uniform heating by microwaves. Control variables and their bounds
for all cases are presented below: p
Case-1: 0.5[m] > h > 0.005[m]; Case-2: 0.2[m] > a > 0, 0.25[m] > b > 0; Case-3: 0.2[m] > c > 0.h COMSOL Multiphysics computational platform was used to perform optimization. The system of equations
was solved with FGMRES Iterative Solver, which uses the restarted flexible generalized minimum residual
method. Mesh built of (minimum) 2,251,507 tetrahedral domain elements and 73,048 triangular boundary
elements. https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 | www.nature.com/scientificreports/ Figure 6. Numerical optimization domains and geometry of microwave chamber. (a) Microwave chambers
with 3 different shapes, namely, a rectangular block shape with a pyramidal hopper-shaped side, a rectangular
block shape, and a cylindrical shape. Microwave emitting waveguide positions variables a,b,c,d. which are used
as control variables in optimization; (b) Positioning of the desiccant wheel in the 3 different chambers, where
the air is colored gray, and the desiccant wheel is colored red to show different domains with different properties
for simulation in 3 cases. Figure 6. Numerical optimization domains and geometry of microwave chamber. (a) Microwave chambers
with 3 different shapes, namely, a rectangular block shape with a pyramidal hopper-shaped side, a rectangular
block shape, and a cylindrical shape. Microwave emitting waveguide positions variables a,b,c,d. which are used
as control variables in optimization; (b) Positioning of the desiccant wheel in the 3 different chambers, where
the air is colored gray, and the desiccant wheel is colored red to show different domains with different properties
for simulation in 3 cases. Experimental apparatus. A microwave generator (Fricke und Mallah, Germany) with an efficiency value
of 0.7 for converting alternative current (AC) electrical (9 kW) power to microwave power was used to generate
the microwaves. A magnetron head (with a circulator and a directional coupler) was used to generate microwaves
and measure forwarded and reflected microwave power. www.nature.com/scientificreports/ A three-stub tuner (Fricke und Mallah, Germany) was
used to tune the microwave phase to perform impedance matching for maximizing energy transfer. Frame and
equipment were grounded with protective grounding to prevent users from high voltage electrical hazards. Experimental apparatus. A microwave generator (Fricke und Mallah, Germany) with an efficiency value
of 0.7 for converting alternative current (AC) electrical (9 kW) power to microwave power was used to generate
the microwaves. A magnetron head (with a circulator and a directional coupler) was used to generate microwaves
and measure forwarded and reflected microwave power. A three-stub tuner (Fricke und Mallah, Germany) was
used to tune the microwave phase to perform impedance matching for maximizing energy transfer. Frame and
equipment were grounded with protective grounding to prevent users from high voltage electrical hazards. q p
g
p
g
g
p
g
g
A specially made double Faraday cage was used in order to avoid microwave leakage to the surrounding area. The first cage is a multi-mode chamber where a desiccant wheel was placed, and its geometry was built based on
numerically optimized results (more detailed information about numerical optimization is provided in Sects. 4
and 5). The first cage was placed in the second cage, and both cages are made of a 3 mm thick aluminum sheet. Aluminum has low resistance and high microwave reflective characteristics. An electrical motor (DKM motors,
Korea) was used to rotate a desiccant wheel with a preset speed to control the microwave process. A three-phase
electrical power meter PowerLogic PM5110 (Schneider Electric, UK), with a measuring range of 3–30 kW and
with (0.5% FS) accuracy, was used to measure electrical power. A calibrated aluminum nozzle was used for
airflow measurements that were made according to the ISO/ANSI standards with high accuracy (RecoV, Italy). A differential pressure sensor Model 264 (Setra, USA) with high (± 0.25% FS) accuracy and with a range of
0–250 Pa was used to measure differential pressure across the nozzle. Pt100 RTD temperature sensors (Omega,
UK) were used to measure dry and wet bulb temperatures with an accuracy of ± 0.1 °C and with a range of
− 20–350 °C. An infrared temperature sensor OS-MINIUSB (Omega, UK) with an accuracy of 1 °C was used to
measure a rotating desiccant wheel temperature that can measure at the range of 0–250 °C. www.nature.com/scientificreports/ An automated data
logging system was created by authors on the software LabVIEW that logs data from Agilent 34970A. An air heat
exchanger was used to recover energy from outlet air, and air-flowing channels, ducts, and microwaving chamber
were thermally insulated with foam rubber. A microwave leak detector was running all the time for safety reasons. A specially made double Faraday cage was used in order to avoid microwave leakage to the surrounding area. The first cage is a multi-mode chamber where a desiccant wheel was placed, and its geometry was built based on
numerically optimized results (more detailed information about numerical optimization is provided in Sects. 4
and 5). The first cage was placed in the second cage, and both cages are made of a 3 mm thick aluminum sheet. Aluminum has low resistance and high microwave reflective characteristics. An electrical motor (DKM motors,
Korea) was used to rotate a desiccant wheel with a preset speed to control the microwave process. A three-phase
electrical power meter PowerLogic PM5110 (Schneider Electric, UK), with a measuring range of 3–30 kW and
with (0.5% FS) accuracy, was used to measure electrical power. A calibrated aluminum nozzle was used for
airflow measurements that were made according to the ISO/ANSI standards with high accuracy (RecoV, Italy). A diff
i l
M d l 264 (S
USA)
i h hi h ( 0 25% FS)
d
i h
f l
A differential pressure sensor Model 264 (Setra, USA) with high (± 0.25% FS) accuracy and with a range of
0–250 Pa was used to measure differential pressure across the nozzle. Pt100 RTD temperature sensors (Omega,
UK) were used to measure dry and wet bulb temperatures with an accuracy of ± 0.1 °C and with a range of
− 20–350 °C. An infrared temperature sensor OS-MINIUSB (Omega, UK) with an accuracy of 1 °C was used to
measure a rotating desiccant wheel temperature that can measure at the range of 0–250 °C. An automated data
logging system was created by authors on the software LabVIEW that logs data from Agilent 34970A. An air heat
exchanger was used to recover energy from outlet air, and air-flowing channels, ducts, and microwaving chamber
were thermally insulated with foam rubber. A microwave leak detector was running all the time for safety reasons. Scientific Reports | (2023) 13:7386 | https://doi.org/10.1038/s41598-023-34542-9 www.nature.com/scientificreports/ Figure 7. View of experimental microwave dehumidification pilot system. References Saputra, D. A. et al. Experimental investigation of desiccant wheel dehumidification control method for changes in regeneration
h
t i
t E
205 118109 (2020) f
g
y
27. Carter, J. W. & Wyszynski, M. L. The pressure swing adsorption drying of compressed air. Chem. Eng. Sci. 38, 1093–1099 (1983). 28 Saputra D A et al Experimental investigation of desiccant wheel dehumidification control method for changes in regeneration 27. Carter, J. W. & Wyszynski, M. L. The pressure swing adsorption drying of compressed air. Chem. Eng. Sci. 38, 1093–1099 (1983).i 8. Saputra, D. A. et al. Experimental investigation of desiccant wheel dehumidification control method for changes in regeneration
heat input. Energy 205, 118109 (2020). gy
29. Bathen, D. Physical waves in adsorption technology: An overview. Sep. Purif. Technol. 33, 163–177 (2003). 0. Lidström, P., Tierney, J., Wathey, B. & Westman, J. Microwave assisted organic synthesis: A review. Tetrahedron 57, 9225–9283
(2001).f 1. Zheng, Y., Meyer, G., Lanagan, M., Agrawal, D. & Cheng, J. A study of water sorption effects on the microwave dielectric properties
of calcium chloride/silica gel composites. Mater. Lett. 95, 157–159 (2013). g
p
32. Cheng, D. K. Field and Wave Electromagnetics (Addison-Wesley, 1991).f g
p
32. Cheng, D. K. Field and Wave Electromagnetics (Addison-Wesley, 1991). 33. Ybyraiymkul, D., Ng, K. C. & Кaltayev, A. Experimental and numerical study of effect of thermal management on storage capacity
of the adsorbed natural gas vessel. Appl. Therm. Eng. 125, 523–525 (2017). Data availability
The presented data is available from the corresponding author on reasonable request. The presented data is available from the corresponding author on reasonable request Received: 20 January 2023; Accepted: 3 May 2023 www.nature.com/scientificreports/ Mode with heat recovery is similar to mode without heat recovery; When the inlet and outlet Scientific Reports | (2023) 13:7386 | https://doi.org/10.1038/s41598-023-34542-9 www.nature.com/scientificreports/ temperatures became the same, air damper-1 and air damper-3 were closed, and air damper-2 and air damper-4
were opened to recover heat from outlet air. www.nature.com/scientificreports/ (a) Pictorial view of the microwave
dehumidification system, composed of the control panel, magnetron head (where microwaves are generated),
3-stub tuner, microwave chamber, and desiccant wheel. Enlarged images show pitch in honeycomb structure;
(b) Schematic diagram of the microwave dehumidification system. Heat recovery is affected by a heat exchanger
between exhaust and inlet air. Figure 7. View of experimental microwave dehumidification pilot system. (a) Pictorial view of the microwave
dehumidification system, composed of the control panel, magnetron head (where microwaves are generated),
3-stub tuner, microwave chamber, and desiccant wheel. Enlarged images show pitch in honeycomb structure;
(b) Schematic diagram of the microwave dehumidification system. Heat recovery is affected by a heat exchanger
between exhaust and inlet air. Desiccant (silica gel) captures moisture from the air (Fig. 1a). Then, the moisture in the desiccant is desorbed
by microwaves (Fig. 1b). The key feature of microwaves is that they can oscillate water molecules and desorb
from the adsorbent’s surface (silica gel). The lab-scale pilot microwave dehumidification system is illustrated in
Fig. 7a, and its schematic diagram is shown in Fig. 7b. A microwave generator (Fricke und Mallah, Germany)
was used to generate the microwaves. Frame and equipment were grounded with protective grounding to prevent
users from high voltage electrical hazards. Two modes were considered: the mode without heat recovering and
the mode with heat recovering from outlet air. Temperatures and differential pressure readings were logged
continuously by software Labview and Agilent 34970A for both modes. The desiccant wheel rotating motor speed
was set to the desired value, running only during desorption. Figure 7b demonstrates a setup diagram. The study
performed the following procedure without heat recovery: Air damper-1 and air damper-3 were opened, and air
damper-2 and air damper-4 were closed, letting the air bypass the heat-recovering device. Then, the honeycomb-
structured desiccant wheel was saturated with moisture at constant relative humidity and temperature at a regular
airflow rate until the inlet and outlet temperatures were the same: the same temperature and humidity indicated
equilibrium conditions. Consequently, microwaves were switched on for the preset time and preset power from
the control panel; the desorption process finishes when the outlet humidity ratio becomes lower than the inlet
humidity ratio. References 1. Davis, R. E., McGregor, G. R. & Enfield, K. B. Humidity: A review and primer on atmospheric moisture and human health. Environ
Res. 144, 106–116 (2016).ih 2. Mazzei, P., Minichiello, F. & Palma, D. HVAC dehumidification systems for thermal comfort: A critical review. Appl. Therm. Eng
25, 677–707 (2005).h 3. Standard 55 – Thermal Environmental Conditions for Human Occupancy. https://www.ashrae.org/technical-resources/bookstore/
standard-55-thermal-environmental-conditions-for-human-occupancy. andard-55-thermal-environmental-conditions-for-human-occupa p
y
4. ISO - ISO 7730:2005 - Ergonomics of the thermal environment — Analytical determination and interpretation of thermal comfort
using calculation of the PMV and PPD indices and local thermal comfort criteria. https://www.iso.org/standard/39155.html.hhi g
p
g
5. Thu, K., Mitra, S., Saha, B. B. & Srinivasa Murthy, S. Thermodynamic feasibility evaluation of hybrid dehumidification: Mechanica
vapour compression systems. Appl. Energy 213, 31–44 (2018). p
p
y
pp
gy
6. Marks, L. S. Mechanical engineers’ handbook (McGraw-Hill Book Company, 1916). 7. Jamil, M. A. et al. Experimental and normalized sensitivity based numerical analyses of a novel h
indirect evaporative cooler. Int. Commun. Heat Mass Transf. 125, 105327 (2021). p
f
8. Muttakin, M., Pal, A., Rupa, M. J., Ito, K. & Saha, B. B. A critical overview of adsorption kinetics for cooling and refrigeration
systems. Adv. Colloid Interface Sci. 294, 102468 (2021).i p
systems. Adv. Colloid Interface Sci. 294, 102468 (2021). y
f
9. Sultan, M., El-Sharkawy, I. I., Miyazaki, T., Saha, B. B. & Koyama, S. An overview of solid desiccant dehumidification and air
conditioning systems. Renew. Sustain. Energy Rev. 46, 16–29 (2015).i 10. Shamim, J. A., Hsu, W. L., Paul, S., Yu, L. & Daiguji, H. A review of solid desiccant dehumidifiers: Current status and near-term
development goals in the context of net zero energy buildings. Renew. Sustain. Energy Rev. 137, 110456 (2021). 11. Gao, D. C., Sun, Y. J., Ma, Z. & Ren, H. A review on integration and design of desiccant air-conditioning systems for overall
performance improvements. Renew. Sustain. Energy Rev. 141, 110809 (2021).i 2. Abd-Elhady, M. M., Salem, M. S., Hamed, A. M. & El-Sharkawy, I. I. Solid desiccant-based dehumidification systems: A critica
review on configurations, techniques, and current trends. Int. J. Refrig. 133, 337–352 (2022). i
3. Shabir, F. et al. Recent updates on the adsorption capacities of adsorbent-adsorbate pairs for heat transformation applications
Renew. Sustain. Energy Rev. 119, 109630 (2020). gy
4. Burhan, M. et al. References A universal mathematical methodology in characterization of materials for tailored design of porous surfaces
Front. Chem. 8, 601132 (2021). 5. Burhan, M., Shahzad, M. W. & Ng, K. C. A universal theoretical framework in material characterization for tailored porous surface
design. Sci. Rep. 9, 8773 (2019). ,
,
,
g,
p
design. Sci. Rep. 9, 8773 (2019). 6 Roussy G & Chenot P Selective energy supply to adsorbed water and nonclassical thermal process during microwave dehydration g
p
6. Roussy, G. & Chenot, P. Selective energy supply to adsorbed water and nonclassical thermal process during microwave dehydration
of zeolite. J. Phys. Chem. 85, 2199–2203 (1981).h y
7. Roussy, G., Zoulalian, A., Charreyre, M. & Thiebaut, J. M. How microwaves dehydrate zeolites. J. Phys. Chem. 88, 5702–5708
(1984).h 8. Polaert, I., Estel, L., Huyghe, R. & Thomas, M. Adsorbents regeneration under microwave irradiation for dehydration and volatile
organic compounds gas treatment. Chem. Eng. J. 162, 941–948 (2010). g
p
g
g
19. Tatsuo, O. & Akiko, W. Simple suppressing method of thermal runaway in microwave heating of zeolite and its application. PhysChemComm 4, 18–20 (2001). y
0. Ito, S. et al. Heat Transfer during microwave-assisted desorption of water vapor from zeolite packed bed. Dry. Technol. 30(15)
1707–1713. https://doi.org/10.1080/07373937.2012.714825 (2012). p
g
1. Kubota, M., Hanada, T., Yabe, S., Kuchar, D. & Matsuda, H. Water desorption behavior of desiccant rotor under microwave
irradiation. Appl. Therm. Eng. 31, 1482–1486 (2011). pph
g
22. Kubota, M., Hanada, T., Yabe, S. & Matsuda, H. Regeneration characteristics of desiccant rotor with microwave and hot-air heating. Appl. Therm. Eng. 50, 1576–1581 (2013).fi h
3. Cherbański, R. Calculation of critical efficiency factors of microwave energy conversion into heat. Chem. Eng. Technol. 34, 2083–
2090 (2011). 4. Reuß, J., Bathen, D. & Schmidt-Traub, H. Desorption by microwaves: Mechanisms of multicomponent mixtures. Chem. Eng
Technol. 25, 381–384 (2002). 5. Goyal, H., Mehdad, A., Lobo, R. F., Stefanidis, G. D. & Vlachos, D. G. Scaleup of a single-mode microwave reactor. Ind. Eng. Chem
Res. 59, 2516–2523 (2020). 6. Shahzad, M. W., Ybyraiymkul, D., Burhan, M., Oh, S. J. & Ng, K. C. An innovative pressure swing adsorption cycle. in AIP
Conference Proceedings vol. 2062 020057 (American Institute of Physics Inc., 2019).h f
g
(
y
,
)
27. Carter, J. W. & Wyszynski, M. L. The pressure swing adsorption drying of compressed air. Chem. Eng. Sci. 38, 1093–1099 (1983). 28. Author contributions
k d
l D. Y. worked on conceptualization, methodology, analysis, investigation, modeling, validation, writing the
original draft, and visualization. Q. C. worked on writing, reviewing, editing, formal analysis, and methodology. M. W. S. worked on writing, reviewing, editing, formal analysis and methodology. R. A. did a formal analysis. K. J. did a formal analysis. M. B. worked on investigation and visualization. F. H. A. worked on investigation and
visualization. K. C. N. worked on conceptualization, methodology, formal analysis, writing the original draft,
supervision, and funding acquisition. All authors reviewed the manuscript. Acknowledgementsh g
The authors gratefully acknowledge the generous funding from (1) the KAUST Cooling Initiative (KCI) project,
REP/1/3988-01-01, and REP/1/3988-04-01, (2) the Water Desalination and Reuse Center (WDRC), King
Abdullah University of Science and Technology (KAUST). Figures 1a,b were drawn by D.Y. https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 | www.nature.com/scientificreports/ Competing interests h p
g
The authors declare no competing interests. © The Author(s) 2023 Additional informationh Additional information
Supplementary Information The online version contains supplementary material available at https://doi.org/
10.1038/s41598-023-34542-9. Correspondence and requests for materials should be addressed to D.Y. Correspondence and requests for materials should be addressed to D.Y. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2023 https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 |
|
https://openalex.org/W4237745806
|
https://www.nature.com/articles/s41598-021-93477-1.pdf
|
English
| null |
Multi-Volume Hemacytometer
|
Research Square (Research Square)
| 2,021
|
cc-by
| 6,762
|
Ravangnam Thunyaporn1, Il Doh2 & Dong Woo Lee1,3* Cell counting has become an essential method for monitoring the viability and proliferation of
cells. A hemacytometer is the standard device used to measure cell numbers in most laboratories
which are typically automated to increase throughput. The principle of both manual and automated
hemacytometers is to calculate cell numbers with a fixed volume within a set measurement
range (105 ~ 106 cells/ml). If the cell concentration of the unknown sample is outside the range
of the hemacytometer, the sample must be prepared again by increasing or decreasing the cell
concentration. We have developed a new hemacytometer that has a multi-volume chamber with
4 different depths containing different volumes (0.1, 0.2, 0.4, 0.8 µl respectively). A multi-volume
hemacytometer can measure cell concentration with a maximum of 106 cells/ml to a minimum of
5 × 103 cells/ml. Compared to a typical hemacytometer with a fixed volume of 0.1 µl, the minimum
measurable cell concentration of 5 × 103 cells/ml on the multi-volume hemacytometer is twenty times
lower. Additionally, the Multi-Volume Cell Counting model (cell concentration calculation with the
slope value of cell number in multi-chambers) showed a wide measurement range (5 × 103 ~ 1 × 106 cells/
ml) while reducing total cell counting numbers by 62.5% compared to a large volume (0.8 µl-chamber)
hemacytometer. Cell counting is a method used to determine the concentration of cells in a sample volume. For example, in
cell culture experiments counting cells is vital to monitor cell viability, proliferation rate, immortalization or
transformation, seeding, or preparing cells in subsequent experiments, transfection, or infection1. Diagnosti-
cally, the concentration of white blood cells and red blood cells can assist those in the medical field to identify
specific diseases and can predict severity of disease states such as coronary atherosclerosis2–4, HIV detection
from whole-blood5, 6, etc.hi Cell counting is a method used to determine the concentration of cells in a sample volume. For example, in
cell culture experiments counting cells is vital to monitor cell viability, proliferation rate, immortalization or
transformation, seeding, or preparing cells in subsequent experiments, transfection, or infection1. Diagnosti-
cally, the concentration of white blood cells and red blood cells can assist those in the medical field to identify
specific diseases and can predict severity of disease states such as coronary atherosclerosis2–4, HIV detection
from whole-blood5, 6, etc.hi The hemacytometer was first invented over a century ago to count blood cells. Ravangnam Thunyaporn1, Il Doh2 & Dong Woo Lee1,3* Presently the hemacytometer
remains the gold standard for cell counting and is used by most laboratories to count cells as it is both inexpen-
sive and versatile7. A hemacytometer is made from optical glass for use under a microscope and consists of 2
parts: a thick glass slide and cover glass with a small gap to contain cell suspension in a 0.1 µl small grid area size
1 × 1 mm8. It cannot be accurately measured in high concentrations because the cells too crowned and difficult
to count. The cells have to be diluted and counting again9. h
g g
Over the last two decades, automated hemacytometers have been developed to increase the throughput of
counting a large number of samples in a short period of time. Automation also reduces human error in the cell
counting process and automatic hemacytometers are commonly used in laboratories for these reasons. In general,
hemacytometers are composed of a digital camera and a slide or cartridge containing the cell suspension to be
counted. The digital camera captures cells on the slide in the fixed volume and analyzes them using specialized
software for cell counters to calculate cell concentration based on the fixed volume6. Both manual and automated
hemacytometers use a fixed volume to measure cell concentration. Though manual and automated hemacytom-
eters are standard in most laboratories, they cannot measure low cell concentration samples and have a narrow
measurement range (105 ~ 106 cells/ml) due to the fixed volume containing cell. Most hemacytometers count
cells in 0.1 µl fixed volume where one cell exists at 104 cells/ml sample. The range of hemacytometer is approxi-
mately 105 cells/ml. For example, the Cedex HiRes Analyzer which is an automatic hemacytometer, can measure
cell density ranging from 3.13 × 105 to approximately 1.0 × 107 cells/ml10. The optimal range of Cellometer cell
counters is approximately 1 × 105 to 1 × 106 cells/ml11.hi pp
y
The previous study has shown that the size of the fixed control volume of cells in the suspension increases, the
number of cells is also increased which can be extended to measure cells in a wide range of concentrations12, 13. The limit of the range of cell concentration measurement is due to the fixed volume of the sample which causes
the minimum and maximum measurable cell concentration to decrease or increase. www.nature.com/scientificreports www.nature.com/scientificreports 1Department of Biomedical Engineering, Konyang University, Daejeon 35365, Republic of Korea. 2Korea Research
Institute of Standards and Science, Daejeon, Korea. 3Central R & D Center, Medical & Bio Decision (MBD) Co., Ltd,
Suwon, Republic of Korea. *email: mems@konyang.ac.kr Scientific Reports | (2021) 11:14106 Multi‑volume hemacytometer
OPEN Ravangnam Thunyaporn1, Il Doh2 & Dong Woo Lee1,3* Ravangnam Thunyaporn1, Il Doh2 & Dong Woo Lee1,3* A typical 0.1 µl fixed vol-
ume (most used volume in hemacytometers) contains 10 ~ 500 cells at 105 ~ 5 × 106 cells/ml while a 0.2 µl fixed
volume contains 10 ~ 500 cells at 5 × 104 ~ 2.5 × 106 cells/ml. Therefore, if a sample of unknown concentration 1Department of Biomedical Engineering, Konyang University, Daejeon 35365, Republic of Korea. 2Korea Research
Institute of Standards and Science, Daejeon, Korea. 3Central R & D Center, Medical & Bio Decision (MBD) Co., Ltd,
Suwon, Republic of Korea. *email: mems@konyang.ac.kr | https://doi.org/10.1038/s41598-021-93477-1 www.nature.com/scientificreports/ Figure 1. Schematic view of the multi-volume hemacytometer. (a) The counting chamber was attached to a
glass slide that had an inlet for pipetting and an outlet for airflow. The cell counting chamber consisted of four
chambers of varying depths including 0.1, 0.2, 0.4, 0.8 mm respectively. (b) The PDMS fabrication device. (c)
Counting cell number in each chamber are proportional to the volume of the chamber when a sample flowed in
the chambers having different volumes. In case of low cell concentration sample, slope which mean cells per unit
volume is small. However, in high cell concentration sample, the slop is large due to large different of counting
cells per chamber. Figure 1. Schematic view of the multi-volume hemacytometer. (a) The counting chamber was attached to a
glass slide that had an inlet for pipetting and an outlet for airflow. The cell counting chamber consisted of four
chambers of varying depths including 0.1, 0.2, 0.4, 0.8 mm respectively. (b) The PDMS fabrication device. (c)
Counting cell number in each chamber are proportional to the volume of the chamber when a sample flowed in
the chambers having different volumes. In case of low cell concentration sample, slope which mean cells per unit
volume is small. However, in high cell concentration sample, the slop is large due to large different of counting
cells per chamber. is out of range of the hemacytometer, the sample must be prepared again by increasing or decreasing the cell
concentration.h To solve this problem, the current study proposed the use of a multi-volume hemacytometer. This device
counts cells in four chambers at volumes of 0.1, 0.2, 0.4, 0.8 µl as shown in Fig. 1. A multi-volume cell counter
can measure cell concentrations ranging from 106 cells/ml to 5 × 103 cells/ml. Ravangnam Thunyaporn1, Il Doh2 & Dong Woo Lee1,3* Compared to a hemacytometer
having a fixed volume of 0.1 µl, the minimum measurable cell concentration is 5 × 103 cells/ml which is twenty
times lower. Additionally, the Multi-Volume Cell Counting model (cell concentration calculation with the slope
value of the cell number in multi-chambers) was also validated to measured cell concentration and increases the
measurement speed as a sample of only 1.5 µl can measure the wide range of cell concentrations. Results To compare the accuracy and range of cell concentration measurement between the conventional hemacytom-
eter and the proposed multi-volume hemacytometer, we measured five samples at 103, 5 × 103, 104, 105, and 106
cells/ml. In the case of the multi-volume hemacytometer, there are two cell concentration calculation models
necessary to determine the cell concentration. In the conventional Fixed-Volume Cell Counting (FVCC), the
cell concentrations were calculated by counting cells in each chamber at different volumes. In the Multi-Volume
Cell Counting (MVCC) model, the cell concentrations were calculated using the slope (cell number growth per
volume) in the graph of cell number by volume as show in Fig. 1c. Fixed‑volume cell counting (FVCC) model. A hemacytometer has a ruled surface below the cover glass
of 0.1 mm which limits the volume of liquid suspension to 0.1 µl per one of nine squares and restricts it to accu- Scientific Reports | (2021) 11:14106 | https://doi.org/10.1038/s41598-021-93477-1 www.nature.com/scientificreports/ Table 1. Comparison of cell concentration measurements between a standard hemacytometer and a multi-
volume hemacytometer. *FVCC Model: Fixed-Volume Cell Counting Model. Results **MVCC Model: Multi-Volume
Sample
Concentration unit
Hemacytometer
(volume: 0.5 µl)
Multi-volume hemacytometer
FVCC Model*
MVCC model **
(volume: 1.5 µl)
Chamber 1 (volume:
0.5 µl)
Chamber 2 (volume:
1 µl)
Chamber 3 (volume:
2 µl)
Chamber 4 (volume:
4 µl)
Mean ± SD (%RSD)
Mean ± SD (%RSD)
Mean ± SD (%RSD)
Mean ± SD (%RSD)
Mean ± SD (%RSD)
Fit values ± SD
(%RSD)
1
106 cells/ml
1.08 ± 0.11 (9.7%)
1.14 ± 0.02 (1.6%)
0.99 ± 0.07 (7.0%)
1.14 ± 0.07 (5.7%)
1.02 ± 0.03 (3.3%)
1.04 ± 0.03 (2.5%)
2
1.18 ± 0.13 (10.8%)
1.14 ± 0.11 (9.3%)
0.94 ± 0.06 (6.7%)
1.08 ± 0.10 (9.6%)
1.14 ± 0.05 (4.5%)
1.12 ± 0.04 (3.6%)
3
1.05 ± 0.09 (8.5%)
1.07 ± 0.16 (14.9%)
1.09 ± 0.15 (17%)
1.08 ± 0.16 (14.4%)
1.07 ± 0.01 (1.3%)
1.07 ± 0.03 (3.1%)
4
1.16 ± 0.12 (10.5%)
0.88 ± 0.06 (6.5%)
0.94 ± 0.04 (4.6%)
1.08 ± 0.15 (14.0%)
1.12 ± 0.07 (6.5%)
1.10 ± 0.03 (3.0%)
5
1.19 ± 0.14 (11.3%)
1.12 ± 0.17 (15.2%)
0.92 ± 0.06 (6.8%)
0.94 ± 0.07 (7.2%)
1.05 ± 0.13 (13.3%)
1.03 ± 0.11 (10.4%)
AVG
1.13 ± 0.06
1.07 ± 0.11
0.98 ± 0.08
1.07 ± 0.08
1.08 ± 0.06
1.07 ± 0.04
%RSD
5.61
10.51
6.89
6.86
4.54
3.64
1
105 cells/ml
1.38 ± 0.35 (25.3%)
1.26 ± 0.27 (21.4%)
1.14 ± 0.20 (17.4%)
0.94 ± 0.12 (11.5%)
1.22 ± 0.10 (8.3%)
1.17 ± 0.02 (1.5%)
2
1.24 ± 0.42 (34.0%)
1.12 ± 0.34 (30.5%)
1.07 ± 0.15 (14.3%)
1.18 ± 0.30 (25.7%)
1.26 ± 0.08 (6.4%)
1.23 ± 0.09 (7.7%)
3
1.20 ± 0.43 (35.8%)
1.16 ± 0.49 (42.1%)
1.28 ± 0.47 (36.9%)
1.20 ± 0.27 (22.3%)
1.21 ± 0.11 (9.0%)
1.21 ± 0.11 (9.4%)
4
1.22 ± 0.35 (28.6%)
1.16 ± 0.43 (37.4%)
1.21 ± 0.19 (15.3%)
1.28 ± 0.29 (22.5%)
1.26 ± 0.12 (9.6%)
1.26 ± 0.13 (10.2%)
5
1.20 ± 0.53 (44.1%)
1.00 ± 0.16 (15.8%)
1.28 ± 0.27 (21.1%)
1.13 ± 0.23 (19.9%)
1.30 ± 0.10 (8.0%)
1.26 ± 0.10 (8.1%)
AVG
1.25 ± 0.08
1.14 ± 0.09
1.20 ± 0.09
1.15 ± 0.13
1.25 ± 0.04
1.23 ± 0.04
%RSD
6.06
8.23
7.63
11.10
6.06
3.17
1
104 cells/ml
1.40 ± 1.14 (81.4%)
1.40 ± 1.14 (81.4%)
1.20 ± 0.57 (47.5%)
1.15 ± 0.14 (11.9%)
1.18 ± 0.14 (12.1%)
1.17 ± 0.10 (8.7%)
2
1.00 ± 0.71 (70.7%)
1.00 ± 1.00 (100%)
1.10 ± 0.65 (59.3%)
1.15 ± 0.42 (36.4%)
1.20 ± 0.19 (15.8%)
1.22 ± 0.12 (9.5%)
3
0.60 ± 0.55 (91.3%)
1.40 ± 0.89 (63.9%)
1.20 ± 0.57 (47.5%)
1.15 ± 0.38 (33.0%)
1.15 ± 0.27 (23.6%)
1.16 ± 0.22 (19.0%)
4
1.00 ± 0.71 (70.7%)
1.20 ± 0.83 (69.7%)
1.20 ± 0.57 (47.5%)
1.10 ± 0.29 (25.9%)
1.28 ± 0.29 (22.4%)
1.24 ± 0.18 (14.3%)
5
1.00 ± 0.71 (70.7%)
0.80 ± 0.45 (55.9%)
1.00 ± 0.50 (50.0%)
1.15 ± 0.52 (45.1%)
1.18 ± 0.59 (22.1%)
1.16 ± 0.23 (19.9%)
AVG
1.00 ± 0.28
1.16 ± 0.26
1.14 ± 0.09
1.14 ± 0.02
1.20 ± 0.05
1.19 ± 0.04
%RSD
28.28
22.48
7.85
1.96
4.02
3.19
1
5 × 103
cells/ml
4.00 ± 5.58
(136.9%)
8.00 ± 4.47
(55.9%)
2.00 ± 2.74
(136.9%)
7.00 ± 3.26
(46.6%)
5.50 ± 1.43
(25.9%)
5.65 ± 1.27
(22.4%)
2
6.00 ± 5.48
(91.3%)
4.00 ± 8.94
(223.6%)
9.00 ± 9.62
(106.9%)
6.00 ± 2.85
(47.5%)
5.25 ± 2.85
(54.3%)
5.54 ± 3.06
(55.3%)
3
2.00 ± 4.47
(223.6%)
4.00 ± 8.94
(223.6%)
4.00 ± 4.18
(104.6%)
7.50 ± 2.50
(33.3%)
6.00 ± 1.63
(27.2%)
6.16 ± 1.17
(19.0%)
4
2.00 ± 4.47
(223.6%)
6.00 ± 8.94
(149.1%)
7.00 ± 8.37
(119.5%)
6.00 ± 1.37
(22.8%)
6.25 ± 1.77
(28.3%)
6.24 ± 1.67
(28.8%)
5
8.00 ± 8.37
(104.6%)
2.00 ± 4.47
(223.6%)
6.00 ± 2.24
(37.3%)
5.50 ± 2.09
(38.0%)
5.25 ± 2.05
(39.1%)
5.29 ± 1.90
(36.0%)
AVG
4.40 ± 2.61
4.80 ± 2.28
5.60 ± 2.70
6.40 ± 0.82
5.65 ± 0.45
5.78 ± 0.41
%RSD
59.27
47.51
48.25
12.84
8.04
7.06
1
103 cells/ml
0
0
0
1.50 ± 1.37
(91.3%)
1.50 ± 0.56
(37.3%)
1.41 ± 0.33
(23.6%)
2
2.00 ± 4.47
(223.6%)
0
0
1.50 ± 1.37
(91.3%)
1.25 ± 0.88
(70.7%)
1.22 ± 0.85
(69.9%)
3
4.00 ± 5.48
(136.9%)
2.00 ± 4.47
(223.6%)
1.00 ± 2.24
(223.6%)
2.00 ± 1.77
(70.7%)
2.00 ± 0.68
(34.2%)
2.05 ± 0.83
(40.6%)
4
2.00 ± 4.47
(223.6%)
2.00 ± 4.47
(223.6%)
1.00 ± 2.24
(223.6%)
2.50 ± 3.26
(163%)
1.75 ± 1.43
(81.4%)
1.76 ± 0.70
(39.4%)
5
0
0
1.00 ± 2.24
(223.6%)
2.50 ± 3.06
(122%)
1.75 ± 2.43
(81.4%)
1.84 ± 0.89
(48.3%)
AVG
1.60 ± 1.67
0.80 ± 1.10
0.60 ± 0.55
2.00 ± 0.50
1.65 ± 0.29
1.66 ± 0.33
%RSD
104.58
136.93
91.28
25.00
17.28
20.10 Table 1. Table 1. Comparison of cell concentration measurements between a standard hemacytometer and a multi-
volume hemacytometer. *FVCC Model: Fixed-Volume Cell Counting Model. **MVCC Model: Multi-Volume
Cell Counting Model. Bold values Cell concentration measurement quality control pass (%RSD < 15%). Results Microscopic cell images in multiple volumes (0.1, 0.2, 0.4, and 0.8 µl) in (a) low concentration at 10
cells/ml, and (b) high concentration at 106 cells/ml liquid sample (under 40 × magnification). Figure 2. Microscopic cell images in multiple volumes (0.1, 0.2, 0.4, and 0.8 µl) in (a) low concentration at 104
cells/ml, and (b) high concentration at 106 cells/ml liquid sample (under 40 × magnification). chamber 1 in area 1 mm2 contained a volume of 0.1 µl equal to the volume of the standard hemacytometer. In
the low volume chamber, the number of cells was not great enough to calculate the cell concentration. In con-
trast, as the volume of the chamber increased, the cells also increased enough to allow the cell concentration to
be calculated. Therefore, chamber 4 has a wide measurement range and was able to be measured at the lowest
concentration at 5 × 103 cells/ml. However, in the maximum cell concentration of 106 cells/ml, it was difficult to
count a high number of cells in a large volume (0.8 µl) and repeat the measurement five times. Therefore, the user
must select the appropriate chamber volume according to the sample. chamber 1 in area 1 mm2 contained a volume of 0.1 µl equal to the volume of the standard hemacytometer. In
the low volume chamber, the number of cells was not great enough to calculate the cell concentration. In con-
trast, as the volume of the chamber increased, the cells also increased enough to allow the cell concentration to
be calculated. Therefore, chamber 4 has a wide measurement range and was able to be measured at the lowest
concentration at 5 × 103 cells/ml. However, in the maximum cell concentration of 106 cells/ml, it was difficult to
count a high number of cells in a large volume (0.8 µl) and repeat the measurement five times. Therefore, the user
must select the appropriate chamber volume according to the sample. Multi‑volume cell counting (MVCC) model. Figure 3 shows the cell number measurement by chamber
volume and the slope in the Multi-Volume Cell Counting (MVCC) model. The slope indicates the cell number
per volume, reflecting the cell concentration. The slope (cell numbers per volume) was calculated by the cell
number in 0.1, 0.2, 0.4, and 0.8 µl chambers. Dotted lines indicate a fit for standard error. As the sample cell
concentration decreased, the spacing between the dotted lines increased. Results Comparison of cell concentration measurements between a standard hemacytometer and a multi-
volume hemacytometer. *FVCC Model: Fixed-Volume Cell Counting Model. **MVCC Model: Multi-Volum
Cell Counting Model. Bold values Cell concentration measurement quality control pass (%RSD < 15%). rately measure the concentration of cells in the range of 2 × 105 ~ 2.5 × 106 cells/ml9. As shown in Table 1, in the
current experiment the hemacytometer shows a large standard deviation (more than 15%) in the samples lower
than 105 cells/ml. The proposed device has several chambers with different volumes such as 0.1, 0.2, 0.4, and
0.8 µl to contain the multi-volume of cells in suspension. Figure 2 shows that the number of cells varied in con-
junction with the volume. The number of cells increased with the volume of the chamber. As shown in Table 1,
chamber 1, 2, 3, 4, and the MVCC model were used to measure cell concentration. In the current experiments, rately measure the concentration of cells in the range of 2 × 105 ~ 2.5 × 106 cells/ml9. As shown in Table 1, in the
current experiment the hemacytometer shows a large standard deviation (more than 15%) in the samples lower
than 105 cells/ml. The proposed device has several chambers with different volumes such as 0.1, 0.2, 0.4, and
0.8 µl to contain the multi-volume of cells in suspension. Figure 2 shows that the number of cells varied in con-
junction with the volume. The number of cells increased with the volume of the chamber. As shown in Table 1,
chamber 1, 2, 3, 4, and the MVCC model were used to measure cell concentration. In the current experiments, https://doi.org/10.1038/s41598-021-93477-1 Scientific Reports | (2021) 11:14106 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ ntificreports/
Figure 2. Microscopic cell images in multiple volumes (0.1, 0.2, 0.4, and 0.8 µl) in (a) low concentration at 104
cells/ml, and (b) high concentration at 106 cells/ml liquid sample (under 40 × magnification). chamber 1 in area 1 mm2 contained a volume of 0.1 µl equal to the volume of the standard hemacytometer
Figure 2. Microscopic cell images in multiple volumes (0.1, 0.2, 0.4, and 0.8 µl) in (a) low concentration at 1
cells/ml, and (b) high concentration at 106 cells/ml liquid sample (under 40 × magnification). Figure 2. Results In the repeated cell concentration
measurement of 5 × 103 cells/ml sample, the MVCC model indicates the standard deviation of cell concentration
is lower than 15%, while the FVCC model indicates a higher standard deviation. As shown in Fig. 4, measure- https://doi.org/10.1038/s41598-021-93477-1 Scientific Reports | (2021) 11:14106 | www.nature.com/scientificreports/ Figure 3. The cell numbers according to chamber volume in different cell concentration. (a) 106 cells/ml, (b)
105 cells/ml, (c) 104 cells/ml, (d) 5 × 103 cells/ml, and (e) 103 cells/ml. Each point is the average of cell number
and error bar is standard deviation in five replications. The slopes from the linear equations were calculated for
measuring cell concentration. Figure 3. The cell numbers according to chamber volume in different cell concentration. (a) 106 cells/ml, (b)
105 cells/ml, (c) 104 cells/ml, (d) 5 × 103 cells/ml, and (e) 103 cells/ml. Each point is the average of cell number
and error bar is standard deviation in five replications. The slopes from the linear equations were calculated for
measuring cell concentration. ment using the conventional hemacytometer shows that when the sample concentration was low, the relative
standard deviation of measuring cell concentration increased significantly. The measurement range of the con-
ventional hemacytometer is 106 ~ 105 cells/ml, and using a 0.8 µl chamber showed a wide measurement range
of 106 ~ 5 × 103 cells/ml. However, in 106 cells/ml, the hemacytometer using a 0.8 µl chamber had too many cells
to count manually (approximately 4000). Figure 4 shows how the proposed MVCC model reduced the num-
ber of cell counts needed to measure cell concentration without reducing the measurement range. The MVCC
model measured 5 × 103 ~ 106 cells/ml within a 10% standard deviation and reduced the counting cell number by
about 62.5%. The MVCC model reduced the counting burden in large volumes as a total volume of only 1.5 µl
(0.1 + 0.2 + 0.4 + 0.8 µl) was needed to measure the cell concentration at a wide range of concentrations. Discussion As well known, the hemacytometer is a basic device used to count cells in most laboratories. Many times a cell
sample needs to be re-prepared by either increasing or decreasing the cell concentration because the unknown
cell concentration is outside the range of the hemacytometer. Thus, our study proposed a novel device to measure
cell concentration using a multi-volume hemacytometer. This device is designed to have a multi-chamber that
can contain multi-volume such as 0.1, 0.2, 0.4, and 0.8 μl, expanding the contained volume for a hemacytometer. https://doi.org/10.1038/s41598-021-93477-1 Scientific Reports | (2021) 11:14106 | www.nature.com/scientificreports/ Figure 4. Comparison of the percent of relative standard deviation of cell number in each concentration
between a standard hemacytometer and a multi-volume hemacytometer with four chambers and the MVCC
model. Figure 4. Comparison of the percent of relative standard deviation of cell number in each concentration
between a standard hemacytometer and a multi-volume hemacytometer with four chambers and the MVCC
model. Compared to a standard hemacytometer with a fixed volume of 0.1 μl, the minimum measurable cell concentra-
tion for the multi-chamber hemacytometer is 5 × 103 cells/ml, which is twenty times lower.hf Compared to a standard hemacytometer with a fixed volume of 0.1 μl, the minimum measurable cell concentra-
tion for the multi-chamber hemacytometer is 5 × 103 cells/ml, which is twenty times lower. Th
b
h
l
h
b
h
h
h
b
h
h ld d ff
f
ll This is because the multi-chamber hemacytometer has 4 chambers that can hold different amounts of cell
samples from low to high volume (0.1, 0.2, 0.4, and 0.8 µl). Cell concentration in each chamber can be calcu-
lated by the average number of cells in the area divided by the fixed volume. The number of cells increases as
the chamber volume increases, i.e. at fixed volume 0.1 μl, there are 10 ~ 500 cells at 105 ~ 5 × 106 cells/ml while at
fixed volumes 0.2 μl, there are 10 ~ 500 cells at 5 × 104 ~ 2.5 × 106 cells/ml. This multi-volume chamber expands
the range of the cell concentration measurements compared to the standard hemacytometer. Discussion When unknown
sample was loaded in multi-volume hemacytometer, the cell concentration can be measured by selecting an
appropriate chamber with the number of cells (10 to 200 cells/chamber) out of four chambers.fh pp
p
In this study, phosphate buffered saline (PBS) is used to dilute the cell concentration. Therefore, the sample
viscosity is similar to PBS, allowing the sample to flow easily into the chip. At low cell concentrations, high-
volume chambers can provide a clearer accurate cell count cell on bottom than high cell concentrations. There-
fore, at high cell concentrations in high volume chambers, we recommend changing the focus to verify that the
cells are completely precipitated before taking images. Basically, we use a cancer cell line which concentrations
5 × 103 ~ 106 cells/ml in PBS. In the case of blood cells, RBC has a concentration of approximately 5 × 1012 cells/L
(5 × 106 cells/µl)14. This sample must be diluted to 104 ~ 106 cells/ml (1:10,000 dilution) with PBS before counting
the cell by using this device. y
g
In addition, the multi-volume hemacytometer proposed the Multi-Volume Cell Counting (MVCC) model for
cell concentration measurement. Chamber 4 contains a maximum volume of 0.8 μl and has a wide measurement
range that able to measure the lowest concentration at 5 × 103 cells/ml. Nonetheless, if cell concentration was high
around 1 × 106 cells/ml, cell counting only in 0.8 μl-chamber was difficult and used a lot of time to determine
the accuracy of the cell concentration. So, we proposed and validated the Multi-Volume Cell Counting (MVCC)
model that cell concentration calculation with the slope value of cell numbers in multi-chambers. The MVCC
showed a wide measurement range of 5 × 103 ~ 1 × 106 cells/ml while reducing total cell counting numbers by
approximately 62.5% compared to a 0.8 μl-chamber hemacytometer. Materials and methods Trypsin (2 ml) was added, and the cells were placed
in an incubator for 3 min. Cells were then washed with phosphate-buffered saline (PBS) and centrifuged. PBS
was removed and 1 ml of DMEM was added. The number of cells in the suspension was determined by manual
counting using a standard hemacytometer. Cell samples were diluted in a 9:1 ratio (PBS: cells suspension) to
almost the target cell concentration range, such as 106, 105, 104, 5 × 103, 103 cells/ml. Cell concentration measurement. For the multi-volume hemacytometer two cell concentration calcu-
lation models are needed. The Fixed-Volume Cell Counting (FVCC) model counted cells in a fixed volume of
0.1 µl which was repeated five times. The FVCC model is the conventional method used in a hemacytometer. The
Multi-Volume Cell Counting (MVCC) model counted cells in multiple different volumes at once by calculating
cells over an increasing slope per volume to measuring cell concentration. A 10 µl sample containing cells were
loaded in the hemacytometer to standardize samples. After direct counting using a microscope with 5 squares
(4 corners and the center squares), we calculated the mean and standard deviation of cell concentrations. The
number of cells is divided by the chamber’s volume of 0.1 µl using the following formula: (1)
concentration (cells/ml) = Number of cells
0.1 µl (1)
concentration (cells/ml) = Number of cells
0.1 µl (1) Cell suspension of 35 µl were loaded in chip and counted after 5 min. For 5 min, most of the cells descend
to the bottom of the chamber. So, all cells were counted from the bottom of the chip with a microscope focused
on the bottom. A microscope with a 40 × objective capture 5 images of different areas in each chamber without
changing focus. Each captured image has an area of 1 mm2. In the current experiment, we designed the four
chambers to have heights of 0.1, 0.2, 0.4, and 0.8 mm. The equation used to calculate cell concentration is below. Chamber 1 (volume: 0.1 µl): concentration (cells/ml) = average of counted cells × 1000
0.1 µl (2) (4)
Chamber 3 (volume: 0.4 µl): concentration (cells/ml) = average of counted cells × 1000
0.4 µl (4) (5) Cell concentrations were calculated by taking five images in a chamber. According to the sample cell con-
centrations, chambers among the four were selected to measure the cell concentration. Materials and methods Multi‑volume hemacytometer design. The multi-volume hemacytometer is designed to address the
problem of loading cell samples in unknown concentrations which can fall outside the range of a standard
hemacytometer. The proposed design defines multiple volumes as it consists of 4 chambers with different depths
as follows: chamber 1 depth 0.1 mm, chamber 2 depth 0.2 mm, chamber 3 depth 0.4 mm, and chamber 4 depth
0.8 mm which are connected within the same sheet. Each chamber has an area of 3 × 3 mm2 and connected by
slope channel. The slope channel has 3 mm wide 1 mm length. The height of slope channel increases gradually
from the lower chamber height to the higher chamber height as shown in Fig. 1. We designed the slope channel
to avoid air bubble trap when the chamber height suddenly increases. The air bubbles make the cells bad disper-
sion in chambers. The slope also helps the cells spread continuously from chamber to chamber without damag-
ing the flow. The counter chamber and slop channel are made using the PDMS production process after which
the PDMS sheets are bonded on a slide glass (Fig. 1). https://doi.org/10.1038/s41598-021-93477-1 Scientific Reports | (2021) 11:14106 | www.nature.com/scientificreports/ Multi‑volume hemacytometer fabrication. This cell counter sheet is made using polydimethylsiloxane
(PDMS) with s SYLGARD 184 silicone elastomer base and SYLGARD 184 silicone elastomer curing agent. The
mold was made of aluminium by computer numerical control (CNC) machining. In the first step silicone elasto-
mer base and silicone curing agent were mixed at a 10:1 ratio and a desiccator was used to burst any bubbles for
15 min before the elastomer mix was poured into a cell counter aluminium mold and placed inside a desiccator
for 15 min. The mold was then baked in the oven at 65 °C for 4 h. The PDMS cell counter sheet was then placed
on a glass slide and sealed to the glass slide with an oxygen plasma bonder for 2 min under a pressure of 80 W. Cell preparation. Cancer cell line A-549 was counted in the current experiment. A-549 cells were cultured
in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1%
Anti-Anti (Antibiotic–Antimycotic (100x)) and incubated at 37 °C in 5% CO2. For harvest, cells were rinsed
with 0.25% Trypsin/0.53 mM EDTA to detach the cell layers. References Huang, L.-C. et al. Validation of cell density and viability assays using Cedex automated cell counter. Biologicals 38(3), 393–400
(2010). 1. Chan, L.L.-Y. et al. A novel image-based cytometry method for autophagy detection in living cells. Autophagy 8(9), 1371–1382
(2012).l (2012). 12. Lee, D. W., Yi, S. & Cho, Y.-H. A flow rate independent cell concentration measurement chip using electrical cell counters across
a fixed control volume. J. Microelectromech. Syst. 17(1), 139–146 (2008). 12. Lee, D. W., Yi, S. & Cho, Y.-H. A flow rate independent cell concentration measurement chip using electrical cell counters a
a fixed control volume. J. Microelectromech. Syst. 17(1), 139–146 (2008).l a fixed control volume. J. Microelectromech. Syst. 17(1), 139 146 (2008). 13. Lee, Y., Kim, B. & Choi, S. On-chip cell staining and counting platform for the rapid detection of blood cells in cerebrospinal fluid. Sensors 18(4), 1124 (2018). i
J
y
( )
(
)
13. Lee, Y., Kim, B. & Choi, S. On-chip cell staining and counting platform for the rapid detection of blood cells in cerebrospinal fluid. Sensors 18(4), 1124 (2018). i
13. Lee, Y., Kim, B. & Choi, S. On-chip cell staining and counting platform for the rapid detection of blood
Sensors 18(4), 1124 (2018). 14. Dean, L. & Dean, L. Blood Groups and Red Cell Antigens Vol. 2 (NCBI Bethesda, 2005). 14. Dean, L. & Dean, L. Blood Groups and Red Cell Antigens Vol. 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 2. Total cell counts from cell concentration measurements. *FVCC Model: Fixed-Volume Cell Counting
Model. **MVCC Model: Multi-Volume Cell Counting Model. Bold values Cell concentration measurement
Measure-ment method
concentration (cells/
ml)
Hema-cytometer
(volume: 0.1 ul *5 )
Multi-volume hemacytometer
FVCC model*
MVCC Model **
(volume: 0.1 ul + 0.2 ul + 0.4 ul + 0.8 ul)
Chamber 1 (volume:
0.1 ul * 5)
Chamber 2 (volume:
0.2 ul * 5)
Chamber 3 (volume:
0.4 ul * 5)
Chamber
4 (volume:
0.8 ul * 5)
106
500
500
1000
2000
4000
1500
105
50
50
100
200
400
150
104
5
5
10
20
40
15
5 × 103
2.5
2.5
5
10
20
7.5
103
0.5
0.5
1
2
4
1.5 Table 2. Total cell counts from cell concentration measurements. *FVCC Model: Fixed-Volume Cell Counting
Model. **MVCC Model: Multi-Volume Cell Counting Model. Bold values Cell concentration measurement
range. too many cells in the high concentration cell sample to be counted. In the proposed MVCC model, the total cell
count was being reduced by more than 50% while the cell concentration measurement range remained the same. too many cells in the high concentration cell sample to be counted. In the proposed MVCC model, the total cell
count was being reduced by more than 50% while the cell concentration measurement range remained the same. Received: 31 January 2021; Accepted: 24 June 2021 Acknowledgementsh g
This work was supported by the Korea Medical Device Development Fund grant funded by the Korea government
(the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare,
the Ministry of Food and Drug Safety) (Project Number: KMDF_PR_20200901_0135-2021). This work was sup-
ported by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of
Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of
Food and Drug Safety) (Project Number: KMDF_PR_20200901_0153-2021). This research was supported by a
grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute,
funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI17C2412). Author contributions R.T. performed experiment and wrote paper. I.D. advised experimental problems and performed data analysis
D.W.L. supervised the experiments and wrote discussion. References 1. Au Ongena, K. et al. Determining cell number during cell culture using the scepter cell counter. JoVE 45, e2204 (2010). 2 B i
C Q
lit
t
N
t
i bl
d
ll
ti
I t J L b H
t l 31(3) 277 297 (2009) 2. Briggs, C. Quality counts: New parameters in blood cell counting. Int. J. Lab. Hematol. 31(3), 277–297 (2009). gg
Q
y
p
g
( )
(
)
3. Ates, A. H. et al. Total white blood cell count is associated with the presence, severity and extent of coronary atherosclerosis d
by dual-source multislice computed tomographic coronary angiography. Cardiol. J. 18(4), 371–377 (2011).l gg
y
g
3. Ates, A. H. et al. Total white blood cell count is associated with the presence, severity and extent of coronary atherosclerosis detected
by dual-source multislice computed tomographic coronary angiography. Cardiol. J. 18(4), 371–377 (2011). y
p
g
p
y
g g
p y
4. Lu, H. et al. High throughput single cell counting in droplet-based microfluidics. Sci. Rep. 7(1), 1366 (2017).l g
g
g
gl
5. Phillips, E. A. et al. Microfluidic rapid and autonomous analytical device (microRAAD) to detect HIV from whole blood samples
Lab Chip 19(20), 3375–3386 (2019). l
5. Phillips, E. A. et al. Microfluidic rapid and autonomous analytical device (microRAAD) to detect HIV from whole blood samples
Lab Chip 19(20), 3375–3386 (2019). p
6. Adams, S. et al. Performance evaluation of the point-of-care INSTITM HIV-1/2 antibody test in early and established HIV infec-
tions. J. Clin. Virol. 91, 90–94 (2017). p
6. Adams, S. et al. Performance evaluation of the point-of-care INSTITM HIV-1/2 antibody test in early and established HIV infec-
tions. J. Clin. Virol. 91, 90–94 (2017). 7. Cadena-Herrera, D. et al. Validation of three viable-cell counting methods: Manual, semi-automated, and automated. Biotechnol
Rep. 7, 9–16 (2015). p
(
)
8. Absher, M. Hemocytometer counting. In Tissue Culture 395–397 (Elsevier, 1973). 8. Absher, M. Hemocytometer counting. In Tissue Culture 395–397 (Elsevier, 1973). 8. Absher, M. Hemocytometer counting. In Tissue Culture 395–397 (Elsevier, 1973). b d
h
h
ll
d
b
h
l
l d 9. Vembadi, A., Menachery, A. & Qasaimeh, M. A. Cell cytometry: Review and perspective on biotechnological advances. F
Bioeng. Biotechnol. 7, 147 (2019). g
10. Huang, L.-C. et al. Validation of cell density and viability assays using Cedex automated cell counter. Biologicals 38(3), 393–400
(2010). g
0. Materials and methods If the concentration was
low, a larger volume chamber was needed. If the concentration was high, 0.1 µl would be enough to measure the
cell concentration. However, there were too many cells to count in a 0.8 µl chamber. Therefore, the volume of
the chamber was chosen according to the cell concentration. g
In the proposed multi-chamber hemacytometer, the Multi-Volume Cell Counting model (MVCC model)
was applied to cell concentration. The cell numbers in each chamber from four images were plotted according to
chamber volume and the slope of a linear equation between counted cell numbers and volume in multi-chambers
was calculated to determine the concentration. The slope indicates the number of cells per volume (ml) in the
graph of cell number by volume. In the current experiment, we counted four pictures in each chamber to find the
cell concentration. The multi-volume model is derived from calculating the slope of the linear equation y = mx;
m = slope and setting the intercept (x, y) at 0 as the counted cell number starts at 0 cell/ml. (Fig. 1) In the FVCC
model, the large volume chamber has a wide cell concentration measurement range (Table 2). However, there are Scientific Reports | (2021) 11:14106 | https://doi.org/10.1038/s41598-021-93477-1 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/. Additional information Correspondence and requests for materials should be addressed to D.W.L. Correspondence and requests for materials should be addressed to D.W.L. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Scientific Reports | (2021) 11:14106 | https://doi.org/10.1038/s41598-021-93477-1 www.nature.com/scientificreports/ Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. fi
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/. 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/. 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. Additional information The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2021 https://doi.org/10.1038/s41598-021-93477-1 Scientific Reports | (2021) 11:14106 |
|
https://openalex.org/W1972453139
|
https://hal.inrae.fr/hal-03325692/file/09-0036.pdf
|
English
| null |
Maternal Antibody Transfer in Yellow-legged Gulls
|
Emerging infectious diseases
| 2,009
|
cc-by
| 3,537
|
Maternal antibody transfer in yellow-legged gulls
Jessica M.C. Pearce-Duvet, Michel Gauthier-Clerc, Elsa Jourdain, Thierry
Boulinier Maternal antibody transfer in yellow-legged gulls
Jessica M.C. Pearce-Duvet, Michel Gauthier-Clerc, Elsa Jourdain, Thierry
Boulinier To cite this version: Jessica M.C. Pearce-Duvet, Michel Gauthier-Clerc, Elsa Jourdain, Thierry Boulinier. Maternal an-
tibody transfer in yellow-legged gulls. Emerging Infectious Diseases, 2009, 15 (7), pp.1147-1149. 10.3201/eid1507.090036. hal-03325692 Distributed under a Creative Commons Attribution 4.0 International License References 1. Daszak P, Berger L, Cunningham AA,
Hyatt AD, Green DE, Speare R. Emerg-
ing infectious diseases and amphibian
population declines. Emerg Infect Dis. 1999;5:735–48. 2. Cunningham AA, Hyatt AD, Russell P,
Bennett PM. Emerging epidemic dis-
eases of frogs in Britain are dependent
on the source of ranavirus agent and the
route of exposure. Epidemiol Infect. 2007;135:1200–12. 3. Docherty DE, Meteyer CU, Wang J, Mao
J, Case ST, Chinchar VG. Diagnostic and
molecular evaluation of three iridovirus-
associated salamander mortality events. J
Wildl Dis. 2003;39:556–66. Japan is located at middle lati-
tudes and has a temperate climate. This
country has long been geologically iso-
lated from Asia. This isolation has re-
sulted in the development of many di-
verse species of amphibians in Japan;
23 species of the order Caudata and 35
species of the order Anura. Of these
species, 49 (84%) are native and 36
(62%) are listed by the Ministry of the
Environment as threatened species (8). R. catesbeiana frogs were introduced
into Japan in 1918 as a food animal,
and raising them by aquaculture was
widely attempted. Although they are
no longer cultured, feral populations
have become established throughout
Japan (9). Ranavirus in R. catesbeiana
frogs represents a serious threat to am-
phibians throughout Japan. 4. Marsh IB, Whittington RJ, O’Rourke B,
Hyatt AD, Chisholm O. Rapid differentia-
tion of Australian, European and Ameri-
can ranavirus based on variation in major
capsid protein gene sequence. Mol Cell
Probes. 2002;16:137–51. DOI: 10.1006/
mcpr.2001.0400 5. Wolf K, Bullock GL, Dunbar CE, Quimby
MC. Tadpole edema virus: a viscerotropic
pathogen for anuran amphibians. J Infect
Dis. 1968;118:253–62. We determined whether eggs of
yellow-legged gulls (Larus micha-
hellis) contained antibodies against
AIVs. Yellow-legged gulls can host
AIVs (C. Lebarbenchon, unpub. data),
are abundant, and nest in large, dense
colonies in coastal areas. In April
2008, we collected 466 eggs from 2
yellow-legged gull colonies located
on the Mediterranean coast: 252 eggs
from Gruissan (43.1099°N, 3.1071°E;
350 breeding pairs over 1.5 hectares),
and 212 from Villeneuve-lès-Mague-
lone (VLM; 43.4895°N, 3.8520°E;
400 pairs over 1 hectare). Villeneuve
nests formed 2 spatially clustered 6. Miller DL, Rajeev S, Gray MJ, Bald-
win CA. Frog virus 3 infection, cultured
American bullfrogs. Emerg Infect Dis. 2007;13:342–3. 7. Majji S, LaPatra S, Long SM, Sample R,
Bryan L, Sinning A, et al. Rana catesbeia-
na virus Z (RCV-Z): a novel pathogenic
ranavirus. Dis Aquat Organ. Yumi Une, Akiko Sakuma,
Hiroki Matsueda, Katsuki Nakai,
and Masaru Murakami
Author affiliations: Azabu University, Kana-
gawa, Japan (Y. Ume, A. Sakuma, H. Mat-
sueda, M. Murakami); and Nature Conser-
vation Division of Shiga Prefecture, Shiga,
Japan (K. Nakai) Yumi Une, Akiko Sakuma,
Hiroki Matsueda, Katsuki Nakai,
and Masaru Murakami
Author affiliations: Azabu University, Kana-
gawa, Japan (Y. Ume, A. Sakuma, H. Mat-
sueda, M. Murakami); and Nature Conser-
vation Division of Shiga Prefecture, Shiga,
Japan (K. Nakai) Yumi Une, Akiko Sakuma,
Hiroki Matsueda, Katsuki Nakai,
and Masaru Murakami
Author affiliations: Azabu University, Kana-
gawa, Japan (Y. Ume, A. Sakuma, H. Mat-
sueda, M. Murakami); and Nature Conser-
vation Division of Shiga Prefecture, Shiga,
Japan (K. Nakai) Live freshwater fish from sev-
eral countries have been imported
into Japan. However, large amounts
(<1,300 tons in 2007) of live aquacul-
ture products, including eels and other
fishes, have been imported from Tai-
wan into Japan (www.customs.go.jp/
tariff/2007_4/data/03.htm). Given that
viruses that originate in Japan and Tai-
wan are similar, the ranavirus we de-
tected was likely imported into Japan
in an infected aquatic organism. How-
ever, an epidemiologic survey will be
necessary to determine the source of
the ranavirus in the pond studied. Like-
wise, this virus may be endemic to Ja-
pan, and a survey of native and foreign
free-ranging amphibians should be
conducted. Molecular analysis of rana-
viruses detected in these surveys will
be necessary to differentiate endemic
viruses from introduced viruses. DOI: 10.3201/eid1507.081636 To the Editor: Avian influenza
viruses (AIVs) are emerging patho-
gens of concern because they can
cause deaths in birds and humans (1). Although wild birds likely contribute
to AIV emergence because they are
the natural reservoir for all known
influenza virus subtypes (1), our un-
derstanding of AIV transmission and
immunology in natural avian popula-
tions is incomplete (2). In this context,
the transfer of maternal antibodies is
a tool that should be used more often
in immunologic analysis. Because an-
tibodies in eggs and hatchlings can
reflect the mother’s past exposure to
pathogens (3,4) and both life stages
are more easily sampled than adults,
quantifying antibodies found in avian
young could help clarify AIV epide-
miology. HAL Id: hal-03325692
https://hal.inrae.fr/hal-03325692v1
Submitted on 25 Aug 2021 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 LETTERS Address for correspondence: Yumi Une,
Laboratory of Veterinary Pathology, School of
Veterinary Medicine, Azabu University, 1-17-
71 Fuchinobe, Sagamihara, Kanagawa 229-
8501, Japan; email: une@azabu-u.ac.jp Address for correspondence: Yumi Une,
Laboratory of Veterinary Pathology, School of
Veterinary Medicine, Azabu University, 1-17-
71 Fuchinobe, Sagamihara, Kanagawa 229-
8501, Japan; email: une@azabu-u.ac.jp and F-081) of the Ministry of the Environ-
ment, Japan, 2008, and The Promotion
and Mutual Aid Corporation for Private
Schools of Japan, Grant in Aid for Match-
ing Fund Subsidy for Private Universities. spp.) that cohabitated the pond, but
showed no external signs of disease,
were positive for ranavirus by PCR us-
ing primers M153 and M154. Further
sequence analyses are ongoing, and
additional investigations of other am-
phibians and fishes are needed.i Jessica M.C. Pearce-Duvet,
Michel Gauthier-Clerc,
Elsa Jourdain,
and Thierry Boulinier Author affiliations: Centre d’Ecologie Fonc-
tionnelle et Evolutive, Montpellier, France
(J.M.C. Pearce-Duvet, T. Boulinier); Centre
de Recherche de la Tour du Valat, Arles,
France (M. Gauthier-Clerc); and Kalmar
University, Kalmar, Sweden (E. Jourdain) y
Overall antibody prevalence was
14% (65/466), indicating exposure to
influenza A viruses in these colonies. As expected, antibody prevalence in
gulls is higher than the viral preva-
lence previously estimated by reverse
transcription–PCR or virus isolation
on fecal samples, i.e., the methods
typically used by avian influenza sur-
veillance networks. A spring and sum-
mer survey performed on feces from
gulls of the Camargue region (east of
our colonies) showed that only 0.9%
of gulls (2 infected of 228 sampled)
were excreting AIVs (C. Lebarben-
chon, unpub. data). DOI: 10.3201/eid1507.090036 References An equal volume of
chloroform was added, the solution
was centrifuged (6,000 × g for 15
min), and the supernatant was used in
the analyses. Extracts were tested for antibod-
ies against the AIV nucleoprotein
by using a commercial competitive
ELISA (IDEXX/Institut Pourquier,
Montpellier, France). The assay has
been validated by using seagull serum
(IDEXX, pers. comm.) and chicken
egg yolk (5). A subset of samples was
tested by using a second commercial
competitive ELISA (IDVet, Montpel-
lier, France). Optical density values
obtained in the 2 assays were signifi-
cantly correlated (r = 0.90, df = 39,
p<0.001), and serostatus was consis-
tent across assays. Overall antibody prevalence was
14% (65/466), indicating exposure to
influenza A viruses in these colonies. As expected, antibody prevalence in
gulls is higher than the viral preva-
lence previously estimated by reverse
colonies of Villeneuve also did not dif
fer: 18/129 (14%) in VLM1 compared
with 12/83 (14.5%) in VLM2 (z =
0.02, p = 0.9). There was no evidence
of spatial autocorrelation in the distri
bution of antibody in eggs by using the
Moran I spatial statistic. This similar
ity of antibody prevalence across and
within colonies suggests that exposure
is dictated by regional rather than loca
conditions, a hypothesis that should be
tested by sampling across a broade
range of nest densities and over time. Our study presents evidence fo
the presence of antibodies agains
AIVs in wild bird eggs, and the find
ings have important practical implica
tions. The difference in prevalence es
timated from virus isolation (0.9%) and
antibody detection (14%), although
expected, highlights the complemen
tary nature of the 2 approaches. Mos
surveys estimate current infection by
virus isolation, which provides infor
mation about disease risk in addition
to phylogeographic tracking of strains
In contrast, information on antibody
prevalence, which shows past and
present population exposure and risk
has largely been ignored with few ex
ceptions (e.g., 6,7). Future work could
benefit from using both approaches in
tandem with modeling to develop an
understanding of avian influenza ecol
ogy in nature. Egg yolks were isolated and fro-
zen at –20°C until analysis. Antibod-
ies were obtained by using chloroform
extraction (4). The yolk was diluted
1:1 in phosphate-buffered saline and
homogenized. An equal volume of
chloroform was added, the solution
was centrifuged (6,000 × g for 15
min), and the supernatant was used in
the analyses. 4. Gasparini J, McCoy KD, Haussy C, Tveraa
T, Boulinier T. Induced maternal response
to the Lyme disease spirochaete Borrelia
burgdorferi sensu lato in a colonial sea-
bird, the kittiwake Rissa tridactyla. Proc
R Soc Lond B Biol Sci. 2001;268:647–50.
DOI: 10.1098/rspb.2000.1411 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 7, July 2009 Acknowledgments We thank P. Peret, P. Mayet, R. Gar-
nier, and E. Charbonnier for egg collection
and V. Staszewski for technical assistance. Sampling permits were obtained from the
Direction Régionale de l’Environnement
de Languedoc-Roussillon. Our study presents evidence for
the presence of antibodies against
AIVs in wild bird eggs, and the find-
ings have important practical implica-
tions. The difference in prevalence es-
timated from virus isolation (0.9%) and
antibody detection (14%), although
expected, highlights the complemen-
tary nature of the 2 approaches. Most
surveys estimate current infection by
virus isolation, which provides infor-
mation about disease risk in addition
to phylogeographic tracking of strains. In contrast, information on antibody
prevalence, which shows past and
present population exposure and risk,
has largely been ignored with few ex-
ceptions (e.g., 6,7). Future work could
benefit from using both approaches in
tandem with modeling to develop an
understanding of avian influenza ecol-
ogy in nature. Extracts were tested for antibod-
ies against the AIV nucleoprotein
by using a commercial competitive
ELISA (IDEXX/Institut Pourquier,
Montpellier, France). The assay has
been validated by using seagull serum
(IDEXX, pers. comm.) and chicken
egg yolk (5). A subset of samples was
tested by using a second commercial
competitive ELISA (IDVet, Montpel-
lier, France). Optical density values
obtained in the 2 assays were signifi-
cantly correlated (r = 0.90, df = 39,
p<0.001), and serostatus was consis-
tent across assays. This study was supported by grants
from the French National Agency for Re-
search, the Agency for Inter-Institutional
Promotion of Research in Developing
Countries, and a Chateaubriand fellowship. Jessica M.C. Pearce-Duvet,
Michel Gauthier-Clerc,
Elsa Jourdain,
and Thierry Boulinier
Author affiliations: Centre d’Ecologie Fonc-
tionnelle et Evolutive, Montpellier, France
(J.M.C. Pearce-Duvet, T. Boulinier); Centre
de Recherche de la Tour du Valat, Arles,
France (M. Gauthier-Clerc); and Kalmar
University, Kalmar, Sweden (E. Jourdain) References 2006;73:1–
11. DOI: 10.3354/dao073001 8. Ota H. Current status of the threatened
amphibians and reptiles of Japan. Popula-
tion Ecology. 2000;42:5–9. DOI: 10.1007/
s101440050003 8. Ota H. Current status of the threatened
amphibians and reptiles of Japan. Popula-
tion Ecology. 2000;42:5–9. DOI: 10.1007/
s101440050003 9. Maeda N, Matsui M. Frogs and toads of
Japan. Tokyo: Bun-Ichi Sogo Shuppan
Publishers; 1989. p. 100–7. 9. Maeda N, Matsui M. Frogs and toads of
Japan. Tokyo: Bun-Ichi Sogo Shuppan
Publishers; 1989. p. 100–7. This study was supported by the
Global Environment Research Fund (F-3 1147 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 7, July 2009 LETTERS LETTERS subcolonies: VLM1 (129 eggs) and
VLM2 (83 eggs). We also collected
global positioning system coordinates
for VLM1 nests. subcolonies: VLM1 (129 eggs) and
VLM2 (83 eggs). We also collected
global positioning system coordinates
for VLM1 nests. mized by taking blood samples from
young nestlings of a standardized age. Finally, such samples provide abundant
material for the simultaneous surveil-
lance of other emerging pathogens of
interest, such as Campylobacter spp. (8) and West Nile virus (9). colonies of Villeneuve also did not dif-
fer: 18/129 (14%) in VLM1 compared
with 12/83 (14.5%) in VLM2 (z =
0.02, p = 0.9). There was no evidence
of spatial autocorrelation in the distri-
bution of antibody in eggs by using the
Moran I spatial statistic. This similar-
ity of antibody prevalence across and
within colonies suggests that exposure
is dictated by regional rather than local
conditions, a hypothesis that should be
tested by sampling across a broader
range of nest densities and over time. colonies of Villeneuve also did not dif-
fer: 18/129 (14%) in VLM1 compared
with 12/83 (14.5%) in VLM2 (z =
0.02, p = 0.9). There was no evidence
of spatial autocorrelation in the distri-
bution of antibody in eggs by using the
Moran I spatial statistic. This similar-
ity of antibody prevalence across and
within colonies suggests that exposure
is dictated by regional rather than local
conditions, a hypothesis that should be
tested by sampling across a broader
range of nest densities and over time. subcolonies: VLM1 (129 eggs) and
VLM2 (83 eggs). We also collected
global positioning system coordinates
for VLM1 nests. Egg yolks were isolated and fro-
zen at –20°C until analysis. Antibod-
ies were obtained by using chloroform
extraction (4). The yolk was diluted
1:1 in phosphate-buffered saline and
homogenized. Prevalence of
Human Bocavirus
in Human Tonsils
and Adenoids 5. Trampel DW, Zhou E-M, Yoon K-J, Koe-
hler KJ. Detection of antibodies in serum
and egg yolk following infection of chick-
ens with an H6N2 avian influenza virus. J
Vet Diagn Invest. 2006;18:437–42. Samples consisted of surgically
removed tonsil or adenoid tissues. DNA was extracted and its concentra-
tion was determined as previously de-
scribed (5). Two primer sets were used
for HBoV detection by using real-time
PCR with SYBR Green detection and
melting-point determination. We de-
signed primers 3097F (5′-GTC-CAA-
TTA-CAT-GAT-CAC-GCC-TAC-TC)
and 3420R (5′-TGC-GTC-CAC-AGT-
ATC-AGG-TTG-TTG) that targeted
the viral protein 1/2 (VP1/VP2) region
of HBoV. The nonstructural protein
1 (NP1) region was targeted by us-
ing primers 188F and 542R from Al-
lander et al. (6) Each 20-μL reaction
contained SYBR Green JumpStart Taq
ReadyMix (Sigma, St. Louis, MO,
USA), 4 mmol/L MgCl2, 250 nmol/L
primers, double-distilled H2O, and 2 μL
of DNA (50–200 ng) cycled on an ABI
PRISM 7900HT (Applied Biosystems,
Foster City, CA, USA) instrument at
94oC for 2 min, followed by 40 cycles
of 94°C for 20 s, 60°C (NP1 primers)
or 68°C (VP1/VP2 primers) for 20 s,
and 72°C for 14 s. Amplification and
melting curves were analyzed with
7900HT version 2.2.1 software (Ap-
plied Biosystems); positive samples
were verified by sequence analysis. Sequenced VP- and NP1-generated
amplicons were 99%–100% identical
to HBoV strain ST1 (6). The detec-
tion sensitivities of the VP and NP1
assays, determined by using a plasmid
construct containing the full HBoV
genome, were 1–5 and 5–10 gene cop-
ies/reaction, respectively.i 6. Krauss S, Obert CA, Franks J, Walker D,
Jones K, Seiler P, et al. Influenza in mi-
gratory birds and evidence of limited in-
tercontinental virus exchange. PLoS Pat-
hog. 2007;3:e167. DOI: 10.1371/journal. ppat.0030167 To the Editor: Recently, Longtin
et al. (1) reported a high rate (43%) of
human bocavirus (HBoV) infection in
a group of children chosen to serve as
controls in a study of HBoV preva-
lence among hospitalized children and
adults. In contrast, previous reports
had found low HBoV prevalence rates
of 0%–1% in control groups (2,3). At-
tempting to explain this surprising dif-
ference in rates, Lu et al. (4) suggested
that selection of control patients may
be related to the difference in rates. References Our results also show the gener-
alizable potential of maternal antibody
transfer for tracking pathogen exposure
in wild birds, notably in the case of rec-
ognized emerging zoonoses. Because
eggs and hatchlings are proxies of
past and present adult pathogen expo-
sure (3,4), the difficult and sometimes
disruptive sampling of adults can be
circumvented by the rapid and cost-ef-
ficient sampling of their young, which
will facilitate monitoring efforts. Due
to the high intranest correlation in egg
antibodies (4), only partial sampling of
clutches (e.g., 1 of 3 eggs) is necessary
to track pathogen presence and preva-
lence through space and time. The
sampling effects could further be mini- 1. Olsen B, Munster VJ, Wallensten A,
Waldenström J, Osterhaus AD, Fouchier
RA. Global patterns of influenza A virus
in wild birds. Science. 2006;312:384–8. DOI: 10.1126/science.1122438 2. Boyce WM, Sandrock C, Kreuder-Johnson
C, Kelly T, Cardona C. Avian influenza in
wild birds: a moving target. Comp Immu-
nol Microbiol Infect Dis. 2009;32:275–86. DOI: 10.1016/j.cimid.2008.01.002 2. Boyce WM, Sandrock C, Kreuder-Johnson
C, Kelly T, Cardona C. Avian influenza in
wild birds: a moving target. Comp Immu-
nol Microbiol Infect Dis. 2009;32:275–86. DOI: 10.1016/j.cimid.2008.01.002 3. Boulinier T, Staszewski V. Maternal trans-
fer of antibodies: raising immuno-ecology
issues. Trends Ecol Evol. 2008;23:282–8. DOI: 10.1016/j.tree.2007.12.006 Egg antibody prevalence did not
differ significantly between colonies. The antibody prevalence of 13.5%
found at Gruissan (34 of 252 eggs)
was comparable with the 14.5% found
at Villeneuve (31 of 214 eggs) (gen-
eralized linear model with binomial
distribution, z = 0.4, p = 0.8). The sub- 4. Gasparini J, McCoy KD, Haussy C, Tveraa
T, Boulinier T. Induced maternal response
to the Lyme disease spirochaete Borrelia
burgdorferi sensu lato in a colonial sea-
bird, the kittiwake Rissa tridactyla. Proc
R Soc Lond B Biol Sci. 2001;268:647–50. DOI: 10.1098/rspb.2000.1411 1148 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 7, July 2009 Prevalence of
Human Bocavirus
in Human Tonsils
and Adenoids The control group used in the Long-
tin study was primarily (71%) made
up of children undergoing elective
surgery; previous studies had selected
control groups from other sources, in-
cluding well children on routine visits
and outpatients with nonrespiratory
symptoms. Because the Longtin study
surgeries were mainly tonsillectomies,
adenoidectomies, and myringotomies,
Lu et al. examined the possibility that
tonsillar tissues, which include the ad-
enoids, are sites of persistent HBoV
infection. When these researchers ex-
tracted DNA from tonsillar lympho-
cytes obtained from pediatric patients
who had undergone tonsillectomies or
adenoidectomies, they detected HBoV
DNA in 32% of the samples (4). These
findings strongly suggest a connec-
tion between HBoV and tonsillar tis-
sue. Therefore, we tested a number of
tonsillar samples for the presence of
HBoV DNA. 7. Stallknecht DE, Shane SM. Host range
of avian influenza in free-living birds. Vet Res Commun. 1988;12:125–41. DOI:
10.1007/BF00362792 8. Sahin O, Zhang Q, Meitzler JC, Harr
BS, Morishita TY, Mohan R. Prevalence,
antigenic specificity, and bactericidal
activity of poultry anti-Campylobacter
maternal antibodies. Appl Environ Mi-
crobiol. 2001;67:3951–7. DOI: 10.1128/
AEM.67.9.3951-3957.2001 9. Baitchman EJ, Tlusty MF, Murphy HW. Passive transfer of maternal antibod-
ies to West Nile virus in flamingo chicks
(Phoenicopterus chilensis and Phoeni-
copterus ruber ruber). J Zoo Wildl Med. 2007;38:337–40. DOI:
10.1638/1042-
7260(2007)038[0337:PTOMAT]2.0.CO;2 Address for correspondence: Jessica M.C. Pearce-Duvet, Centre d’Ecologie Fonctionnelle
et Evolutive, Centre National de la Recherche
Scientifique–Unité de Mixte de Recherche 5175,
1919 Route de Mende, F-34293 Montpellier,
France; email: jessica.pearce@cefe.cnrs.fr Letters Letters commenting on recent ar-
ticles as well as letters reporting
cases, outbreaks, or original re-
search are welcome. Letters com-
menting on articles should contain
no more than 300 words and 5
references; they are more likely to
be published if submitted within 4
weeks of the original article’s publi-
cation. Letters reporting cases, out-
breaks, or original research should
contain no more than 800 words
and 10 references. They may have 1
Figure or Table and should not be
divided into sections. All letters
should contain material not previ-
ously published and include a word
count. Our testing identified HBoV
DNA in 5 (5.5%) of the 91 children
who underwent elective tonsillecto-
my/adenoidectomy. Ages ranged from
1.9 to 4.6 years (median 3.4 years). The reason for the much lower HBoV
prevalence in this group of children,
compared with prevalences found in
studies by Longtin et al. and Lu et al.,
is unclear. Lu et al. (4) reported a much
higher HBoV rate of lymphocytes
from adenoids (56%) than from ton-
sils (16%). Although we did not know Sample acquisition was approved
by the Nationwide Children’s Hospi-
tal Institutional Review Board. Tonsils
and adenoids were obtained from 91
patients who underwent elective sur-
gery at Nationwide Children’s Hos-
pital from June through September
2004. Patients’ ages ranged from 1 to
16 years (median 5.9 years; age was
unknown for 4 patients). 1149 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 7, July 2009
|
https://openalex.org/W2038757318
|
https://www.repo.uni-hannover.de/bitstream/123456789/634/1/srep03770.pdf
|
English
| null |
Irreversibility of Pressure Induced Boron Speciation Change in Glass
|
Scientific reports
| 2,014
|
cc-by
| 6,884
|
OPEN SUBJECT AREAS:
MATERIALS SCIENCE
CHEMISTRY
PHYSICS Morten M. Smedskjaer1, Randall E. Youngman2, Simon Striepe3, Marcel Potuzak2, Ute Bauer4,
Joachim Deubener3, Harald Behrens4, John C. Mauro2 & Yuanzheng Yue1 1Section of Chemistry, Aalborg University, DK-9000 Aalborg, Denmark, 2Science and Technology Division, Corning Incorporated,
Corning, NY 14831, USA, 3Institute of Non-Metallic Materials, Clausthal University of Technology, 38678 Clausthal-Zellerfeld,
Germany, 4Institute of Mineralogy, Leibniz University Hannover, 30167 Hannover, Germany. Received
30 August 2013
Accepted
30 December 2013
Published
20 January 2014 It is known that the coordination number (CN) of atoms or ions in many materials increases through
application of sufficiently high pressure. This also applies to glassy materials. In boron-containing glasses,
trigonal BO3 units can be transformed into tetrahedral BO4 under pressure. However, one of the key
questions is whether the pressure-quenched CN change in glass is reversible upon annealing below the
ambient glass transition temperature (Tg). Here we address this issue by performing 11B NMR
measurements on a soda lime borate glass that has been pressure-quenched at ,0.6 GPa near Tg. The results
show a remarkable phenomenon, i.e., upon annealing at 0.9Tg the pressure-induced change in CN remains
unchanged, while the pressurised values of macroscopic properties such as density, refractive index, and
hardness are relaxing. This suggests that the pressure-induced changes in macroscopic properties of soda
lime borate glasses compressed up to ,0.6 GPa are not attributed to changes in the short-range order in the
glass, but rather to changes in overall atomic packing density and medium-range structures. A
ffi A
ny natural or man-made material will exhibit a significant change in microstructure and properties when
subjected to high pressure1–5; particularly the coordination number (CN) of atoms or ions will generally
increase with pressure6–9. It has long been known that when a glassy material or its liquid state is subjected
to sufficiently high pressure, significant changes can take place in the local and medium-range structure, vibra-
tional density of states, and physical properties4–8,10,11. In recent years, theoretical modeling approaches have
provided insights regarding the structural response of various non-crystalline materials to composition, temper-
ature, and pressure11–15. Nevertheless, the relationship between structure and dynamics in glass and glass-forming
melts under high pressures remains a challenging problem in condensed matter science. The difficulties in
addressing this problem are due to both experimental limitations at high pressures and the inadequacy of
computer simulations under such conditions. Correspondence and
requests for materials
should be addressed to
Y.Z.Y. (yy@bio.aau.
dk) OPEN While Vickers hardness has
essentially decreased to its original value prior to compression
(5.0 GPa) after annealing for 6 h, density has only decreased to
,2.47 g/cm3 after annealing for 24 h, which is significantly higher
than the value prior to compression (2.438 g/cm3), i.e., density has
relaxed by only 43% of the total possible relaxation. In this work we investigate the microscopic and macroscopic res-
ponses of the soda-lime borate glass to comparatively low pressure at
an elevated temperature around the Tg, since numerous high pres-
sure studies at room temperature have already been reported in
literature and it is also our aim to detect the sensitivity of glass
structure and properties to the low pressure. Moreover, these condi-
tions are chosen to be able to prepare compressed samples, which are
large enough to allow for subsequent characterization of mac-
roscopic properties. The as-produced glass has a nonzero equilib-
rium concentration of BO4 due to the presence of network modifiers
(Na2O and CaO) that act to charge balance the tetrahedral boron
species. Only recently has it been found that boron speciation (i.e. the
CN) changes occur at rather low pressure (,0.6 GPa) near Tg and
can be frozen-in under pressure18. Here the key questions arise: Is the
pressure-quenched CN change reversible upon annealing at tem-
peratures slightly below Tg at atmospheric pressure? Are the pres-
sure-induced changes in the macroscopic properties also reversible
upon annealing? What is the microscopic origin of the pressure-
induced changes in the macroscopic properties? We answer these
questions by investigating the structure and property responses of
the sodium-calcium-borate glass to pressurization at ,0.6 GPa and
subsequent annealing at 0.9Tg under ambient pressure. We thereby
hope to obtain a better understanding of the pressure-structure-
property relationship of this and other glass systems, which can be
used for tailoring both microscopic and macroscopic properties of
glassy materials. With increasing isostatic pressure, the overshoot in the isobaric
heat capacity (Cp) above Tg increases as illustrated in Fig. 2a, where
the evolution of Cp with temperature during the first DSC upscan is
shown. This overshoot is considered to be a direct consequence of the
nonexponentiality of the relaxation process26, i.e., due to broadening
of the relaxation time distribution. OPEN fraction suddenly drops at pressures just above 4 GPa and then
approaches zero as the pressure is further increased17. A structural
response of vitreous B2O3 to pressure below 4 GPa was not detected. After decompression from high pressure the boron coordination
reverts back from tetrahedral to trigonal, however, the densification
is apparently permanent17. It has been attempted to correlate these
changes in microscopic structure with the macroscopic properties. For example, the viscosity of the B2O3 liquid along the melting curve
has been shown to decrease by 4 orders of magnitude upon a pressure
increase up to 5.5 GPa but then remains unchanged upon further
increase of the pressure22. However, a generally accepted viewpoint
about the microscopic origin of the pressure-induced changes in the
macroscopic properties is still lacking. The abovementioned studies were carried out on vitreous B2O3 at
room temperature and rather high pressure up to 22.5 GPa. It should
be noted that the structure of glasses compressed at room temper-
ature (in diamond anvil cell) is different from that obtained by pres-
sure-quenching liquids from above Tg
21, even though the main
structural changes upon compression (i.e., decreased fraction of bor-
oxol rings and increased fraction of tetrahedral boron) are identical. It should also be noted that modified borate and pure B2O3 glasses
have different initial concentration of boroxol rings and may exhibit
different densification mechanisms. However, Lee et al. have shown
that the pressure dependent structural changes of a lithium borate
glass are similar to those of vitreous B2O3
24. In particular, they
showed that the pressure-induced CN change in Li2B4O7 glass from
three-coordinated to four-coordinated boron at room temperature
begins around 5 GPa and the BO4 fraction increases with pressure
from about 50% at 1 atm to more than 95% at 30 GPa. Figure 1 | Relaxation of macroscopic properties. Evolution of density (r)
and Vickers hardness (HV) with annealing duration (ta) at 0.9Tg 5 688 K
of the borate glass compressed at 570 MPa. The dashed lines indicate the
values of r and HV prior to compression. Inset: impact of isostatic pressure
(p) on r and HV. The errors of r and HV are around 60.005 g/cm3 and
60.2 GPa, respectively. the compressed glass are relaxing during annealing towards those of
the glass prior to compression. OPEN This also implies that subjecting
the glass to high pressure forces the glass into a lower region of the
enthalpy landscape compared to the glass under ambient pressure,
even though it should be noted that the enthalpy landscape itself is
changing as a function of pressure. In agreement with previous stud-
ies19,27, we find that an increase in isostatic pressure enhances the
nonexponentiality of the enthalpy relaxation as measured ex situ. With increasing pressure on the glass, the density of the glass
increases, and at the same time the topological degree of atomic
freedom decreases as well due to the BO3RBO4 conversion, i.e.,
the increase of network connectivity. Upon heating through the glass
transition region, the glass compressed at higher pressures should
exhibit a larger jump in configurational entropy in order to approach
the liquid state. This is also shown in Fig. 2b, in which the pressure
dependence of the fictive temperature (Tf) is plotted. The procedure
for determining Tf based on DSC data is described elsewhere28,29. g
f
The compressed glass relaxes in the glass transition region during
the first upscan and recovers back to its original state with respect to
enthalpy, i.e., that of the uncompressed glass. In other words, the
enthalpy level of the glass reaches that of the glass cooled under
standard conditions, e.g., at 10 K/min and ambient pressure. This
is seen from the second DSC upscans as shown in the inset of Fig. 2a
and in Supplementary Figure S1. Enthalpy recovery of the com-
pressed glass during the DSC upscan is a ‘‘structural depression’’
process, induced by thermal excitation under ambient pressure27. The structural depression leads to an increase in enthalpy, which is
manifested as an enhancement of the overshoot during the first DSC
upscan. This is also evident from the relaxation of refractive index
before and after the first DSC upscan (Fig. 3). With increasing iso-
static pressure, the refractive index at 633 nm increases due to den-
sification, but the measured value on the sample following the first OPEN It is crucial to determine the link between the microscopic structure
and macroscopic properties of glasses under high pressure from both scientific and technological perspectives,
since the glass structures frozen-in under elevated pressure may give rise to properties unattainable under
ambient pressure. Borate glass is the quintessential example of glass capable of dramatic changes in short-range
order as a function of composition, pressure, and thermal history, and hence we select a simple ternary borate
system, namely, sodium-calcium-borate glass, for this study in order to address the above mentioned challenging
problem. Boric oxide (B2O3) is also widely used as a network forming constituent in many high-tech glass
materials owing to its contribution to high glass forming ability and low melting temperature, and for its favorable
impact on thermal, mechanical, and optical properties. One of the most striking structural features of borate glasses is the transformation of the coordination number
(CN) of boron from three to four upon compression16–19, which is associated with anomalous pressure depend-
ence of viscosity6,16 and topological disorder8,12. Densification of borate glasses can be achieved by applying
isostatic pressure to glass directly at room temperature16,17, or by pressure-quenching from the molten state18–21. However, the elastic part of the densification relaxes during pressure release, i.e., the density of a compressed glass
at ambient pressure decreases to the value before compression upon reheating below the glass transition tem-
perature (Tg)17. For vitreous B2O3, it has been shown that the fraction of boroxol rings decreases with increasing
pressure16,20,22, leading to densification of the glass16 and increase of elastic moduli20. When the pressure applied at
room temperature is released, such densification is irreversible since the local structures may be topologically/
stereochemically unfavorable for the reformation of boroxol rings16. However, it is not clear whether this irre-
versibility of densification is linked to the CN change of boron23. More recently, it has been found that the BO3 SCIENTIFIC REPORTS | 4 : 3770 | DOI: 10.1038/srep03770 1 Figure 1 | Relaxation of macroscopic properties. Evolution of density (r)
and Vickers hardness (HV) with annealing duration (ta) at 0.9Tg 5 688 K
of the borate glass compressed at 570 MPa. The dashed lines indicate the
values of r and HV prior to compression. Inset: impact of isostatic pressure
(p) on r and HV. The errors of r and HV are around 60.005 g/cm3 and
60.2 GPa, respectively. Results Isotropic projections fr
these data (Supplementary Figure S4a) show very little change w
annealing time, and thus, the estimated quadrupolar coupling pr
uct (PQ) and isotropic chemical shifts (diso) for 23Na also do
indicate any detectable change in sodium environment in the co
pressed glass upon annealing (Supplementary Figure S4b). This s
gests that the Na–O bond length does not change with anneal
time, and hence it does not determine the recovery of the m
roscopic properties upon annealing. Discussion
Our work has shown that upon annealing at 0 9T of a soda l
Figure 2 | Pressure history dependence of calorimetric glass transition. (a) Evolution of endothermic overshoot in isobaric heat capacity (Cp) at
the glass transition for samples with different pressure history. DSC scans
were performed at 10 K/min at ambient pressure. Inset: second DSC
upscans at 10 K/min of all samples following prior up- and downscans at
10 K/min. (b) Pressure history dependence of the fictive temperature (Tf)
evaluated from the first and second DSC upscan, respectively. Figure 3 | Recovery of refractive index during DSC scan. Pressure
history dependence of the refractive index (n) at 633 nm before and a
the first DSC scan, as shown in Fig. 2a. DSC upscan is independent of the initially applied pressure during
compression. Structural response. 11B magic-angle spinning (MAS) NMR spectra
obtained at 16.4 T (700 MHz) for the 0.57 GPa compressed glass
Figure 2 | Pressure history dependence of calorimetric glass transition. (a) Evolution of endothermic overshoot in isobaric heat capacity (Cp) at
the glass transition for samples with different pressure history. DSC scans
were performed at 10 K/min at ambient pressure. Inset: second DSC
upscans at 10 K/min of all samples following prior up- and downscans at
10 K/min. (b) Pressure history dependence of the fictive temperature (Tf)
evaluated from the first and second DSC upscan, respectively. Figure 3 | Recovery of refractive index during DSC scan. Pressure
history dependence of the refractive index (n) at 633 nm before and after
the first DSC scan, as shown in Fig. 2a. remains almost constant within the error range when extending
the annealing time up to 25 hours at the annealing temperature of
688 K (0.9Tg) and at the ambient pressure. In other words, the glass
structure, as defined by boron coordination number, does not return
to its original state prior to compression upon annealing. Results (b) Pressure history dependence of the fictive temperature (Tf)
evaluated from the first and second DSC upscan, respectively. DSC upscan is independent of the initially applied pressure during
compression. Structural response. 11B magic-angle spinning (MAS) NMR spectra
obtained at 16.4 T (700 MHz) for the 0.57 GPa compressed glass
annealed for different durations are shown in Fig. 4a. These spectra
are
characterised
by
a
broad
peak
centered
at
115 ppm,
corresponding to BIII sites, and a relatively narrow peak centered
around 12 ppm, corresponding to BIV sites. The spectra vary
slightly with annealing time, which reflects minor changes in either
the relative proportions of BIII and BIV, or changes in bond angles and
distances involving boron and oxygen. We quantify these differences
by accurate simulation of the spectral lineshapes and subsequent
determination of the fraction of tetrahedral to total boron (N4)
through integration. The simulation parameters are given in
Supplementary Table S1, and two examples of the deconvolution
are shown in Supplementary Figure S2. Prior to annealing, we find
that with increasing isostatic pressure, N4 increases from 44.2 to 46.5
at% (inset of Fig. 4b and Supplementary Figure S3a). N4 also
increases upon compression in pure B2O3 glass17 and in boron-
containing multicomponent E-glass18. In Fig. 4b we observe an
interesting phenomenon, viz., N4 of the compressed sample Results Physical properties and heat capacity. The glass composition under
study is 25Na2O – 10CaO – 65B2O3 (mol%). As reported
previously25, the density and Vickers hardness of this glass increase
approximately linearly with increasing isostatic pressure (inset of
Fig. 1). The glass isostatically compressed at 570 MPa is then
annealed under ambient pressure at 688 K, i.e., 0.9 times its
ambient Tg for various durations (ta). This results in a decrease of
both density and Vickers hardness with increasing annealing
duration (Fig. 1). In other words, these macroscopic properties of SCIENTIFIC REPORTS | 4 : 3770 | DOI: 10.1038/srep03770 2 Figure 3 | Recovery of refractive index during DSC scan. Pressure
history dependence of the refractive index (n) at 633 nm before and after
the first DSC scan, as shown in Fig. 2a. www.nature.com/scientificreports DSC upscan is independent of the initially applied pressure during
compression. Structural response. 11B magic-angle spinning (MAS) NMR spectra
obtained at 16.4 T (700 MHz) for the 0.57 GPa compressed glass
annealed for different durations are shown in Fig. 4a. These spectra
are
characterised
by
a
broad
peak
centered
at
115 ppm
remains almost constant within the error range when extend
the annealing time up to 25 hours at the annealing temperatur
688 K (0.9Tg) and at the ambient pressure. In other words, the g
structure, as defined by boron coordination number, does not ret
to its original state prior to compression upon annealing. Altho
N4 does not appear to change, the11B MAS NMR spectra do sh
differences with annealing time. For example, the base of
tetrahedral boron resonances in Fig. 4a exhibit some anneal
time dependence, signifying a change in environment of the
groups, possibly due to changes in bond angle distribution arou
these boron atoms. The BIII features in Fig. 4a do not show m
change with annealing time, indicating that annealing has li
impact on B-O bond distance or angles for these structural eleme
Supplementary Figure S3b shows 23Na MAS NMR spectra of
samples prior to annealing. We see a small but systematic increas
frequency shift with increasing pressure, which is due to a decreas
the mean Na–O bond distance upon compression18. Fig. 4c shows
23Na MAS NMR spectra for the compressed-annealed glasses. Th
MAS NMR spectra overlap completely, and to further study
sodium speciation,
23Na triple-quantum magic-angle spinn
(3QMAS) NMR data were also collected. Results Although
N4 does not appear to change, the11B MAS NMR spectra do show
differences with annealing time. For example, the base of the
tetrahedral boron resonances in Fig. 4a exhibit some annealing
time dependence, signifying a change in environment of the BIV
groups, possibly due to changes in bond angle distribution around
these boron atoms. The BIII features in Fig. 4a do not show much
change with annealing time, indicating that annealing has little
impact on B-O bond distance or angles for these structural elements. Supplementary Figure S3b shows 23Na MAS NMR spectra of the
samples prior to annealing. We see a small but systematic increase in
frequency shift with increasing pressure, which is due to a decrease in
the mean Na–O bond distance upon compression18. Fig. 4c shows the
23Na MAS NMR spectra for the compressed-annealed glasses. These
MAS NMR spectra overlap completely, and to further study the
sodium speciation,
23Na triple-quantum magic-angle spinning
(3QMAS) NMR data were also collected. Isotropic projections from
these data (Supplementary Figure S4a) show very little change with
annealing time, and thus, the estimated quadrupolar coupling prod-
uct (PQ) and isotropic chemical shifts (diso) for 23Na also do not
indicate any detectable change in sodium environment in the com-
pressed glass upon annealing (Supplementary Figure S4b). This sug-
gests that the Na–O bond length does not change with annealing
time, and hence it does not determine the recovery of the mac-
roscopic properties upon annealing. Figure 2 | Pressure history dependence of calorimetric glass transition. (a) Evolution of endothermic overshoot in isobaric heat capacity (Cp) at
the glass transition for samples with different pressure history. DSC scans
were performed at 10 K/min at ambient pressure. Inset: second DSC
upscans at 10 K/min of all samples following prior up- and downscans at
10 K/min. (b) Pressure history dependence of the fictive temperature (Tf)
evaluated from the first and second DSC upscan, respectively. Figure 2 | Pressure history dependence of calorimetric glass transition. Figure 2 | Pressure history dependence of calorimetric glass transition. (a) Evolution of endothermic overshoot in isobaric heat capacity (Cp) at
the glass transition for samples with different pressure history. DSC scans
were performed at 10 K/min at ambient pressure. Inset: second DSC
upscans at 10 K/min of all samples following prior up- and downscans at
10 K/min. Discussion Our work has shown that upon annealing at 0.9Tg of a soda lime
borate glass compressed up to ,0.6 GPa, the pressure-induced
change in CN remains unchanged while the pressurised values of
macroscopic properties such as density, refractive index, and hard-
ness are relaxing. This suggests that the pressure-induced changes in
macroscopic properties of such glasses are not attributed to changes
in the short-range order, in agreement with previous studies at rela-
tively low pressure30. In sodium boroaluminosilicate glasses it has
been shown that while pressure-induced density changes are related
to changes in boron coordination, the relatively small difference in
partial molar volume of the BO4 and BO3 structural units cannot
account for the dominant part of the density change18. Instead it was
suggested that the shortening of the Na-O bond upon compression is
a more likely factor governing the density change18,31. Such changes
were observed at considerably higher pressures than the current
study and thus may not in fact account for the findings reported SCIENTIFIC REPORTS | 4 : 3770 | DOI: 10.1038/srep03770 3 www.nature.com/scientificreports Figure 4 | Structural response to annealing. (a) Solid state 11B MAS NMR
spectra at 16.4 T of the samples compressed at 570 MPa (solid line) and
then annealed for various durations (ta) at 0.9Tg 5 688 K. (b) Evolution of
fraction of tetrahedral to total boron (N4), which is obtained through
integration of peaks in Fig. 4a, with annealing duration. Inset: impact of
isostatic pressure on N4 in absence of annealing (corresponding 11B spectra
are shown in Supplementary Figure 2a). The error of N4 is 60.5 at% for the
variable pressure series (inset) and 61 at% for the variable annealing time
study. The latter has higher error due to changes in lineshape and thus
more difficulty in fitting the 11B MAS NMR data. (c) Solid state 23Na MAS
NMR p
tr
t 16 4 T f th
mpl
mpr
d t 570 MP
nd th n herein. For example, Allwardt et al. reported a change in 23Na chem-
ical shift of 3.5 ppm at 10 GPa, roughly corresponding to a 0.005 nm
decrease in Na-O distance32. The chemical shifts for sodium in these
soda-lime borates vary slightly with annealing, but within roughly 1–
2 ppm and due to measurement uncertainty, appear to indicate a
negligible contribution of Na-O bond distance changes to the density
change. Discussion Shannon & Prewitt33 indicate that Na-O bonds should be
more compressible than Ca-O bonds. Since Na-O bonds do not
change in the detectable range, the contribution of Ca-O bond con-
traction under pressure could thus likely be ruled out, and therefore
the expansion of these bonds does not occur upon annealing, i.e.,
should not be the reason for the density decrease. y
Although boron speciation and modifier-oxygen bond lengths do
not change with increased annealing time, density partially
decreases. This might be explained as follows. In addition to coordi-
nation numbers and bond distances, bond angle distributions (B-O-
B and O-B-O) and superstructures (e.g., boroxol rings34) may change
upon annealing16. The change in bond angle and configuration of BIV
groups with annealing time could be related to the evolution of the
BIV resonance around the frequency shift of 12 ppm in the 11B NMR
spectra (Fig. 4a). By isostatic compression, not only the connections
of neighbored BO4 tetrahedra, but possibly also the linkages between
boroxol rings, become stronger or tighter, and this is accompanied by
the narrowing down of the bond angle distribution. Consequently,
the total potential energy in the glass decreases with increasing the
compression, which is why the glass transition overshoot becomes
larger upon DSC upscanning (see Fig. 2a). Once the compressed glass
is subjected to annealing around Tg, the ‘‘tightened state’’ of glass will
relax, leading to the decrease of density and hardness. However, the
thermal energy at 0.9Tg could be insufficient for breaking the B-O
bonds in favor of formation of BO3 units, despite the fact that struc-
tural relaxation generally occurs even at temperatures below 0.9Tg
35. It should be noted that changes in boron speciation due to annealing
at 0.92Tg have previously been reported for non-compressed boro-
silicate glasses36 and the thermomechanically trapped state of the
glass studied herein thus appears to be different from that of the
thermally trapped glass. It is known that the a relaxation is decoupled from the b and c
relaxation below Tg
37. Generally, for network glass systems, the a
relaxation is dominated by the change of the network connectivity
(e.g., the boron speciation), and the b relaxation is controlled by the
local motion of structural units (e.g., the change of bond angles). Discussion In
this context, it is understandable that the density relaxes upon
annealing slightly below Tg, whereas the CN remains unchanged
since the former is of the b relaxation feature requiring low temper-
ature and the latter is of the a relaxation feature demanding the
temperature to exceed Tg. According to literature the BIII-to-BIV ratio
increases with increasing temperature above Tg
36. Thus, it is expected
that the thermomechanically induced BIV units would be converted
to BIII units when the temperature is well above Tg. In contrast to the
density decay, the hardness is fully recovered by annealing at 0.9Tg to
the original value more quickly, as shown in Fig. 1. This could be
related to the dominant contribution of the c relaxation process, or to
contributions of hydrated structure relaxations of a near surface
volume (OH-groups and molecular water) to the relaxation of hard-
ness. The latter has been found recently to proceed more rapidly than
both a and b relaxations38. This implies that the hardness relaxation
be governed by the fast local network relaxation of the surface layer39. Figure 4 | Structural response to annealing. (a) Solid state 11B MAS NMR
spectra at 16.4 T of the samples compressed at 570 MPa (solid line) and
then annealed for various durations (ta) at 0.9Tg 5 688 K. (b) Evolution of
fraction of tetrahedral to total boron (N4), which is obtained through Figure 4 | Structural response to annealing. (a) Solid state 11B MAS NMR
spectra at 16.4 T of the samples compressed at 570 MPa (solid line) and
then annealed for various durations (ta) at 0.9Tg 5 688 K. (b) Evolution of
fraction of tetrahedral to total boron (N4), which is obtained through
integration of peaks in Fig. 4a, with annealing duration. Inset: impact of
isostatic pressure on N4 in absence of annealing (corresponding 11B spectra
are shown in Supplementary Figure 2a). The error of N4 is 60.5 at% for the
variable pressure series (inset) and 61 at% for the variable annealing time
study. The latter has higher error due to changes in lineshape and thus
more difficulty in fitting the 11B MAS NMR data. (c) Solid state 23Na MAS
NMR spectra at 16.4 T of the samples compressed at 570 MPa and then
annealed for various durations at 0.9Tg 5 688 K. Methods
l Rev. B 76,
014202 (2007). 20. Carini Jr, G., Gilioli, E., Tripodo, G. & Vasi, C. Structural changes and elastic
characteristics of permanently densified vitreous B2O3. Phys. Rev. B 84, 024207
(2011). 21. Lee, S. K., Mibe, K., Fei, Y. W., Cody, G. D. & Mysen, B. O. Structure of B2O3 Glass
at High Pressure: A 11B Solid-State NMR Study. Phys. Rev. Lett. 94, 165507 (2005). Differential scanning calorimetry. Isobaric heat capacities (Cp) of the investigated
samples were determined using a differential scanning calorimeter (DSC Netzsch
404C). DSC runs included measurements of the baseline (two empty Pt–Rh crucibles,
6 mm in diameter, 0.1 mm wall thickness covered with a lid), a sapphire standard
(with one crucible containing the standard and the other empty), and finally the
sample (with one crucible containing the sample and the other empty). The glass
samples were polished to within 1 mm to ensure an accurate fit with the bottom of the
crucible and to reach a mass comparable to that of the sapphire standard (,56 mg). Calorimetry was performed under argon flow of 40 ml/min and the glasses were
placed on the DSC sample holder at room temperature. Subsequently they were
heated to an initial temperature of 313 K, held isothermally for 15 min, and then
heated further at a rate of 10 K/min to a temperature 60 K above the Tg in order to
fully relax the sample. At the next step, the sample was cooled at a rate of 10 K/min to
313 K, and then held for one hour prior to the following heating, which occurred at a
rate of 10 K/min. 22. Brazhkin, V. V. et al. Structural Transformations and Anomalous Viscosity in the
B2O3 Melt under High Pressure. Phys. Rev. Lett. 105, 115701 (2010). 23. Nicholas, J. D., Youngman, R. E., Sinogeikin, S. V., Bass, J. D. & Kieffer, J. Structural changes in vitreous boron oxide. Phys. Chem. Glasses 44, 249–251
(2003). 24. Lee, S. K., Eng, P. J., Mao, H.-K., Meng, Y. & Shu, J. F. Structure of Alkali Borate
Glasses at High Pressure: B and Li K-Edge Inelastic X-Ray Scattering Study. Phys. Rev. Lett. 98, 105502 (2007). 25. Striepe, S. et al. Elastic and micromechanical properties of isostatically
compressed soda-lime-borate glasses. J. Non-Cryst. Solids 364, 44–52 (201 26. Hodge, I. M. Enthalpy relaxation and recovery in amorphous materials. J. Non-
Cryst. Solids 169, 211–266 (1994). 27. Yue, Y. Methods
l 13. Klug, D. D. et al. Ab initio molecular dynamics study of the pressure-induced
phase transformations in cristobalite. Phys. Rev. B 63, 104106 (2001). Sample preparation. Glass with composition (in mol%) of 25Na2O – 10CaO –
65B2O3 was prepared using melt-quenching technique in an inductively heated
furnace, as described in details elsewhere25,43. Sample rods (4 3 4 3 30 mm3) were
then isostatically compressed using a cold seal pressure vessel under argon gas. For
details of the setup, see Ref. 25. The samples were heated under pressure to a
temperature around Tg 1 20 K (Tg 5 764 K), followed by equilibration at this
temperature for ,3 min, and finally cooled to room temperature at an initial cooling
rate of ,3 K/min. The experiments were performed at the following pressures (p):
0.1, 100, 200, 300, 400, 500, and 570 MPa. Relaxation studies were performed on the
sample compressed at 570 MPa. This was done by isothermal heat-treatment at 0.9Tg
5 688 K for the following durations (ta): 15, 30, 60, 120, 180, 360, and 1440 min. 14. Huang, L. P. et al. Transformation pathways of silica under high pressure. Nat. Mater. 5, 977–981 (2006). 15. Bauchy, M. & Micoulaut, M. Transport anomalies and adaptative pressure-
dependent topological constraints in tetrahedral liquids: evidence for a
reversibility window analogue. Phys. Rev. Lett. 110, 095501 (2013). 16. Grimsditch, M., Polian, A. & Wright, A. C. Irreversible structural changes in
vitreous B2O3 under pressure. Phys. Rev. B 54, 152–155 (1996). 17. Lee, S. K. et al. Probing of bonding changes in B2O3 glasses at high pressure with
inelastic X-ray scattering. Nat. Mater. 4, 851 (2005). 18. Wu, J. et al. Structural response of a highly viscous aluminoborosilicate melt to
isotropic and anisotropic compressions. J. Chem. Phys. 131, 104504 (2009). Physical property measurements. Density (r) was measured in ethanol using
Archimedes’ principle. Vickers hardness (HV) was determined using a micro-
indentor (HMV2000, Shimadzu) operated at a load of 9.81 N at ambient conditions
using a dwell time of 15 s. The diagonals of the indentation were measured using a 3D
laser scanning microscope (VK-9700K, Keyence). Refractive index (n) of samples
before and after the DSC measurements (see below) were performed at 633 nm using
a low range Precision Refractometer (Bausch & Lomb). 19. Wondraczek, L., Sen, S., Behrens, H. & Youngman, R. E. Structure-energy map of
alkali borosilicate glasses: Effects of pressure and temperature. Phys. www.nature.com/scientificreports enthalpy landscape view of glasses40. At high temperatures (i.e., well
above Tg), the system can flow freely among its configurational
microstates, corresponding to the case of an ergodic, equilibrium
liquid. As the system is cooled, the configurational transitions occur
less frequently owing to the loss of thermal energy. At the glass
transition, there is a continuous breakdown of ergodicity as the sys-
tem gradually becomes trapped in a subset of the available config-
urational phase space known as a ‘‘metabasin’’, i.e., a group of
configurations that are mutually accessible at a given temperature
and for a given observation time41. The relatively slow configura-
tional transitions among different metabasins typically involve a
concerted series of such transitions. During annealing of the borate
glass in this work, there is more thermal energy available for the
system to sample the phase space. However, since there is no decrease
of N4 during annealing, the boron coordination state is thermome-
chanically trapped in a basin in the energy landscape as a con-
sequence of the isostatic compression although the macroscopic
properties relax on the time scale of the annealing time. This kinetic
effect is caused by a high activation barrier in the enthalpy landscape,
preventing the boron coordination number from decreasing. Another possibility is the lack of a thermodynamic driving force
for the coordination change. According to the random pair model
of Gupta, the equilibrium N4 value of the studied composition is 49.2
at%42,43. Hence, the N4 of the compressed glass is closer to the ambi-
ent equilibrium value than that of the as-prepared glass (see inset of
Fig. 4b), i.e., there is no thermodynamic driving force to convert BIV
into BIII units. determined from integration of these resonances and with consideration of a small determined from integration of these resonances and with consideration of a small
correction in BIV intensity due to overlapping satellite transitions45. 23Na 3QMAS
experiments were conducted also at 16.4 T (185.10 MHz resonance frequency) using
a 3.2 mm MAS NMR probe and sample spinning of 20 kHz. The pulse sequence used
two hard rf pulses (2.3 and 0.9 ms), followed by a z-filter echo using a delay of 10 ms
and a soft reading pulse of 15 ms. 1. Dubrovinsky, L., Dubrovinskaia, N., Prakapenka, V. B. & Abakumov, A. M. Implementation of micro-ball nanodiamond anvils for high-pressure studies
above 6 Mbar. Nat. Commun. 3, 1163 (2012). 2. Yang, W. G. www.nature.com/scientificreports et al. Coherent diffraction imaging of nanoscale strain evolution in a
single crystal under high pressure. Nat. Commun. 4, 1680 (2013). 3. Mao, H. K. et al. Phonon density of states of iron up to 153 Gigapascals. Science
292, 914–916 (2001). 4. Mishima, O., Calvert, L. D. & Whalley, E. An apparently first-order transition
between two amorphous phases of ice induced by pressure. Nature 314, 76–78
(1985). 5. Greaves, G. N. et al. Identifying vibrations that destabilize crystals and
characterize the glassy state. Science 308, 1299–1302 (2005). g
y
6. Elliot, S. R. Physics of Amorphous Materials (Wiley, New York, 1988 7. Yarger, J. L. et al. Al coordination changes in high-pressure aluminosilicate
liquids. Science 270, 1964–1967 (1995). q
8. Guthrie, M. et al. The formation and structure of a dense octahedral glass. Phys. Rev. Lett. 93, 115502 (2004). 9. Meng, Y. et al. The formation of sp(3) bonding in compressed BN. Nat. Mater. 3,
111–114 (2004). 10. Monaco, A. et al. Effect of densification on the density of vibrational states of
glasses. Phys. Rev. Lett. 97, 135501 (2006). g
y
11. Bouhadja, M., Jakse, N. & Pasturel, A. Structural and dynamic properties of
calcium aluminosilicate melts: A molecular dynamics study. J. Chem. Phys. 138,
224510 (2013). 12. Angell, C. A., Cheeseman, P. A. & Tamaddon, S. Pressure enhancement of ion
mobilities in liquid silicates from computer simulations studies to 800 kbar. Science 218, 885–887 (1982). Discussion The irreversibility of the thermomechanically induced change in
boron speciation upon annealing at 0.9Tg implies that the free energy
barrier for forming a given structural state (i.e., boron speciation) can
be overcome not only chemically, but also thermomechanically. This
allows for tailoring of both microscopic and macroscopic properties,
since the increased network connectivity that remains after anneal-
ing should increase, e.g., chemical durability and thermal shock
resistance. To further understand this effect, we consider the integration of peaks in Fig. 4a, with annealing duration. Inset: impact of
isostatic pressure on N4 in absence of annealing (corresponding 11B spectra
are shown in Supplementary Figure 2a). The error of N4 is 60.5 at% for the
variable pressure series (inset) and 61 at% for the variable annealing time
study. The latter has higher error due to changes in lineshape and thus
more difficulty in fitting the 11B MAS NMR data. (c) Solid state 23Na MAS
NMR spectra at 16.4 T of the samples compressed at 570 MPa and then
annealed for various durations at 0.9Tg 5 688 K. SCIENTIFIC REPORTS | 4 : 3770 | DOI: 10.1038/srep03770 4 Author contributions Y.Z.Y. conceived the study. M.M.S., R.E.Y., S.S., M.P. and U.B. prepared the samples,
performed the measurements, and contributed to analysis of the data. M.M.S. and Y.Z.Y. wrote the manuscript with inputs from R.E.Y., J.D., H.B. and J.C.M. All authors were
involved in the discussions. 37. Deubener, J., Yue, Y. Z., Bornho¨ft, H. & Ya, M. Decoupling between birefringence
decay, enthalpy relaxation and viscous flow in calcium boroalumosilicate glasses. Chem. Geol. 256, 298–304 (2008). 38. Reinsch, S, Mu¨ller, R., Deubener, J. & Behrens, H. Internal friction of hydrated
soda-lime-silicate glasses. J. Chem. Phys. 139, 174506 (2013). Methods
l Z., Wondraczek, L., Behrens, H. & Deubener, J. Glass transition in an
isostatically compressed calcium metaphosphate glass. J. Chem. Phys. 126, 144902
(2007). 28. Yue, Y. Z., von der Ohe, R. & Jensen, S. L. Fictive temperature, cooling rate, and
viscosity of glasses. J. Chem. Phys. 120, 8053–8059 (2004). Solid state NMR. 11B and 23Na MAS NMR experiments were conducted at 16.T using
a commercial spectrometer and MAS NMR probes. Resonance frequencies for 11B
and 23Na at this external magnetic field strength were 224.51 and 185.10 MHz,
respectively. Samples were crushed using an agate mortar and pestle, packed into
3.2 mm zirconia rotors and spun at frequencies of nominally 20 kHZ. 11B and 23Na
MAS NMR spectra were collected using short radio-frequency pulses (0.6 ms,
equivalent to p/12 tip angles), relaxation delays of 2 seconds and signal averaging of
1000 to 2000 acquisitions. Data were frequency referenced to aqueous boric acid at
19.6 ppm and aqueous NaCl at 0 ppm for 11B and 23Na, respectively. 11B MAS NMR
spectra were fit using DMfit44 to reproduce BIII and BIV lineshapes, and N4 values were 29. Yue, Y. Z. & Angell, C. A. Clarifying the glass-transition behaviour of water by
comparison with hyperquenched inorganic glasses. Nature 427, 717–720 (2004). 30. Wolf, G. H. & McMillan, P. F. Pressure effects on silicate melt – Structure and
properties. Rev. Mineral. 32, 505–561 (1995). 31. Wondraczek, L., Krolikowski, S. & Behrens, H. Kinetics of pressure relaxation in a
compressed alkali borosilicate glass. J. Non-Crys. Solids 356, 1859–1862 (2010). 31. Wondraczek, L., Krolikowski, S. & Behrens, H. Kinetics of pressure relaxation in a
compressed alkali borosilicate glass. J. Non-Crys. Solids 356, 1859–1862 (2010). 32. Allwardt, J. R. et al. Aluminum coordination and the densification of high-
pressure aluminosilicate glasses. Am. Mineral. 90, 1218–1222 (2005). 32. Allwardt, J. R. et al. Aluminum coordination and the densification of high-
pressure aluminosilicate glasses. Am. Mineral. 90, 1218–1222 (2005). SCIENTIFIC REPORTS | 4 : 3770 | DOI: 10.1038/srep03770 5 44. Massiot, D. et al. Modelling one- and two-dimensional solid-state NMR spectra. Magn. Reson. Chem. 40, 70–76 (2002). 33. Shannon, R. D. & Prewitt, C. T. Effective ionic radii in oxides and fluorides. Acta
Crystallogr. B 25, 925–946 (1969). y
g
34. Youngman, R. E. & Zwanziger, J. W. Network Modification in Potassium Borate
Glasses: Structural Studies with NMR and Raman Spectroscopies. J. Phys. Chem. 100, 16720–16728 (1996). 45. Methods
l Massiot, D., Bessada, C., Coutures, J. P. & Taulelle, F. A quantitative study of 27Al
MAS NMR in crystalline YAG. J. Magn. Reson. 90, 231–242 (1990). 35. Ya, M., Deubener, J. & Yue, Y. Z. Enthalpy and anisotropy relaxation of glass
fibers. J. Am. Ceram. Soc. 91, 745–752 (2008). 36. Sen, S., Topping, T., Yu, P. & Youngman, R. E. Atomic-scale understanding of
structural relaxation in simple and complex borosilicate glasses. Phys. Rev. B 75,
094293 (2007). Additional information g
y
39. Smedskjaer, M. M., Mauro, J. C. & Yue, Y. Z. Prediction of Glass Hardness Using
Temperature-Dependent Constraint Theory. Phys. Rev. Lett. 105, 115503 (2010). 40. Stillinger, F. H. & Weber, T. A. Hidden structure in liquids. Phys. Rev. A 25,
978–989 (1982). 39. Smedskjaer, M. M., Mauro, J. C. & Yue, Y. Z. Prediction of Glass Hardness Using
Temperature-Dependent Constraint Theory. Phys. Rev. Lett. 105, 115503 (2010). Supplementary information accompanies this paper at http://www.nature.com/
scientificreports 40. Stillinger, F. H. & Weber, T. A. Hidden structure in liquids. Phys. Rev. A 25,
978–989 (1982). Competing financial interests: The authors declare no competing financial interests. How to cite this article: Smedskjaer, M.M. et al. Irreversibility of Pressure Induced Boron
Speciation Change in Glass. Sci. Rep. 4, 3770; DOI:10.1038/srep03770 (2014). 41. Gupta, P. K. & Mauro, J. C. The laboratory glass transition. J. Chem. Phys. 126,
224504 (2007). 42. Gupta, P. K. In Proceedings of the International Congress on Glass (New Delhi,
India, 1986). This work is licensed under a Creative Commons Attribution 3.0 Unported license. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0 This work is licensed under a Creative Commons Attribution 3.0 Unported license. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0 43. Smedskjaer, M. M., Mauro, J. C., Sen, S. & Yue, Y. Z. Quantitative design of glassy
materials using temperature-dependent constraint theory. Chem. Mater. 22,
5358–5365 (2010). SCIENTIFIC REPORTS | 4 : 3770 | DOI: 10.1038/srep03770 6
|
https://openalex.org/W4377043257
|
https://www.frontiersin.org/articles/10.3389/fpls.2023.1125560/pdf
|
English
| null |
Impact of meteorological conditions, canopy shading and leaf removal on yield, must quality, and norisoprenoid compounds content in Franciacorta sparkling wine
|
Frontiers in plant science
| 2,023
|
cc-by
| 12,812
|
OPEN ACCESS OPEN ACCESS
EDITED BY
Alessandra Ferrandino,
University of Turin, Italy
REVIEWED BY
Silvia Guidoni,
University of Turin, Italy
Daniela Farinelli,
University of Perugia, Italy
Maurizio Petrozziello,
Council for Agricultural and Economics
Research (CREA), Italy
*CORRESPONDENCE
Isabella Ghiglieno
isabella.ghiglieno@unibs.it
†PRESENT ADDRESS
Isabella Ghiglieno,
Department of Civil, Environmental,
Architectural Engineering and Mathematics,
Agrofood Research Hub, University of
Brescia, Brescia, Italy
‡These authors have contributed
equally to this work and share
first authorship
RECEIVED 16 December 2022
ACCEPTED 17 April 2023
PUBLISHED 17 May 2023
CITATION
Ghiglieno I, Carlin S, Cola G, Vrhovsek U,
Valenti L, Garcia-Aloy M and Mattivi F
(2023) Impact of meteorological
conditions, canopy shading and leaf
l
i ld
t
lit
d University of Turin, Italy
REVIEWED BY
Silvia Guidoni,
University of Turin, Italy
Daniela Farinelli,
University of Perugia, Italy
Maurizio Petrozziello,
Council for Agricultural and Economics
Research (CREA), Italy 1Department of Agricultural and Environmental Sciences - Production, Landscape, Agroenergy,
University of Milan, Milan, Italy, 2Metabolomic Unit, Food Quality and Nutrition Department, Research
and Innovation Center, Edmund Mach Foundation, S. Michele all’Adige, Italy Climate change is a major concern in agriculture; in grapevine production,
climate change can affect yield and wine quality as they depend on the
complex interactions between weather, plant material, and viticultural
techniques. Wine characteristics are strongly influenced by microclimate of the
canopy affecting primary and secondary metabolites of the grapevine. Air
temperature and water availability can influence sugar and acid concentration
in grapes and relative wines, and their content of volatile compounds such as
norisoprenoids. This becomes relevant in sparkling wine production where
grapes are generally harvested at a relatively low pH, high acidity, and low
sugar content and where the norisoprenoids significantly contributes to the
final aroma of the wine. The effect of climate change on grapevine and wine,
therefore, calls for the implementation of on-field adaptation strategies. Among
them canopy management through leaf removal and shading have been largely
investigated in the wine growing sector. The present study, conducted over 4
years (2010-2013) aims at investigating how leaf removal and artificial shading
strategies affect grape maturation, must quality and the production of
norisoprenoids, analyzed using an untargeted approach, in sparkling wine. Specifically, this paper investigates the effect of meteorological conditions (i.e.,
water availability and temperatures) and the effect of leaf removal and shading on
Vitis vinifera L. cv. COPYRIGHT
© 2023 Ghiglieno, Carlin, Cola, Vrhovsek,
Valenti, Garcia-Aloy and Mattivi. 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. TYPE Original Research
PUBLISHED 17 May 2023
DOI 10.3389/fpls.2023.1125560 TYPE Original Research
PUBLISHED 17 May 2023
DOI 10.3389/fpls.2023.1125560 1 Introduction lower soluble sugar content than those for table wine production
(Alfonzo et al., 2020). The synthesis of these compounds tends to
increase during the herbaceous phase of the berry, while their
degradation is enhanced after veraison and during the final stages
of ripening. Carotenoids are the precursors of the norisoprenoids,
volatile compounds. Norisoprenoids can be generated by the direct
degradation of carotenoids such as b-carotene and neoxanthin, and
can be stored as glycoconjugates, which can then release their
volatile aglycones during wine fermentation or ageing; hydrolysis
is often slow and depends on various factors, such as storage
duration, temperature, and pH (Winterhalter and Schreier, 1994;
Deluc et al., 2009; Song et al., 2012). The olfactory perception
thresholds of these compounds are very low and, therefore, they
have a high sensorial impact on the wine aroma (Mendes-Pinto,
2009). The most important C13 norisoprenoids are: TDN,
vitispirane, actinidols, b-damascenone, b-ionone, and E-1-(2,3,6-
trimethylphenyl) buta-1,3diene (TBP-1). Climate change means any changes in weather patterns caused
by natural events and human activities during a certain period
(IPCC, 2007). The temperature increase that has characterized
Europe and Italy since the end of the 1980s has relevant
consequences on the quantity and quality of crops, representing a
major concern in agriculture (Mariani et al., 2012; Suter et al., 2021). In grape-growing for wine production, climate change can
significantly affect yield and, indirectly, wine quality (Fraga et al.,
2012), as they depend on the complex interactions between weather
variables, plant material, and viticultural techniques (Nesbitt et al.,
2016; van Leeuwen et al., 2019; Mirás-Avalos and Araujo, 2021). Air
temperature, water availability, and sunlight are the most relevant
factors that, influencing grape and must quality, indirectly affect
wine quality. Grapes and musts characteristics are, indeed, strongly
influenced by climatic conditions as changes in the microclimate of
the canopy have an impact on the primary and secondary
metabolites of the grapevine. Although the concentration of norisoprenoids in grapes
depends on many factors, such as variety and stage of ripeness,
the environmental conditions, have a fundamental role in
influencing their synthesis. This is confirmed by the behavior of
norisoprenoid b-damascenone that decreases in white wines in
conditions of exposure to light and higher temperature (Marais
et al., 1992; Kwasniewski et al., 2010). OPEN ACCESS Chardonnay and Pinot noir, which are suitable to produce
sparkling wine in the DOCG Franciacorta wine growing area (Lombardy, Italy). The effect of leaf removal and shading practices on norisoprenoids has been the
focus of the study. No defoliation and artificial shading treatments play an
important role in the preservation of the acidity in warm seasons and this
suggests calibrating defoliation activities in relation to the meteorological trend
without standardized procedures. This is particularly relevant in the case of
sparkling wine, where the acidity is essential to determine wine quality. The CITATION
Ghiglieno I, Carlin S, Cola G, Vrhovsek U,
Valenti L, Garcia-Aloy M and Mattivi F
(2023) Impact of meteorological
conditions, canopy shading and leaf
removal on yield, must quality, and
norisoprenoid compounds content in
Franciacorta sparkling wine. Front. Plant Sci. 14:1125560. doi: 10.3389/fpls.2023.1125560 01 Frontiers in Plant Science frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 enhanced norisoprenoid aromas obtained with a total defoliation represent a
further element to direct defoliation and shading strategies. The obtained results
increase knowledge about the effect of different defoliation and artificial shading
applications in relation to meteorological condition supporting the management
decision-making in the Franciacorta wine growing area. Frontiers in Plant Science vine (Vitis vinifera L.), leaf removal, grapevine shading, must quality, yield, sparkling
wine, aroma compounds, norisoprenoid frontiersin.org KEYWORDS Ghiglieno et al. 2.1 Experimental setting This study was carried out in the winegrowing area of Franciacorta
DOCG, a famous Italian sparkling wine production area located in the
Lombardy Region. The experiment was repeated for four consecutive
years (from 2010 to 2013) in a vineyard (45.572850° Lat, 9.967622°
Lon) belonging to Azienda Agricola Castello Bonomi Tenute in
Franciacorta. This company is located in the southern part of the
Franciacorta region (Supplementary Figure 1). The soil of experimental vineyard is grass-covered and
characterized by a loamy texture, high total organic carbon
content and CEC (Cation Exchange Capacity). The vineyard, with
row orientation from north to south, was planted in 2004 with two
international Vitis vinifera L. cultivars, Chardonnay (clone
ENTAV-INRA® 96) and Pinot noir (clone 292), both grafted
onto Kober 5BB rootstock. Both cultivars are cordon trained. The experimental plan included five different treatments: a
control test without defoliation and shading (ND), a test where
about six basal leaves, equal to about 35% of total leaf area, were
removed, east and west side (TD), and three different systems
adopting shading nets applied along the bunch zone; two of the
shaded treatments were defoliated as for TD and covered with one
layer of shading net (TD1) or two layers of shading net (TD2), while
a third treatment was covered by only one layer of shading net, but
not defoliated (ND1L). The present 4-year (from 2010 to 2013) study aims to
investigate how leaf removal and artificial shading strategies affect
grape maturation, must quality and the production of
norisoprenoids in sparkling wine. Wine volatiles compounds were
analyzed using an untargeted approach aimed at a holistic view
rather than restricted only to a list of target compounds (Cozzolino,
2016). Specifically, the paper investigates the effect of
meteorological conditions (i.e., water availability and
temperatures) on grapevine production and must quality. Moreover, the practice of application of different canopy shading
levels on Vitis vinifera L. cv. Chardonnay and Pinot noir is studied
to understand their effect on grapevine production and must quality
as well as the on the aroma of sparkling wine in the DOCG
Franciacorta wine growing area (Lombardy, Italy). 2.1 Experimental setting Understanding
the response of musts and wine quality to leaf removal and shading,
in relation to the meteorological variability of each year, is useful to
direct agronomical management in tackling the issue of climate
change (e.g., early ripening, acidity degradation, wine aroma profile
variation) that is affecting sparkling wine quality in particular. The
leaf removal and shading practices on norisoprenoids compounds
shall also be examined. The concentrations of these aromatic
compounds tend to increase during wine aging, especially in
sparkling wines, and can reach the level above the threshold of
the characteristic aroma of aged wine which is sometimes
detrimental for the wine quality. Since there aren’t, to our
knowledge, studies that had been conducted on the modulation of
norisoprenoids in relation to canopy management in finished
sparkling wine, this work could be useful to understand the effect
of these treatments in the final product. The treatments were replicated on both the Chardonnay and
Pinot noir cultivars. All treatments were set in two replicates
represented by two rows of the vineyard for each cultivar. Each
treatment was extended for 25 vines for each of the two replicates. All treatments took place at about 20% veraison, corresponding to
July 19th, July 14th, July 19th, July 29th, respectively, in 2010, 2011,
2012 and 2013. Both manual leaf removal and shading net
application were carried out along the bunch zone; shading was
realized through a UVstabilized polyethylene net of approximately
95 g m-2 (shading net OF50N supplied by Retes srl). The
transmittance of global solar radiation of the single layer and
double layer nets was preliminarily tested in order to evaluate the
percentage of global solar radiation passing through the nets. A
reduction by 50% and 70% was detected for single- and double-
layer shading nets, respectively. 1 Introduction Water availability and water stress lead to different effects
depending on the grapevine developmental stage, cultivar and
wine target (Deloire et al., 2004; Santos et al., 2020; Mirás-Avalos
and Araujo, 2021). During post-veraison excessive humidity tends
to promote sugar dilution (Reynolds and Naylor, 1994), delaying
the harvesting time (Tonietto, 1999) and avoiding an excessive
accumulation of total soluble solids (Intrigliolo et al., 2016). In
general, the water status correlates positively with berry size, total
acidity, and malic acid concentration (Mirás-Avalos and
Intrigliolo, 2017). The effect of climate change on grapes, musts and,
consequently, on wine quality, therefore, calls for the
implementation of on-field adaptation strategies. Among these,
the management of the canopy through leaf removal and shading
have been largely investigated in the wine growing sector (Downey
et al., 2006; Caravia et al., 2016; Ghiglieno et al., 2020; Martı́nez-
Lüscher et al., 2020). The influence of these agronomic practices on
canopy microclimate has been widely studied (Crippen and
Morrison, 1986; Jackson and Lombard, 1993; Downey et al., 2006;
Ghiglieno et al., 2020). The direct solar radiation on grape caused by
leaf removal and the consequent increase in berry temperature
during maturation, influence berry ripening and metabolism,
promoting sugar contents accumulation (Riou et al., 1994),
reducing titratable acidity, and increasing malic acid degradation
(Lakso and Kliewer, 1975; Conde et al., 2006; de Oliveira et al.,
2019). Moreover, excessive sunlight inhibits the development of
flavor and aroma components (Jones et al., 2005). Much has been
written about how canopy management practices affect the content Many studies focused on the effect of temperature increases on
grapevine and wine quality (Biasi et al., 2019; Santos et al., 2020). The mean air temperatures during the growing season are directly
related to the length of the growing season for each variety (Jones
et al., 2005); the different stages of development generally take place
earlier and the time between veraison and ripening is shorter
(Schultz, 2000; Jones et al., 2005). Excessively hot weather during
the veraison–maturity period can significantly influence sugar
accumulation (Greer, 2013), reducing the level of acidity in
grapevines and, consequently, in wines (Pons et al., 2017). This
becomes relevant in grapevines suitable to produce sparkling wines. In sparkling wines production grapevines are in fact generally
harvested at a relatively low pH, higher titratable acidity, and 02 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 2 Materials and methods in norisoprenoids, especially TDN and vitispirane (Marais et al.,
1992; Chen et al., 2017; Asproudi et al., 2018; Wang et al., 2018). Some authors (Kwasniewski et al., 2010) have indeed demonstrated
that the timing of leaf removal can alter the carotenoid profile, as
well as TDN and vitispirane precursors in mature Riesling grapes: if
the leaves were removed one month after the berry set, they
observed elevated total amounts in both must and wine. With
regard to grapevine shading, although previous studies reported
that artificial shading protects grapes from sunlight exposure but
leads to an increase of air temperature in the fruit zone (Chorti et al,
2010), other researches carried out in Franciacorta wine growing
area demonstrated that this agronomical practice also allows to
decrease inner berry temperature (Ghiglieno et al., 2020). This effect
of artificial on canopy microclimate makes this practice a good
adaptation strategy to climate change. Many authors (Reynolds
et al., 1986; Smart et al., 1990; Dokoozlian and Kliewer, 1996;
Filippetti et al., 2015; Martin et al., 2016) have in fact demonstrated
the effect of shading on delaying ripening and preserving acidity, in
terms of both titratable acidity and malate concentration. The
response of pH and potassium to artificial shading is less clear:
some authors demonstrated a positive effect of shading on pH and
potassium (Smart et al., 1985; Scafidi et al., 2013; Toda and Balda,
2014), while more recent studies reported that this treatment does
not significantly affect these parameters (Filippetti et al., 2015). in norisoprenoids, especially TDN and vitispirane (Marais et al.,
1992; Chen et al., 2017; Asproudi et al., 2018; Wang et al., 2018). Some authors (Kwasniewski et al., 2010) have indeed demonstrated
that the timing of leaf removal can alter the carotenoid profile, as
well as TDN and vitispirane precursors in mature Riesling grapes: if
the leaves were removed one month after the berry set, they
observed elevated total amounts in both must and wine. With
regard to grapevine shading, although previous studies reported
that artificial shading protects grapes from sunlight exposure but
leads to an increase of air temperature in the fruit zone (Chorti et al,
2010), other researches carried out in Franciacorta wine growing
area demonstrated that this agronomical practice also allows to
decrease inner berry temperature (Ghiglieno et al., 2020). This effect
of artificial on canopy microclimate makes this practice a good
adaptation strategy to climate change. 2 Materials and methods Many authors (Reynolds
et al., 1986; Smart et al., 1990; Dokoozlian and Kliewer, 1996;
Filippetti et al., 2015; Martin et al., 2016) have in fact demonstrated
the effect of shading on delaying ripening and preserving acidity, in
terms of both titratable acidity and malate concentration. The
response of pH and potassium to artificial shading is less clear:
some authors demonstrated a positive effect of shading on pH and
potassium (Smart et al., 1985; Scafidi et al., 2013; Toda and Balda,
2014), while more recent studies reported that this treatment does
not significantly affect these parameters (Filippetti et al., 2015). Frontiers in Plant Science 2.2.1 Water stress * When the hourly air temperature is below LC: LHH=1,
NHH=0, HHH=0, meaning that the whole hour was
spent by the plant under stressing conditions caused by
low temperatures. In order to provide a description of the relationship between
plant growth and water resources, a single-layer reservoir model
with a daily time step was created (Cola et al., 2014), considering a
reference soil reservoir of 120 mm. The daily reference
evapotranspiration (ET0) was calculated by means of the
Hargreaves-Samani method and the daily maximum
evapotranspiration of grapevine (ETM) was obtained by means of
a dynamic crop multiplicative coefficient (Kc), as a function of the
phenological stage of the plant, modelled on the base of air
temperature-based course (Mariani et al., 2013). The daily real
grapevine evapotranspiration (ETR) was obtained by means of the
water limiting factor (WLFR) response curve, that relates the soil
water content with the plant stress. When the soil water level is
between the field capacity and the easily available water limit, the
plant does not face stress, WLFR is equal to 1 and ETR=ETM. Otherwise, when the water content moves above the field capacity
up to saturation or when it moves from the easily available water
limit down to the wilting point, WLFR linearly decreases toward 0. As a consequence, the stress increases and ETR decreases. * When the hourly air temperature is above UC: LHH=0,
NHH=0, HHH=1 meaning that the whole hour was spent
by the plant under stressing conditions caused by high
temperatures. * When the hourly air temperature sits between LOC and
UOC: LHH=0, NHH=1, HHH=0 meaning that the whole
hour was spent by the plant under restful conditions and
the development was non limited. * Between LC and LOC the low temperature stress decreases
linearly moving from LC to LOC, so that LHH moves from
1 to 0 and NHH from 0 to 1 (always with LHH+NHH=1
and HHH=0). * Between UOC and UC the high temperature stress increases
linearly moving from UOC to UC so that NHH moves from
1 to 0 and HHH from 0 to 1 (always with NHH+HHH=1
and LHH=0). In this work, the seasonal water stress (WSTR) was obtained
cumulating the daily values of 1-WLFR, so that a day without water
stress weights 0 and a day with maximum water stress weights 1. 2.2.2 Thermal regime In this work, NHH, LHH and HHH were cumulated from fruit-
set to harvest to describe the thermal course of the season. Phenological stages of each plot were determined on the base of
weekly phenological monitoring. In order to describe the air thermal condition during fruit
development and ripening, thermal resources and limitations were
obtained on the basis of the air temperature, applying the Normal
Heat Hours (NHH) response curve (Mariani et al., 2012; Mariani
et al., 2013; Cola et al., 2020). This method tries to overcome the
overestimation of high temperature in terms of plant development
that characterizes the classic growing degree-days approach of the
Winkler Index (Amerine and Winkler, 1944) in which a very warm
day is translated into a high value of the index, meaning a strong
positive contribution to plant growth when high air temperature
values are detrimental for the plant development. Additionally, the stress caused by heat waves (33STR) was
obtained by counting the number of days between fruit set and
physiological maturity with a maximum temperature above 33°C
(Cola et al., 2020). 2.2 Meteorological data and
agrometeorological indices During the four years, weather conditions were monitored by
means of the weather station of Rovato (BS) of the
Agrometeorological Network of the Province of Brescia, which is
located at a distance of 5 km from the experimental vineyard. To provide a more precise description of the thermal conditions
in the experimental vineyard a four-channel data logger (Onset Frontiers in Plant Science 03 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 10.3389/fpls.2023.1125560 NHH indicates the total hourly thermal resources useful for
berry maturation from fruit set to physiological maturity (Cola
et al., 2017; Cola et al., 2020) and represent thermal resources. Low
Heat Hours (LHH) represents the stress caused by hourly
temperatures below the optimal level and High Heat Hours
(HHH) is the stress caused by hourly temperatures above the
optimal level. NHH indicates the total hourly thermal resources useful for
berry maturation from fruit set to physiological maturity (Cola
et al., 2017; Cola et al., 2020) and represent thermal resources. Low HOBO U12) equipped with an air temperature sensor (thermistor
Onset HOBO S-THC), protected by a solar shield was placed in
the vineyard. Precipitation data recorded by the Rovato weather station and
temperature data from the vineyard thermometer were used to
characterize the general meteorological features of the four years of
investigation and to calculate thermal indices, the grapevine water
balance and the derived water stress indices, as described
further below. Heat Hours (LHH) represents the stress caused by hourly
temperatures below the optimal level and High Heat Hours
(HHH) is the stress caused by hourly temperatures above the
optimal level. More in detail, air temperature is weighed on the base of four
parameters, namely, low cardinal - LC, low optimal cardinal – LOC,
upper optimal cardinal – UOC and upper cardinal UC, respectively: 2.2.1 Water stress Additionally, the cumulated evapotranspiration deficit (DET) was
obtained, on the base of the daily difference [mm] between ETM
and ETR throughout the season. The parametrization of the four parameters (LC=12°C,
LOC=24°C, UOC=26°C, UC=33°C) proved to perform very well
in describing the development of several cultivars (Cabernet-
Sauvignon, Chardonnay, Barbera and Georgian cultivars Mtsvane
Kakhuri, Rkatsiteli, Ojaleshi and Saperavi) (Mariani et al., 2013;
Cola et al., 2014; Cola et al., 2017). Frontiers in Plant Science frontiersin.org 2.3 Yield components and the
composition of must Indices were calculated considering meteorological conditions
along the berry development period (i.e., from berry set stage to
harvest) and specifically: from June 1st to August 26th for 2010; from
May 20th to August 1st 2011; from May 29th to August 8th 2012;
from June 6th to August 22nd 2013. With the aim to compare musts and wine composition at a
standard potential alcohol degree, for both cultivars and all
treatments, the harvesting time was established on the basis of the 04 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 strategy adopted by the wine company for the specific year and
ranging from 10 to 10.7% of potential alcohol. each sample, 2 ml of wine with 50 ml of internal standard solution (2-
octanol in ethanol at a concentration of 1 mg l-1) was mixed with 1 g
NaCl in a 20 ml headspace vial. A Gerstel MultiPurpose Sampler
autosampler (Gerstel GmbH & Co. KG Mülheim an der Ruhr
Germany) with an agitator and SPME fiber 2 cm (50/30 DVB/
CAR/PDMS), from Supelco Merk KGaA (Darmstadt, Germany), was
used to extract the volatiles from the sample vial headspace. The
sample was pre-incubated for 5 min at 35°C. Adsorption lasted
20 min, at the same temperature, Then, desorption took place in the
injector in splitless mode (4 min) at 250°C. The fiber was then
reconditioned for 10 min at 250°C. The GC×GC system was the
Agilent 7890 A (Agilent Technologies, Santa Clara, CA). Injections
were performed in splitless mode. Equipped columns were the VF-
Wax column (100% polyethylene glycol; 30 m × 0.25 mm × 0.25 mm,
Agilent J&W Scientific Inc., Folsom, CA) as the 1st dimension and
Rxi-17Sil MS 1.50 m × 0.15 mm × 0.15 mm, Restek Bellefonte, USA)
as the 2nd dimension. The GC system was equipped with a secondary
column oven and non-moving quadjet dual-stage thermal modulator. The injector/transfer line was maintained at 250°C. Oven
temperature program conditions were as follows: initial
temperature of 40°C for 4 min, programmed at 6°C min−1 at 250°
C, where it remained for 5 min. The secondary oven was kept at 5°C
above the primary oven throughout the chromatographic run. The
modulator was offset by +15°C in relation to the secondary oven; the
modulation time was 7 s and 1.4 s of hot pulse duration. Helium
(99.9995% purity) was used as carrier gas at a constant flow of 1.2 ml
min-1. 2.4 Wine production About 100 kg of grapes were harvested for each cultivar and
treatment upon reaching the standard potential alcohol. Grapes
were harvested in small boxes weighing maximum 15 kg, and
stocked in a cold room at about 10°C. Microvinifications was
then initiated according to a standardized protocol. The wine-
making protocol was organized in different steps described in
Supplementary Figure 2. For GC×GC-MS data, LECO ChromaTOF Version 4.22
software was used for all acquisition control, data processing and
Fisher ratio calculations. Automated peak detection and spectral
deconvolution with a baseline offset of 0.8 and signal-to-noise of
100 were used during data treatment. Before proceeding with the
data analysis a quality control of the data sets, checking the
distribution of the QC injections was carried out (Arapitsas et al.,
2016). The identification of the wine volatile compounds was
achieved by comparing the mass spectrometric information for
each chromatographic peak with NIST 2.0, Wiley 8 and the FFNSC
2 mass spectral library (Chromaleont, Messina, Italy), with a library
similarity match factor of 750 and comparing also the experimental
linear temperature retention index (LTPRI) with retention indices
reported in the literature for 1D-GC (VCF Volatile Compounds in
Food 16.1 database). Retention data for a series of n-alkanes (C10–
C30), under the same experimental conditions employed for
chromatographic analysis of wine volatiles, were used for
experimental LTPRI calculation. The content of norisoprenoids
were expressed as peak areas. The wine so obtained was stocked in 15 l demijohns and
periodically monitored for free sulfur content, using the
WineScan™SO2 (FOSS) instrument, to avoid undesired malic
fermentation. After about five months the wine was stabilized by
adding 10 ml hl-1 silica sol and 1 ml hl-1 gelatin. The wine was then
bottled using 0.75 l bottles (for a total of 18 bottles); nine bottles
were then stocked and 0.3 g l-1 of yeast and 22 g l-1 of sugar were
added to the other nine bottles to induce refermentation, thus
allowing to produce a final sparkling wine. After this last step, all
bottles were stocked and manually disgorged, after a manual
remuage to remove the residue of yeasts without any further
addition. Disgorgement was performed for all wine samples at the
time of the volatile aroma compounds analysis in 2016 and 3
different bottles for each treatment were used as replicates. 2.3 Yield components and the
composition of must The MS signal was obtained with a Pegasus IV time-of flight
mass spectrometer (Leco Corporation, St. Joseph, MI) with electron
ionization at 70 eV and the ion source temperature at 230°C, detector
voltage of 1317 V, mass range of m/z 35–450 and acquisition rate of
200 spectra s−1. The evolution of ripening for both Chardonnay and Pinot noir
was monitored weekly (maintaining an interval of 7-5 days between
each sampling), in order to identify the relevant harvesting time for
each treatment. Experimental harvesting was organized by selecting 5
grapevines from the two 25 vine replicates for each treatment, for
a total of 10 vines per treatment (ND, TD, TD1, TD2 and ND1L). For each grapevine, the average bunch weight (ABW) was
calculated counting the number of bunches and weighing the
entire grapevine production. A sample of three bunches was
collected from each grapevine to check must quality. These
samples were then crushed and the total soluble solids
concentration (TSS), pH, titratable acidity (TA) and malic acid
(MA) concentration were analyzed in the resulting musts. These
measurements were determined using a traditional handheld
refractometer for soluble solids concentration, a Crison compact
titrator analyser for both pH and TA, and the enzymatic method
(Hyperlab wine analyser) to determine MA concentration. 2.5 Analysis of volatile aroma compounds
by GC×GC-TOF-MS All the analyses were carried out following the method described
in our previous works (Carlin et al., 2016; Carlin et al., 2022). In order
to monitor instrument stability, pooled quality controls (QC)
consisting of equal proportion of each sample were placed at the
beginning of the run (n=5) and thereafter every 10th sample as a
common practice in metabolomics studies (Arapitsas et al., 2016). For Frontiers in Plant Science frontiersin.org 3.1 Meteorological characterization Considering the limited number of biological replicates used for
the untargeted volatile compounds analyses (i.e., 3 replicates for
each cultivar, treatment, and year) a different approach was
outlined. In this case, it was checked whether there were any
statistically significant differences associated with each treatment
compared to the untreated thesis (ND) by means of a linear model
on log-scaled data using R software (R Core Team, 2022). The year 2012 was characterized by the highest water stress
value over the four years (as shown by WSTR and DET values),
mainly due to the limited precipitation during June, July and
August that led to a long period of a limited water soil content. 3.1 Meteorological characterization Table 1 shows the monthly variation of temperature and
precipitation by season, respectively, while the agrometeorological
indices are shown in Table 2. The relationships between year and agrometeorological indices
and AWB and must quality were also investigated through a
principal component analysis (PCA) on autoscaled data (i.e.,
mean-centered and divided by the standard deviation of each
variable), through R software (R Core Team, 2022). In the PCA
analysis for each cultivar, five points for each year, corresponding to
the average value of the single treatment in a specific year, were
considered. This approach was used because one single value of
agrometeorological indices was available for each year without
having the possibility to differentiate between treatments. The year 2010 was characterized by optimal water conditions. The scarce precipitation of the winter period (Jan-Mar) was
preceded by good precipitations in the 2009 Oct-Dec period and
followed by abundant spring precipitations. From a thermal point
of view, 2010 showed lower maximum temperatures during spring
than 2011 and 2012, resulting in the highest NHH value in the
series. However, the indices for this year were strongly affected by
a longer interval between fruit set and harvest, than in the other
three years. Low temperature stress (LHH) was the second highest
over the four years and high temperature stress (HHH) was the
highest. The 33°C limit was exceeded for 65 days during
the season. Additional ANOVA and Siegel-Tukey’s post-hoc test were
performed to determine the influence of the treatment on
variables related to ABW and musts quality (TSS, TA, MA, pH). In this analysis, both the cultivars and the four years were
considered separately. The decision to process the different years
separately was determined by the meteorological differences in the
individual years and the effect on the AWB and must quality
revelead by the results obtained from the analysis of the
relationship between year and these variables. The year 2011 was still characterized by very low water stress,
thanks to the refilling of the water reservoir during winter and the
good distribution of precipitation over the months. As compared to
other years, low temperatures caused a high level of NHH (second
highest), the highest value of low temperature stress (LHH) and the
lowest value of high temperature stress (HHH), confirmed by only 2
days with maximum temperatures above 33°C. 2.6 Statistical analysis In the statistical analysis, Chardonnay and Pinot noir were kept
separated in relation to the different composition of their grapes and 05 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 3 Results their different sensory characterization in wines (Herrero
et al., 2016). The effect of the year was initially investigated for variables
related to ABW and musts quality through an analysis of variance
(ANOVA) and Siegel-Tukey’s post-hoc test using the SPSS software
(Statistical Package for Social Science) (IBM Corp, 2021). Frontiers in Plant Science frontiersin.org 3.1 Meteorological characterization From a thermal point of view, the year 2012 featured the lowest
levels in terms of NHH accumulation (though very close to 2013), Month
Air Temperature [°C]
Monthly Precipitation [mm]
Minimum
Maximum
2010
2011
2012
2013
2010
2011
2012
2013
2010
2011
2012
2013
Jan
-1.3 (2.2)
-0.6 (2.2)
-1.4 (2.7)
0.4 (1.7)
4.9 (2.8)
6.1 (2.3)
8.8 (3.8)
7.3 (3.8)
32
29
26
59
Feb
0.9 (3.1)
1.7 (2.7)
-2.8 (4.8)
-0.1 (2.0)
8.9 (3.2)
11.4 (3.0)
7.3 (6.8)
7.6 (3.1)
93
62
12
61
Mar
3.7 (4.4)
5.2 (3.5)
6.8 (2.6)
3.8 (2.3)
13.5 (4.8)
14.7 (4.2)
19.3 (3.9)
11.3 (3.6)
49
46
11
137
Apr
8.1 (3.2)
10.1 (2.0)
7.9 (2.6)
9.4 (3.3)
19.9 (4.2)
23.0 (3.3)
17.8 (3.7)
18.5 (5.0)
71
13
146
96
May
12.2 (2.1)
12.9 (3.1)
12.1 (3.0)
10.9 (2.3)
23.1 (3.9)
26.5 (3.2)
24.5 (4.0)
22.1 (3.3)
165
44
128
180
Jun
16.8 (2.9)
16.0 (1.5)
17.4 (2.8)
15.5 (3.2)
28.7 (3.7)
26.9 (2.9)
30.0 (3.8)
28.7 (3.9)
94
122
33
53
Jul
19.2 (2.8)
16.7 (2.5)
18.6 (1.9)
19.2 (1.9)
32.0 (2.6)
28.7 (2.2)
32.2 (1.9)
32.1 (1.9)
91
79
39
50
Aug
16.7 (2.8)
18.2 (2.3)
19.7 (2.4)
17.5 (2.3)
29.2 (3.2)
31.0 (2.3)
33.5 (3.2)
30.9 (3.2)
154
93
35
109
Sep
13.1 (2.4)
16.4 (2.4)
14.4 (2.6)
14.3 (2.5)
24.8 (3.0)
28.3 (2.4)
25.9 (3.1)
26.1 (3.2)
105
143
125
23
Oct
8.1 (3.3)
8.5 (3.9)
10.5 (3.5)
11.8 (2.4)
17.5 (3.4)
20.2 (5.5)
19.9 (4.5)
19.0 (2.3)
128
60
146
91
Nov
6.0 (3.9)
4.6 (4.6)
6.8 (2.5)
5.6 (4.5)
12.6 (3.6)
13.9 (2.9)
14.3 (2.5)
13.4 (4.2)
178
63
140
111
Dec
-1.7 (3.7)
1.6 (3.0)
-0.9 (2.5)
1.4 (3.0)
5.3 (3.5)
9.4 (2.3)
7.2 (3.6)
10.9 (2.7)
151
32
68
62 06 10.3389/fpls.2023.1125560 Ghiglieno et al. TABLE 2 Environmental indices for the characterization of the four seasons. TABLE 2 Environmental indices for the characterization of the four seasons. WSTR
DET
NHH
LHH
HHH
33STR
2010
3.50
4.30
1276
412
324
65
2011
2.86
8.62
1151
468
140
2
2012
24.83
79.18
1020
288
315
93
2013
18.95
52.91
1075
315
321
99 and LHH and the highest level in HHH (thermal excess, with 93
days of maximum temperature above 33°C). the cv. Chardonnay, and from 26/08 to 6/09 for Pinot noir. 3.2.3 Must characterization Considering the thermal behavior in the four seasons in relative
terms, in 2010 the percentages of NHH, LHH and HHH versus their
total (NHH+LHH+HHH) were 63.85, 18.23 and 17.92 respectively. 2012 and 2013 showed a similar behavior with 62.85, 17.78, 19.38
and 62.81, 18.41, 18,77 respectively. The year 2011, with 65.41,
26.62, 7.97 showed a strongly different repartition, again
emphasizing its unique environmental conditions. The results obtained in must characterization (Figure 2)
underlined a similar response concerning TSS when comparing
Chardonnay and Pinot noir, while some differences were observed
in terms of acidity and pH. TSS was higher in 2012, while 2010 recorded the lowest value; in
the case of Pinot noir no significant differences were recorded
between 2012 and 2011. The year 2012 also showed a high TA value for both cultivars, as
in 2011 for Chardonnay and in 2013 for Pinot noir. The year 2010
recorded the lowest TA value for Pinot noir, while no significant
differences were recorded compared to the 2013 value
for Chardonnay. 3.2.1 Average bunch weight The ABW varies over the years equally for both cultivars
(Figure 1). The year 2012 showed a significantly lower value of
ABW than that of the other years. The year 2013 showed an
intermediate response, while 2011 and 2010 recorded higher values. Results related to MA were similar to TA: for Pinot noir, the
year 2012 differed significantly from 2010, recording the highest
and the lowest values, respectively; in the case of Chardonnay the
highest value was recorded in 2011 and the lowest value in 2013. pH
values were high in 2010 for both cultivars, as in 2012 for
Chardonnay. Pinot noir showed no differences over 2011, 2012
and 2013 for this variable, while Chardonnay showed the lowest
value in the case of 2013. 3.1 Meteorological characterization The
delay in ripening was further confirmed by the lower average sugar
content reached at harvesting time (i.e., 18°Brix). The years 2011
and 2012 had the earliest vintages, while year 2013 can be
considered in between, having recorded harvesting times from 28/
08 to 2/09 for Chardonnay and from 20/08 to 24/08 for Pinot noir. The year 2013 was very similar to 2012, with a certain amount
of water stress (second highest in both indices), the second lowest in
NHH and LHH, and the second highest in HHH and the first in the
number of days with a maximum temperature above 33°C
(99 days). 3.2.4 Years general responses concerning vectors related to average bunch weight (ABW) and
musts quality variables (pH, TSS, TA, MA) confirms the findings
previously reported in Sections 3.2.1 and 3.2.3. The biplots obtained from PCA analysis allowed to visualize the
distributions of the data collected for Chardonnay and Pinot noir
during the four-year 2010-2013 period (Figure 3). The pattern in
the sample distribution highlighted a differentiation, especially
between the years 2010, 2011 and 2012-2013. In both PCAs, the
first two dimensions explained around 75% of variance. Vectors
made it possible to display the inner relationship between years and
variables (i.e., meteorological variables and ABW and must quality). Supporting the results described in the previous Section 3.1, the
years 2012 and 2013 were associated, for both cultivar, to stress
caused by water scarcity, higher evapotranspiration (DET) and
temperatures (HHH and 33STR), while 2011 was associated with
lower temperatures (LHH). The pattern in the distribution of years 3.2.2 Harvesting time The four years were characterized by different harvesting times
due to varying ripening rates. The year 2010 was characterized by a
delayed maturation. Harvesting times ranged from 24/08 to 2/09 for FIGURE 1
Boxplots showing average bunch weight (ABW in grams) over the 4 different vintages for Chardonnay (CH) and Pinot noir (PN): different letters
indicate that values are significantly different at p < 0.05 obtained by one-way ANOVA and Siegel-Tukey’s post-hoc test. FIGURE 1
Boxplots showing average bunch weight (ABW in grams) over the 4 different vintages for Chardonnay (CH) and Pinot noir (PN): different letters
indicate that values are significantly different at p < 0.05 obtained by one-way ANOVA and Siegel-Tukey’s post-hoc test. 07 07 Frontiers in Plant Science frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 FIGURE 2
Boxplots showing data of total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA) and pH over the 4 different vintages for
Chardonnay (CH) and Pinot noir (PN): different letters indicate that values are significantly different at p < 0.05 obtained by one-way ANOVA and
Siegel-Tukey’s post-hoc test. Boxplots showing data of total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA) and pH over the 4 different vintages for
Chardonnay (CH) and Pinot noir (PN): different letters indicate that values are significantly different at p < 0.05 obtained by one-way ANOVA and
Siegel-Tukey’s post-hoc test. 3.3.1 Average bunch weight The results obtained for Chardonnay ABW by comparing
treatments showed no homogeneous response over the years
(Figure 4). In general, no significant differences were found
between treatments. A significant difference emerged in 2011
between the less shaded (i.e., TD and TD1) and high shaded (i.e.,
ND1L, and TD2) treatments. Pinot noir recorded a generally FIGURE 3
Biplots showing the distribution of data along of the two first principal component dimensions for Chardonnay (CH) and Pinot noir (PN). Stress
caused by heat waves (33STR), High Heat Hours (HHH), daily evapotranspiration deficit (DET), seasonal water stress (WSTR), Normal Heat Hours
(NHH), Low Heat Hours (LHH), average bunch weight (ABW), total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA), pH are
shown as vectors. Data from a single year is identified by different colored dots. Each dot represents the average value of the single treatment in a
specific year. FIGURE 3
Biplots showing the distribution of data along of the two first principal component dimensions for Chardonnay (CH) and Pinot noir (PN). Stress
caused by heat waves (33STR), High Heat Hours (HHH), daily evapotranspiration deficit (DET), seasonal water stress (WSTR), Normal Heat Hours
(NHH), Low Heat Hours (LHH), average bunch weight (ABW), total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA), pH are
shown as vectors. Data from a single year is identified by different colored dots. Each dot represents the average value of the single treatment in a
specific year. 08 Frontiers in Plant Science frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 this year, the defoliated treatment recorded a higher sugar
concentration than ND1L. Regarding titratable acidity (TA) and
malic acid concentration (MA), the treatments revealed similar
trends in 2010 and 2011. Considering these two years, the totally
defoliated (TD) and defoliated and shaded (TD1 and TD2)
treatments recorded the lowest values in both titratable acidity
and malic acid concentration, with the exception of the results
obtained for TD1, which in 2010 were similar to those obtained for
the not defoliated treatments (ND and ND1L). Significant
differences were highlighted only in the case of titratable acidity
in 2012 and in the case of malic acid concentration in 2013; in these
cases, ND1L and TD recorded the highest values. pH didn’t show
significant differences in 2010. As described in the case of
Chardonnay, the trend observed for pH was not homogeneous
over the years. 3.3.1 Average bunch weight negative effect of defoliation without shading (TD) on ABW in 2012
and 2013 (Figure 4). The years 2010 and 2011 showed a different
response, reporting an intermediate value for TD. Other treatments
related to defoliation and shading (TD1 and TD2) showed the
opposite response in 2010 and 2011, resulting in a higher ABW in
2010 compared to no defoliated treatment (ND and ND1L) and a
lower ABW in 2011 compared to ND and ND1L. 3.3.3 Effect on sparkling wine norisoprenoids As indicated in the Section 2.6 statistical analyses performed on
norisoprenoids abundances were restricted to check whether,
within a specific cultivar and year, there were statistically
significant differences associated to each treatment compared to
reference treatment, i.e. the not defoliated thesis (ND). In order to
display the data, a matrix plot was performed (Figure 7) by
indicating with colored cells the statistically significant
comparisons, using a red color when the treatment mean value
was statistically higher than the mean value of the reference
treatment, or a blue color in the opposite case (grey cells indicate
those cases of no statistical significance). 3.3.3.1 Chardonnay In the 2010 vintage, TDN, vitispirane, ethoxy actinidol 1 and
the unknown norisoprenoid were present in greater quantities in
the total defoliated treatment (TD), the unknown norisoprenoid
was also greater in the TD2 treatment, while the lowest contents
were observed in ND1L treatment. Vitispirane was high also in 3.3.2 Must characterization
3.3.2.1 Chardonnay Concerning sugar concentration (TSS), no significant
differences were highlighted in 2013, while differences among
treatments emerged in the other years (Figure 5). Specifically, the
ND1L generally recorded high TSS values compared to other
treatments. This can be observed by comparing ND1L and ND in
2010, ND1L and all other treatments in 2011 and comparing ND1L
and TD2 in 2012. Titratable acidity (TA) and malic acid showed, in
general, the same response (Figure 5). Not defoliated treatments
(ND and ND1L) tended to preserve the acidity better than
defoliated treatments (TD, TD1 and TD2). This can be seen in all
the years considered, except in 2011, when no significant differences
were found. The efficiency of the not defoliation and shading effect
on acidity preservation is more evident in 2012 and 2013; in these
years, the not defoliated and shaded treatment (ND1L) recorded the
highest level of both titratable acidity and malic acid concentration. Significant differences can be observed in 2010, 2012 and 2013 for
pH, even though the response was generally not uniform between
years (Figure 5). In 2012 and 2013 the highest values recorded
concerned TD2, while in 2010 the highest values can be associated
to TD1. 3.3.2.2 Pinot noir Figure 6 shows the results obtained for Pinot noir musts
characterization. Sugar concentration (TSS) showed no significant
differences among treatments with the exception of year 2011. In FIGURE 4
Boxplots showing average bunch weight (ABW in grams) for each treatment considering 4 years, for Chardonnay (CH) and Pinot noir (PN): different
letters indicate that values are significantly different at p < 0.05; NS indicates no significant differences among treatments at p < 0.05 obtained by
one-way ANOVA and Siegel-Tukey’s post-hoc test. FIGURE 4
Boxplots showing average bunch weight (ABW in grams) for each treatment considering 4 years, for Chardonnay (CH) and Pinot noir (PN): different
letters indicate that values are significantly different at p < 0.05; NS indicates no significant differences among treatments at p < 0.05 obtained by
one-way ANOVA and Siegel-Tukey’s post-hoc test. 09 Frontiers in Plant Science frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 FIGURE 5
Boxplots showing total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA) and pH for each treatment considering 4 years,
2010, 2011, 2012, 2013, for Chardonnay: different letters indicate that values are significantly different at p < 0.05; NS indicates no significant
differences among treatments at p < 0.05 obtained by one-way ANOVA and Siegel Tukey’s post-hoc test. Each column is referred to one variable
(each variable is reported in the upper part of the graph); each row is referred to one year (each year is reported in the left part of the graph). Boxplots showing total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA) and pH for each treatment considering 4 years,
2010, 2011, 2012, 2013, for Chardonnay: different letters indicate that values are significantly different at p < 0.05; NS indicates no significant
differences among treatments at p < 0.05 obtained by one-way ANOVA and Siegel Tukey’s post-hoc test. Each column is referred to one variable
(each variable is reported in the upper part of the graph); each row is referred to one year (each year is reported in the left part of the graph). defoliated and shaded (TD1 and TD2) treatments. In 2011, it was
observed that all the norisoprenoids considered were higher in TD
and some others, such as ethoxy actinidol 1 also increased in the
TD1 and TD2 treatments. The ND1L treatment showed lower
contents for ethoxy actinidol 1, vitispirane and TDN. 4.1 Effect of meteorological conditions The results obtained in this study highlight the influence of
temperature and water availability on grapevine yield, musts quality
and on the composition of aromas in wine. This confirms the results
obtained by other authors that underlined the effect of
meteorological conditions on grape and wine quality (Mariani
et al., 2009). The average weight of the bunches was higher when
water needs were satisfied; 2010 and 2011 were, indeed,
characterised by lower water stress indices (WSTR and DET) and
the highest values of ABW. The grapevine yield positive response to
higher water availability was confirmed by other authors (Ramos 3.3.2.2 Pinot noir In 2012, a
greater quantity was observed in TD for the unknown
norisoprenoid, ethoxy actinidol 1 and vitispirane; ethoxy actinidol
1 was also more abundant in the TD1 treatment. In 2013, only
vitispirane was higher in TD, and ethoxy actinidol 1 for TD1
treatment, while ethoxy actinidol 1 was present in less quantities
in the ND1L treatment. Chardonnay wine seems to be highly
influenced by the agronomic interventions on the canopy, indeed,
it is clear that for the main norisoprenoids the total defoliated
treatment (TD) was often higher followed by the artificial shading
and defoliated treatments. The boxplots of norisoprenoids for each
year and treatment are included in the supplementary material
(Supplementary Figure 3). this treatment. In 2012 vitispirane was the highest in TD treatment
while ethoxy actinidol 1 and the unknown norisoprenoid were the
lowest in ND1L treatment. In the 2010 and 2013 vintages, safranal,
ethoxy actinidol 1 and the unknown norisoprenoids were the highest in
the TD1 treatment. In 2013 also the unknown norisoprenoid were
highest in the TD treatment as well. The boxplots of norisoprenoids for
each year and treatment are reported in the supplementary material
(Supplementary Figure 4). Frontiers in Plant Science frontiersin.org 3.3.3.2 Pinot noir Conversely, Pinot Noir wine seems to be less influenced by the
treatments (Figure 7), although showing similar trends to that seen for
Chardonnay, in all the years surveyed ethoxy actinidol 1 was prevailing
in the defoliated treatment. In 2010 all compounds except TPB-1 were
prevailing in the TD2 treatment while in 2011 only TDN was highest in 10 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 FIGURE 6
Boxplots showing mean and unit variance of total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA) and pH for each
treatment considering 4 years, 2010, 2011, 2012, 2013, for Pinot noir: different letters indicate that values are significantly different at p < 0.05; NS
indicates no significant differences among treatments at p < 0.05 obtained by one-way ANOVA and Siegel-Tukey’s post-hoc test. Each column is
referred to one variable (each variable is reported in the upper part of the graph); each row is referred to one year (each year is reported in the left
part of the graph). Boxplots showing mean and unit variance of total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA) and pH for each
treatment considering 4 years, 2010, 2011, 2012, 2013, for Pinot noir: different letters indicate that values are significantly different at p < 0.05; NS
indicates no significant differences among treatments at p < 0.05 obtained by one-way ANOVA and Siegel-Tukey’s post-hoc test. Each column is
referred to one variable (each variable is reported in the upper part of the graph); each row is referred to one year (each year is reported in the left
part of the graph). and Mulligan, 2005; Ramos and Martı́nez-Casasnovas, 2006;
Intrigliolo et al., 2016; Pérez-Á lvarez et al., 2021; Ohana-Levi
et al., 2022). On the other hand, water scarcity in 2012 lead to a
reduction of the bunch weight, causing in turn the typical
phenomena of sugar and acidity concentration (Intrigliolo et al.,
2016; Pérez-Á lvarez et al., 2021) and pH level increase (Reynolds
and Naylor, 1994). values for meteorological indicators were reported, the reference
sugar level was reached later (24 August - 6 September), leading to
lower acidic levels, and higher pH levels. Moreover, the year 2010
reported the highest total amount of cumulated precipitation
between berry set and harvest. 3.3.3.2 Pinot noir The low level of titratable acidity
and the high value of pH in musts can therefore be related to the
effect of dilution of acidic components (Keller, 2006) and the
increase of K absorption (Marais et al., 1992) caused by water
absorption. The negative effect of high temperatures on the must
acidic content was evident in 2013, when a value of the stress caused
by heat waves (33STR) was recorded. This was particularly evident
in the case of malic acid in Chardonnay musts that showed low
values during this vintage. Many previous studies reported the
increase of malic acid degradation kinetics when high temperatures
were recorded (Michelini et al., 2021). Previous studies reported how the ripening of grapes for
sparkling wine production was positively influenced by a cool
climate (Jones et al., 2014). The optimum temperature for the
accumulation of malic acid during grape ripening was estimated
to be around 20°C (Lakso and Kliewer, 1975), while the negative
correlation between high temperatures and MA content after
véraison was reported by many studies (Rienth et al., 2016; Blank
et al., 2019). Preserving malic acid degradation made it possible to
contrast the decrease of the acidic level in must (Volschenk et al.,
2006), which is a desirable condition for sparkling wine production
(Jones et al., 2014). In the present study, the positive effect of cooler
temperatures on must quality was revealed in 2011, when high
values of indicators of suboptimal temperature and optimal
temperature were recorded. This led in 2011 to an early ripening
(1 August – 12 August), while preserving the must sugar-acid
balance, and a low pH. On the contrary in 2010, where similar Frontiers in Plant Science 4.2 The effect of leaf removal and
artificial shading Leaf removal and artificial shading effects were differentiated by
year and cultivar. In general, years characterized by satisfying water
availability and cooler temperatures (i.e., 2010 and 2011) 11 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 FIGURE 7
Matrix plot colored cells showing the statistically significant comparison of some norisoprenoids using a red color when mean value of treatment
was statistically higher than the mean value of the reference treatment (ND) or a blue color in the opposite case over the 4 years (2010, 2011, 2012,
2013) for Chardonnay and Pinot noir. NS means not significant, NA means not available data. GU
Matrix plot colored cells showing the statistically significant comparison of some norisoprenoids using a red color when mean value of treatment
was statistically higher than the mean value of the reference treatment (ND) or a blue color in the opposite case over the 4 years (2010, 2011, 2012,
2013) for Chardonnay and Pinot noir. NS means not significant, NA means not available data. two vintages of Pinot noir, and would suggest that it is the light that
most influences the biosynthesis of this compound. TDN with a
very low olfactory threshold (2 µg l-1), can sometimes represent a
problem, especially in sparkling wines intended for a long period of
aging, because it increases over time. It has been seen that the
olfactory threshold of TDN in sparkling wine is slightly higher and
goes from 2.1 µg l-1 in still Riesling to 4.0 µg l-1 in sparkling Riesling
(Ziegler et al., 2019); however, this is a compound to pay attention
to when deciding on agronomic practices. Several glycosylated
precursors have also been reported to originate these compounds
during fermentation and wine ageing, through acid-catalysed
reactions, and these reactions are certainly promoted in a wine
with a low pH, such as sparkling wine (Schneider et al., 2001; Janusz
et al., 2003). emphasized the difference in ripening delay between the non-
artificial shaded and the artificial shaded treatments. This is in
agreement with previous papers that reported how shading makes it
possible to slow down grape maturation (Caravia et al., 2016;
Ghiglieno et al., 2020). The effect of no defoliation and artificial
shading on acidity preservation was more evident in years
characterized by high temperatures and high-water stress (i.e.,
2012 and 2013). Frontiers in Plant Science frontiersin.org 4.2 The effect of leaf removal and
artificial shading This can be related to the positive relationships
between malic acid degradation and high temperature and water
scarcity (Blank et al., 2019) that suggest the need for shading during
warm and dry vintages. In the case of ABW and pH there was no
unique response. The analysis of the main norisoprenoids showed how the
canopy treatments greatly affect their quantity (boxplots in
Supplementary Figures 3, 4). A general increasing effect of total
defoliation can be highlighted, especially concerning Chardonnay. Even though Oliveira et al. (2004) reported that carotenoid contents
were consistently higher in grapes in the shade than in those
exposed to direct sunlight, the positive effect of exposure on
norisoprenoids was underlined by many previous studies (Marais
et al., 1991; Marais et al., 1992; Asproudi et al., 2016; Asproudi et al.,
2020). The results obtained in the present study, in terms of TDN
content in wines, showed that this aromatic compound, even if not
always in a statistically significant way, was produced in larger
quantities in TD while the shaded theses TD1 and TD2 contained
less TDN; this behavior was clearly visible in Chardonnay and in The content in vitispirane was always much higher in TD theses
and also in this case, although in Pinot noir there was no statistical
proof, the defoliated and shaded theses presented a lower content of
this compound. The olfactory descriptor of vitispirane is “camphor”
or “eucalyptus”. Some studies (Silva Ferreira and Guedes de Pinho,
2004) showed that in aged wines the presence of vitispirane can
reach the threshold of perception (800 µg l-1) and, consequently,
that it participates in the aroma of wine. As with the TDN, pH and
temperature are the two factors that have the greatest influence on
its formation over time. (E)-1-(2,3,6-trimethylphenyl) buta-1,3-diene (TPB-1) was
tendentially higher in the TD and less present in the shady theses 12 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 10.3389/fpls.2023.1125560 TD1-TD2; this compound, like TDN at high concentrations, could
lead to an unpleasant chemical note at higher content (40 ng l-1
sensory threshold) (Janusz et al., 2003). The behavior of actinidols
and their ethoxy forms was very similar to that seen previously with
a higher content in TD, a little lower in the shaded theses and even
lower in the not defoliated ones. For this compound the situation
was very clear in both Chardonnay and Pinot Noir. 5 Conclusions The results obtained in the present study showed the
importance of canopy management, in terms of defoliation and
shading, on must and wine quality. This increases our knowledge
about the effect of different defoliation and artificial shading
application in relation to meteorological condition, supporting the
management decision-making in the Franciacorta wine-growing
area. The results obtained with no defoliation and artificial shading
in the preservation of the acidic composition in warmer vintages
suggest that defoliation activities should be calibrated in relation to
the meteorological trends, without standardized procedures. This is
particularly relevant in the case of sparkling wine production, where
the acidic composition is essential to determine wine quality. The
enhanced values of some norisoprenoids obtained with the total
defoliation treatment represent a further element to direct
defoliation and shading strategies. The very low sensory
thresholds of some norisoprenoids place them among the
important compounds for the final characteristics of the wine. Moreover, the concentrations of these aromatic compounds tend
to increase during ageing, especially in sparkling wines, and can
reach levels above the threshold, thus developing the characteristic
aroma of aged wine, which is sometimes a detrimental wine quality. For example, TDN and TPB-1 found in wine, have a pleasant aroma Author contributions Another compound that has been increasingly found in TD
treatments and less in the other not defoliated and shaded ones was
safranal. This compound is responsible for the characteristic smell of
saffron and can in some cases exceed the olfactory threshold and be
considered too intense, leading to possible depreciation in sparkling
wine. It is known that the principal monoterpene glycoside precursor
of safranal in saffron is picrocrocin (Zougagh et al., 2006). We don’t
know if picrocrocin is a precursor also in wine or if safranal is formed
starting from some other precursor or by the rearrangement of some
other molecules, but previous studies reported that the content in this
compound tends to increase over time, especially at low pH and if the
temperature during the storage of the wine is high. Very similar
trends in both Chardonnay and Pinot noir, with high levels in the TD
thesis and less content in other thesis, were also observed for an
unidentified norisoprenoid. On the other hand, we did not observe
any clear treatment effects for b- damascenone. IG, SC, GC, LV and FM contributed to the conception and
design of the study. IG, SC, GC and UV performed the experiments
and analyzed the data. IG, SC, GC and MG-A organized the
databases. IG, SC and MG-A performed the statistical analysis. IG, SC and GC wrote the first draft of the manuscript. IG, SC and
MG-A prepared figures and tables UV, LV and FM reviewed the
drafts of the manuscript. All authors contributed to the article and
approved the submitted version. Acknowledgments Our thanks go to Castello Bonomi Tenute in Franciacorta,
which hosted the experimental tests and the ‘Consorzio per la tutela
del Franciacorta’, which promoted and inspired a collaboration
between the universities and the wine sector. Our heartful thanks
also go to Pietro Franceschi for his helpful discussion. 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. Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. 4.2 The effect of leaf removal and
artificial shading Actinidols have
an odor that has been described as camphoraceous or as woody and
resinous, but their contribution to wine aroma is considered limited
at best, as their concentrations are usually much lower than their
detection threshold. Perhaps the corresponding ethyl ethers that
have been found with the GC-O technique by Schneider et al. (2001), with fruity, citric and eucalyptus notes offer a greater
contribution to the aroma. at low concentrations, but they can reduce the quality of the wine at
high concentrations, and it is assumed that other compounds such
as safranal, if present at high concentrations, may also be
undesirable. However, further studies are needed to establish the
olfactory threshold of this compounds in the wine, and which are
their precursors in the sparkling wine base before refermentation. Data availability statement The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation. Frontiers in Plant Science References Alfonzo, A., Francesca, N., Mercurio, V., Prestianni, R., Settanni, L., Spanò, G., et al. (2020). Use of grape racemes from grillo cultivar to increase the acidity level of
sparkling base wines produced with different saccharomyces cerevisiae strains. Yeast
37, 475–486. doi: 10.1002/yea.3505 Downey, M. O., Dokoozlian, N. K., and Krstic, M. P. (2006). Cultural practice and
environmental impacts on the flavonoid composition of grapes and wine: a review of
recent research. Am. J. Enol. Vitic. 57, 257–268. doi: 10.5344/ajev.2006.57.3.257 Filippetti, I., Movahed, N., Allegro, G., Valentini, G., Pastore, C., Colucci, E., et al. (2015). Effect of post-veraison source limitation on the accumulation of sugar,
anthocyanins and seed tannins in vitis vinifera cv. sangiovese berries. Aust. J. Grape
Wine Res. 21, 90–100. doi: 10.1111/ajgw.12115 Amerine, M., and Winkler, A. (1944). Composition and quality of musts and wines
of California grapes. Hilgardia 15, 493–675. doi: 10.3733/hilg.v15n06p493 Arapitsas, P., Ugliano, M., Perenzoni, D., Angeli, A., Pangrazzi, P., and Mattivi, F. (2016). Wine metabolomics reveals new sulfonated products in bottled white wines,
promoted by small amounts of oxygen. J. Chromatogr. A 1429, 155–165. doi: 10.1016/
j.chroma.2015.12.010 Fraga, H., Malheiro, A. C., Moutinho-Pereira, J., and Santos, J. A. (2012). An
overview of climate change impacts on European viticulture. Food Energy Secur. 1, 94–
110. doi: 10.1002/fes3.14 Ghiglieno, I., Mattivi, F., Cola, G., Trionfini, D., Perenzoni, D., Simonetto, A., et al. (2020). The effects of leaf removal and artificial shading on the composition of
Chardonnay and pinot noir grapes. OENO One 54, 761–777. doi: 10.20870/oeno-
one.2020.54.4.2556 Asproudi, A., Ferrandino, A., Bonello, F., Vaudano, E., Pollon, M., and Petrozziello,
M. (2018). Key norisoprenoid compounds in wines from early-harvested grapes in view
of climate change. Food Chem. 268, 143–152. doi: 10.1016/j.foodchem.2018.06.069 Asproudi, A., Petrozziello, M., Cavalletto, S., Ferrandino, A., Mania, E., and Guidoni,
S. (2020). Bunch microclimate affects carotenoids evolution in cv. nebbiolo (V. vinifera
l.). Appl. Sci. 10, 3846. doi: 10.3390/app10113846 Greer, D. H. (2013). The impact of high temperatures on vitis vinifera cv. semillon
grapevine performance and berry ripening. Front. Plant Sci. 4. doi: 10.3389/
fpls.2013.00491 Asproudi, A., Petrozziello, M., Cavalletto, S., and Guidoni, S. (2016). Grape aroma
precursors in cv. nebbiolo as affected by vine microclimate. Food Chem. 211, 947–956. doi: 10.1016/j.foodchem.2016.05.070 Herrero, P., Sáenz-Navajas, P., Culleré, L., Ferreira, V., Chatin, A., Chaperon, V.,
et al. (2016). Chemosensory characterization of Chardonnay and pinot noir base wines
of champagne. References two very different varieties for a common product. Food Chem. 207,
239–250. doi: 10.1016/j.foodchem.2016.03.068 Biasi, R., Brunori, E., Ferrara, C., and Salvati, L. (2019). Assessing impacts of climate
change on phenology and quality traits of vitis vinifera l.: the contribution of local
knowledge. Plants 8, 121. doi: 10.3390/plants8050121 IBM Corp (2021). IBM SPSS Statistics for windows, version 28.0 (Armonk, NY: IBM
Corp). Blank, M., Hofmann, M., and Stoll, M. (2019). Seasonal differences in vitis vinifera l. cv. pinot noir fruit and wine quality in relation to climate. OENO One 53, 189–203. doi: 10.20870/oeno-one.2019.53.2.2427 Intrigliolo, D. S., Lizama, V., Garcı́a-Esparza, M. J., Abrisqueta, I., and Á lvarez, I. (2016). Effects of post-veraison irrigation regime on Cabernet sauvignon grapevines in
Valencia, Spain: yield and grape composition. Agric. Water Manag 170, 110–119. doi: 10.1016/j.agwat.2015.10.020 Caravia, L., Collins, C., Petrie, P., and Tyerman, S. (2016). Application of shade
treatments during Shiraz berry ripening to reduce the impact of high temperature. Aust. J. Grape Wine Res. 22, 422–437. doi: 10.1111/ajgw.12248 IPCC (2007). “Climate change 2007: the physical science basis,” in Contribution of
working group I to the fourth assessment report of the intergovernmental panel on
climate change. Eds. S. Solomon, D. Qin, M. Manning, Z. Chen, M. Marquis, K. B. Averyt, M. Tignor and H. L. Miller (Cambridge: Cambridge University Press). Carlin, S., Mattivi, F., Durantini, V., and Panagiotis, A. (2022). Flint glass bottles
cause white wine aroma identity degradation. PNAS 119 (29), e2121940119. doi: 10.1073/pnas.2121940119 Jackson, D., and Lombard, P. (1993). Environmental and management practices
affecting grape composition and wine quality - a review. Am. J. Enol. Vitic. 44, 409–430. doi: 10.5344/ajev.1993.44.4.409 Carlin, S., Vrhovsek, U., Franceschi, P., Lotti, C., Bontempo, L., Camin, F., et al. (2016). Regional features of northern Italian sparkling wines, identified using solid-
phase micro extraction and comprehensive two-dimensional gas chromatography
coupled with time-of-flight mass spectrometry. Food Chem. 208, 68–80. doi: 10.1016/j.foodchem.2016.03.112 Janusz, A., Capone, D. L., Puglisi, C. J., Perkins, M. V., Elsey, G. M., and Sefton, M. A. (2003). (E)-1-(2,3,6-Trimethylphenyl) buta-1,3-Diene: a potent grape-derived odorant
in wine. J. Agric. Food Chem. 51 (26), 7759–7763. doi: 10.1021/jf0347113 Chen, W.-K., Yu, K.-J., Liu, B., Lan, Y.-B., Sun, R.-Z., Li, Q., et al. (2017). Comparison of transcriptional expression patterns of carotenoid metabolism in
‘Cabernet sauvignon’ grapes from two regions with distinct climate. J. Plant Physiol. 213, 75–86. doi: 10.1016/j.jplph.2017.03.001 Jones, G. References V., Duchêne, E., Tomasi, D., Yuste, J., Braslavska, O., Schultz, H., et al. (2005). Changes in European winegrape phenology and relationships with climate. XIV
Int. GESCO Vitic. Congr. Geisenh. Ger. 23-27 August 2005, 54–61. Chorti, E., Guidoni, S., Ferrandino, A., and Novello, V. (2010). Effect of different
cluster sunlight exposure levels on ripening and anthocyanin accumulation in nebbiolo
grapes. Am. J. Enol. Vitic. 61, 23–30. doi: 10.5344/ajev.2010.61.1.23 Jones, J. E., Kerslake, F. L., Close, D. C., and Dambergs, R. G. (2014). Viticulture for
sparkling wine production: a review. Am. J. Enol. Vitic. 65, 407–416. doi: 10.5344/
ajev.2014.13099 Cola, G., Failla, O., Maghradze, D., Megrelidze, L., and Mariani, L. (2017). Grapevine
phenology and climate change in Georgia. Int. J. Biometeorol. 61, 761–773. doi: 10.1007/s00484-016-1241-9 Keller, M. (2006). Ripening grape berries remain hydraulically connected to the
shoot. J. Exp. Bot. 57, 2577–2587. doi: 10.1093/jxb/erl020 Kwasniewski, M. T., Vanden Heuvel, J. E., Pan, B. S., and Sacks, G. L. (2010). Timing
of cluster light environment manipulation during grape development affects C13
norisoprenoid and carotenoid concentrations in Riesling. J. Agric. Food Chem. 58,
6841–6849. doi: 10.1021/jf904555p Cola, G., Mariani, L., Maghradze, D., and Failla, O. (2020). Changes in thermal
resources and limitations for Georgian viticulture. Aust. J. Grape Wine Res. 26, 29–40. doi: 10.1111/ajgw.12412 Lakso, A. N., and Kliewer, W. M. (1975). The influence of temperature on malic acid
metabolism in grape berries: i. enzyme responses. Plant Physiol. 56, 370–372. doi: 10.1104/pp.56.3.370 Cola, G., Mariani, L., Salinari, F., Civardi, S., Bernizzoni, F., Gatti, M., et al. (2014). Description and testing of a weather-based model for predicting phenology, canopy
development and source–sink balance in vitis vinifera l. cv. barbera. Agric. For. Meteorol. 184, 117–136. doi: 10.1016/j.agrformet.2013.09.008 Marais, J., Wyk, C., and Rapp, A. (1991). Carotenoid levels in maturing grapes as
affected by climatic regions, sunlight and shade. South Afr. J. Enol. Vitic. 12, 64–69. doi: 10.21548/12-2-2209 Conde, C., Silva, P., Fontes, N., Dias, A., Tavares, R., Sousa, M., et al. (2006). Biochemical changes throughout grape berry development and fruit and wine quality. Food 1, 5–6. Marais, J., Wyk, C. J., and Rapp, A. (1992). Effect of sunlight and shade on
norisoprenoid levels in maturing weisser Riesling and chenin blanc grapes and
weisser Riesling wines. South Afr. J. Enol. Vitic. 13, 23–32. doi: 10.21548/13-1-2191 Cozzolino, D. (2016). Metabolomics in grape and wine: definition, current status and
future prospects. Food Anal. Methods 9, 2986–2997. Supplementary material The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1125560/
full#supplementary-material 13 frontiersin.org 10.3389/fpls.2023.1125560 Ghiglieno et al. Ghiglieno et al. Frontiers in Plant Science References 13, 175. doi: 10.1186/1471-2229-13-175 Michelini, S., Tomada, S., Kadison, A. E., Pichler, F., Hinz, F., Zejfart, M., et al. (2021). Modeling malic acid dynamics to ensure quality, aroma and freshness of pinot
blanc wines in south tyrol (Italy). OENO One 55, 159–179. doi: 10.20870/oeno-
one.2021.55.2.4570 Schneider, R., Razungles, A., Augier, C., and Baumes, R. (2001). Monoterpenic and
norisoprenoidic glycoconjugates of vitis vinifera l. cv. melon b. as precursors of
odorants in muscadet wines. J. Chromatogr. A 936, 145–157. doi: 10.1016/S0021-
9673(01)01150-5 Mirás-Avalos, J. M., and Araujo, E. S. (2021). Optimization of vineyard water
management: challenges, strategies, and perspectives. Water 13, 746. doi: 10.3390/
w13060746 Schultz, H. (2000). Climate change and viticulture: a European perspective on
climatology, carbon dioxide and UV-b effects. Aust. J. Grape Wine Res. 6, 2–12. doi: 10.1111/j.1755-0238.2000.tb00156.x Mirás-Avalos, J. M., and Intrigliolo, D. S. (2017). Grape composition under abiotic
constrains: water stress and salinity. Front. Plant Sci. 8. doi: 10.3389/fpls.2017.00851 Silva Ferreira, A. C., and Guedes de Pinho, P. (2004). Norisoprenoids profile during
port wine ageing–influence of some technological parameters. Anal. Chim. Acta 513,
169–176. doi: 10.1016/j.aca.2003.12.027 Nesbitt, A., Kemp, B., Steele, C., Lovett, A., and Dorling, S. (2016). Impact of recent
climate change and weather variability on the viability of UK viticulture - combining
weather and climate records with producers’ perspectives. Aust. J. Grape Wine Res. 22,
324–335. doi: 10.1111/ajgw.12215 Smart, R. E., Dick, J. K., Gravett, I. M., and Fisher, B. M. (1990). Canopy
management to improve grape yield and wine quality - principles and practices. South Afr. J. Enol. Vitic. 11, 3–17. doi: 10.21548/11-1-2232 Ohana-Levi, N., Mintz, D. F., Hagag, N., Stern, Y., Munitz, S., Friedman-Levi, Y.,
et al. (2022). Grapevine responses to site-specific spatiotemporal factors in a
Mediterranean climate. Agric. Water Manage. 259, 107226. doi: 10.1016/
j.agwat.2021.107226 Smart, R. E., Robinson, J. B., Due, G. R., and Brien, C. J. (1985). Canopy
microclimate modification for the cultivar Shiraz II. effects on must and wine
composition. Vitis - J. Grapevine Res. 24, 119–128. doi: 10.5073/vitis.1985.24 Oliveira, C., Ferreira, A. C., Costa, P., Guerra, J., and Guedes de Pinho, P. (2004). Effect of some viticultural parameters on the grape carotenoid profile. J. Agric. Food
Chem. 52, 4178–4184. doi: 10.1021/jf0498766 Song, J., Shellie, K. C., Wang, H., and Qian, M. C. (2012). Influence of deficit
irrigation and kaolin particle film on grape composition and volatile compounds in
merlot grape (Vitis vinifera l.). Food Chem. 134, 841–850. References doi: 10.1007/s12161-016-0502-x Mariani, L., Alilla, R., Cola, G., Monte, G. D., Epifani, C., Puppi, G., et al. (2013). IPHEN–a real-time network for phenological monitoring and modelling in Italy. Int. J. Biometeorol. 57, 881–893. doi: 10.1007/s00484-012-0615-x Crippen, D., and Morrison, J. (1986). The effects of sun exposure on the
compositional development of Cabernet-sauvignon berries. Am. J. Enol. Vitic. 37,
235–242. doi: 10.5344/ajev.1986.37.4.235 Deloire, A., Carbonneau, A., Wang, Z., and Ojeda, H. (2004). Vine and water: a short
review. OENO One 38, 1–13. doi: 10.20870/oeno-one.2004.38.1.932 Mariani, L., Parisi, S. G., Cola, G., and Failla, O. (2012). Climate change in Europe
and effects on thermal resources for crops. Int. J. Biometeorol. 56, 1123–1134. doi: 10.1007/s00484-012-0528-8 Deluc, L. G., Quilici, D. R., Decendit, A., Grimplet, J., Wheatley, M. D., Schlauch, K. A., et al. (2009). Water deficit alters differentially metabolic pathways affecting
important flavor and quality traits in grape berries of Cabernet sauvignon and
Chardonnay. BMC Genomics 10, 212. doi: 10.1186/1471-2164-10-212 Mariani, L., Parisi, S., Failla, O., Cola, G., Zoia, G., and Bonardi, L. (2009). Tiran
(1624-1930): a long time series of harvest dates for grapevine. Italain J. Agrometeorogy
1, 7–16. Martin, D., Grose, C., Fedrizzi, B., Stuart, L., Albright, A., and McLachlan, A. (2016). Grape cluster microclimate influences the aroma composition of sauvignon blanc wine. Food Chem. 210, 640–647. doi: 10.1016/j.foodchem.2016.05.010 de Oliveira, J. B., Egipto, R., Laureano, O., de Castro, R., Pereira, G. E., and Ricardo-
da-Silva, J. M. (2019). Climate effects on physicochemical composition of syrah grapes
at low and high altitude sites from tropical grown regions of Brazil. Food Res. Int. 121,
870–879. doi: 10.1016/j.foodres.2019.01.011 Martı́nez-Lüscher, J., Chen, C. C. L., Brillante, L., and Kurtural, S. K. (2020). Mitigating heat wave and exposure damage to “Cabernet sauvignon” wine grape
with partial shading under two irrigation amounts. Front. Plant Sci. 11. doi: 10.3389/
fpls.2020.579192 Dokoozlian, N., and Kliewer, W. (1996). Influence of light on grape berry growth
and composition varies during fruit development. J. Am. Soc Hortic. Sci. 121 (5), 869–
874. doi: 10.21273/jashs.121.5.869 14 Frontiers in Plant Science frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 10.3389/fpls.2023.1125560 Mendes-Pinto, M. M. (2009). Carotenoid breakdown products the–norisoprenoids
—in wine aroma. Arch. Biochem. Biophys. 483, 236–245. doi: 10.1016/j.abb.2009.01.008 Scafidi, P., Pisciotta, A., Patti, D., Tamborra, P., Di Lorenzo, R., and Barbagallo, M. G. (2013). Effect of artificial shading on the tannin accumulation and aromatic
composition of the grillo cultivar (Vitis vinifera l.). BMC Plant Biol. References doi: 10.1016/
j.foodchem.2012.02.193 Pérez-Á lvarez, E. P., Intrigliolo Molina, D. S., Vivaldi, G. A., Garcı́a-Esparza, M. J.,
Lizama, V., and Á lvarez, I. (2021). Effects of the irrigation regimes on grapevine cv. bobal in a Mediterranean climate: i. water relations, vine performance and grape
composition. Agric. Water Manage. 248, 106772. doi: 10.1016/j.agwat.2021.106772 Suter, B., Destrac Irvine, A., Gowdy, M., Dai, Z., and van Leeuwen, C. (2021). Adapting wine grape ripening to global change requires a multi-trait approach. Front. Plant Sci. 12. doi: 10.3389/fpls.2021.624867 Pons, A., Allamy, L., Schüttler, A., Rauhut, D., Thibon, C., and Darriet, P. (2017). What is the expected impact of climate change on wine aroma compounds and their
precursors in grape? OENO One 51, 141–146. doi: 10.20870/oeno-one.2017.51.2.1868 Toda, M. D., and Balda, P. (2014). Reducing the pH of wine by increasing grape
sunlight exposure: a method to mitigate the effects of climate warming. Vitis - J. Grapevine Res. 53, 17–20. doi: 10.5073/vitis.2014.53 Ramos, M. C., and Martı́nez-Casasnovas, J. A. (2006). Trends in precipitation
concentration and extremes in the Mediterranean penedès-anoia region, Ne Spain. Clim. Change 74, 457–474. doi: 10.1007/s10584-006-3458-9 Tonietto, J. (1999). Les Macroclimats viticoles mondiaux et l’influence du mésoclimat
sur la typicité de la syrah et du Muscat de hambourg dans le sud de la France:
méthodologie de caractérisation (Montpellier: PhD dissertation, Ecole Nationale
Supérieure Agronomique). Ramos, M. C., and Mulligan, M. (2005). Spatial modelling of the impact of climate
variability on the annual soil moisture regime in a mechanized Mediterranean
vineyard. J. Hydrol. 306, 287–301. doi: 10.1016/j.jhydrol.2004.09.013 van Leeuwen, C., Destrac-Irvine, A., Dubernet, M., Duchêne, E., Gowdy, M.,
Marguerit, E., et al. (2019). An update on the impact of climate change in viticulture
and potential adaptations. Agronomy 9, 514. doi: 10.3390/agronomy9090514 R Core Team (2022). R: a language and environment for statistical computing
(Vienna, Austria: R Foundation for Statistical Computing). Available at: https://www. R-project.org/. Volschenk, H., Vuuren, H., and Viljoen-Bloom, M. (2006). Malic acid in wine:
origin, function and metabolism during vinification. South Afr. J. Enol. Vitic. 27, 123–
136. doi: 10.21548/27-2-1613 Reynolds, A. G., and Naylor, A. P. (1994). `Pinot noir’ and `Riesling’ grapevines
respond to water stress duration and soil water-holding capacity. HortScience 29, 1505–
1510. doi: 10.21273/HORTSCI.29.12.1505 Wang, Y., He, L., Pan, Q., Duan, C., and Wang, J. (2018). Effects of basal defoliation
on wine aromas: a meta-analysis. Molecules 23, 779. doi: 10.3390/molecules23040779 Reynolds, A., Pool, R., and Mattick, R. (1986). References Influence of cluster exposure on fruit
composition and wine quality of seyval blanc grapes. Vitis 25, 85–95. Winterhalter, P., and Schreier, P. (1994). C13-norisoprenoid glycosides in plant
tissues: an overview on their occurrence, composition and role as flavour precursors. Flavour Fragr. J. 9, 281–287. doi: 10.1002/ffj.2730090602 Rienth, M., Torregrosa, L., Sarah, G., Ardisson, M., Brillouet, J.-M., and Romieu, C. (2016). Temperature desynchronizes sugar and organic acid metabolism in ripening
grapevine fruits and remodels their transcriptome. BMC Plant Biol. 16, 164. doi: 10.1186/s12870-016-0850-0 Ziegler, M., Gök, R., Bechtloff, P., Winterhalter, P., Schmarr, H.-G., and Fischer,
U. (2019). Impact of matrix variables and expertise of panelists on sensory
thresholds of 1,1,6-trimethyl-1,2-dihydronaphthalene known as petrol off-flavor
compound in Riesling wines. Food Qual. Prefer. 78, 103735. doi: 10.1016/
j.foodqual.2019.103735 Riou, C., Pieri, P., and Clech, B. L. (1994). Consommation d’eau de la vigne en
conditions hydriques non limitantes. formulation simplifiée de la transpiration. VITIS
-. J. Grapevine Res. 33, 109–109. doi: 10.5073/vitis.1994.33.109-115 Santos, J. A., Fraga, H., Malheiro, A. C., Moutinho-Pereira, J., Dinis, L.-T., Correia,
C., et al. (2020). A review of the potential climate change impacts and adaptation
options for European viticulture. Appl. Sci. 10, 3092. doi: 10.3390/app10093092 Zougagh, M., Rı́os, A., and Valcárcel, M. (2006). Determination of total safranal by
in situ acid hydrolysis in supercritical fluid media: application to the quality control of
commercial saffron. Anal. Chim. Acta 578, 117–121. doi: 10.1016/j.aca.2006.06.064 15 Frontiers in Plant Science frontiersin.org
|
https://openalex.org/W3101434244
|
https://hal.archives-ouvertes.fr/hal-02066786/file/aa32433-17.pdf
|
English
| null |
The<i>Gaia</i>-ESO Survey: impact of extra mixing on C and N abundances of giant stars
|
Astronomy & astrophysics
| 2,018
|
cc-by
| 12,024
|
The Gaia-ESO Survey: impact of extra mixing on C and
N abundances of giant stars
N. Lagarde, C. Reylé, A. C. Robin, G. Tautvaišienė, A. Drazdauskas, Š. Mikolaitis, R. Minkevičiūtė, E. Stonkutė, Y. Chorniy, V. Bagdonas, et al. The Gaia-ESO Survey: impact of extra mixing on C and
N abundances of giant stars
N. Lagarde, C. Reylé, A. C. Robin, G. Tautvaišienė, A. Drazdauskas, Š. Mikolaitis, R. Minkevičiūtė, E. Stonkutė, Y. Chorniy, V. Bagdonas, et al. To cite this version: N. Lagarde, C. Reylé, A. C. Robin, G. Tautvaišienė, A. Drazdauskas, et al.. The Gaia-ESO Survey:
impact of extra mixing on C and N abundances of giant stars. Astronomy and Astrophysics - A&A,
2019, 621, pp.A24. 10.1051/0004-6361/201732433. hal-02066786 1. Introduction stars (e.g. Gilroy 1989; Gilroy & Brown 1991; Gratton et al. 2000;
Luck
1994;
Tautvaišien˙e et al. 2000,
2001,
2005;
Smiljanic et al. 2009; Mikolaitis et al. 2010, 2012). Over the last decade, the understanding of our Galaxy
has dramatically increased thanks to the development of
large spectroscopic surveys that provide fundamental prop-
erties of a large number of stars in different regions of
our Galaxy (e.g. RAVE: Steinmetz et al. 2006; SEGUE:
Yanny et al. 2009; Gaia-ESO: Gilmore et al. 2012; APOGEE:
Blanton et al. 2017; LAMOST: Cui et al. 2012; GALAH:
De Silva et al. 2015). Some of them provide abundances
deduced from high-resolution spectra, allowing the determina-
tion of light chemical elements such as carbon, nitrogen, or
lithium. Different transport processes have been discussed in the lit-
erature to explain the abundance anomalies in giants. Paramet-
ric computations have been proposed to better understand the
behaviour of chemical abundances at the stellar surface in low-
and intermediate-mass stars. After showing that the hot bot-
tom burning (HBB) process, which was previously proposed by
Cameron & Fowler (1971) to allow Li production in AGB stars,
can explain the oxygen isotopic ratios in AGB stars with ini-
tial stellar mass between 4.5 and 7.0 M, Boothroyd et al. (1995)
introduced the notion of cool bottom processing (i.e. ad hoc
transport material from the cool bottom of stellar convective
envelope to deeper and hotter radiative regions where nuclear
reactions occur) to explain surface abundances of giant stars
with masses lower than 2.0 M⊙(see also Wasserburg et al. 1995;
Boothroyd & Sackmann 1999; Sackmann & Boothroyd 1999). Denissenkov & Weiss (1996) suggested, as did Wasserburg et al. (1995), the presence of non-standard mixing of unknown phys-
ical origin, between the hydrogen burning and the base of
convective envelope after the bump to explain abundances
anomalies in red giant stars. To understand variations in The
carbon
and
nitrogen
abundances,
and
also
the
carbon isotopic ratio, are key chemical tracers used to
constrain the stellar evolution of giant stars. Indeed, low-
mass stars experience the well-known first dredge-up at
the
bottom
of
the
red
giant
branch
(RGB),
implying
changes of the surface abundances of C and N (Iben
1967). Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Overall, the current model including thermohaline mixing is able to reproduce very well the C and N
metallicity range investigated by the Gaia-ESO survey data. he current model including thermohaline mixing is able to reproduce very well the C and N abundances over the wh
gated by the Gaia-ESO survey data. Key words. stars: abundances – stars: evolution – Galaxy: stellar content – Galaxy: abundances Received 7 December 2017 / Accepted 5 June 2018 Received 7 December 2017 / Accepted 5 June 2018 ABSTRACT Context. The Gaia-ESO Public Spectroscopic Survey using FLAMES at the VLT has obtained high-resolution UVES spectra for a large number of
giant stars, allowing a determination of the abundances of the key chemical elements carbon and nitrogen at their surface. The surface abundances
of these chemical species are known to change in stars during their evolution on the red giant branch (RGB) after the first dredge-up episode, as a
result of the extra mixing phenomena. ffects of thermohaline mixing on C and N abundances using the first comparison between the Gaia-ESO survey [C/N]
ions of the observed fields using a model of stellar population synthesis. s. We investigate the effects of thermohaline mixing on C and N abundances using the first comparison between the G
rminations with simulations of the observed fields using a model of stellar population synthesis. s
g
g
g
p
y [ / ]
determinations with simulations of the observed fields using a model of stellar population synthesis. Methods. We explore the effects of thermohaline mixing on the chemical properties of giants through stellar evolutionary models computed with
the stellar evolution code STAREVOL. We include these stellar evolution models in the Besançon Galaxy model to simulate the [C/N] distributions
determined from the UVES spectra of the Gaia-ESO survey and to compare them with the observations. Methods. We explore the effects of thermohaline mixing on the chemical properties of giants through stellar evolutionary models computed with
the stellar evolution code STAREVOL. We include these stellar evolution models in the Besançon Galaxy model to simulate the [C/N] distributions
determined from the UVES spectra of the Gaia-ESO survey and to compare them with the observations. Results. Theoretical predictions including the effect of thermohaline mixing are in good agreement with the observations. However, the field stars
in the Gaia-ESO survey with C and N abundance measurements have a metallicity close to solar, where the efficiency of thermohaline mixing is
not very large. The C and N abundances derived by the Gaia-ESO survey in open and globular clusters clearly show the impact of thermohaline
mixing at low metallicity, which explains the [C/N] value observed in lower mass and older giant stars. Using independent observations of carbon
isotopic ratio in clump field stars and open clusters, we also confirm that thermohaline mixing should be taken into account to explain the behaviour
of 12C/13C as a function of stellar age. g
Conclusions. HAL Id: hal-02066786
https://hal.science/hal-02066786v1
Submitted on 11 Nov 2020 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. A&A 621, A24 (2019)
https://doi.org/10.1051/0004-6361/201732433
c⃝ESO 2018 Astronomy
&
Astrophysics Astronomy
&
Astrophysics Astronomy
&
Astrophysics The Gaia-ESO Survey: impact of extra mixing on C and N
abundances of giant stars
N. Lagarde1, C. Reylé1, A. C. Robin1, G. Tautvaišien˙e2, A. Drazdauskas2, Š. Mikolaitis2, R. Minkeviˇci¯ut˙e2,
E. Stonkut˙e2, Y. Chorniy2, V. Bagdonas2, A. Miglio3, G. Nasello1, G. Gilmore4, S. Randich5, T. Bensby6,
A. Bragaglia7, E. Flaccomio8, P. Francois9, A. J. Korn10, E. Pancino5,11, R. Smiljanic12, A. Bayo13, G. Carraro14,
M. T. Costado15, F. Jiménez-Esteban16,17, P. Jofré18, S. L. Martell19, T. Masseron20,21, L. Monaco22, L. Morbidelli23,
L. Sbordone24, S. G. Sousa25, and S. Zaggia26
(Affiliations can be found after the references)
Received 7 December 2017 / Accepted 5 June 2018 N. Lagarde1, C. Reylé1, A. C. Robin1, G. Tautvaišien˙e2, A. Drazdauskas2, Š. Mikolaitis2, R. Minkeviˇci¯ut˙e2,
E. Stonkut˙e2, Y. Chorniy2, V. Bagdonas2, A. Miglio3, G. Nasello1, G. Gilmore4, S. Randich5, T. Bensby6,
A. Bragaglia7, E. Flaccomio8, P. Francois9, A. J. Korn10, E. Pancino5,11, R. Smiljanic12, A. Bayo13, G. Carraro14,
M. T. Costado15, F. Jiménez-Esteban16,17, P. Jofré18, S. L. Martell19, T. Masseron20,21, L. Monaco22, L. Morbidelli23,
L. Sbordone24, S. G. Sousa25, and S. Zaggia26
(Affiliations can be found after the references)
Received 7 December 2017 / Accepted 5 June 2018 N. Lagarde1, C. Reylé1, A. C. Robin1, G. Tautvaišien˙e2, A. Drazdauskas2, Š. Mikolaitis2, R. Minkeviˇci¯ut˙e2,
E. Stonkut˙e2, Y. Chorniy2, V. Bagdonas2, A. Miglio3, G. Nasello1, G. Gilmore4, S. Randich5, T. Bensby6,
A. Bragaglia7, E. Flaccomio8, P. Francois9, A. J. Korn10, E. Pancino5,11, R. Smiljanic12, A. Bayo13, G. Carraro14,
M. T. Costado15, F. Jiménez-Esteban16,17, P. Jofré18, S. L. Martell19, T. Masseron20,21, L. Monaco22, L. Morbidelli23,
L. Sbordone24, S. G. Sousa25, and S. Zaggia26
(Affiliations can be found after the references)
Received 7 December 2017 / Accepted 5 June 2018 2. Data set and simulations (2017), improvements have
been extended to all populations other than the thin disc, imply-
ing the computation of stellar evolution models with different
α-enhancements ([α/Fe] = 0.15 and 0.30), following the obser-
vational [α/Fe] versus [Fe/H] trend observed by Data Release 12
of APOGEE (Majewski et al. 2017). Namely, for [Fe/H] < 0.1, To exploit all the potential of the spectroscopic data,
Lagarde et al. (2017; hereafter L17) improved the Besançon
Galaxy model (hereafter BGM) including the stellar evolution
models that provide surface chemical and seismic properties of
stars during their life. In addition to global properties, the BGM
is now able to explore the effects of extra mixing on the sur-
face abundances of different chemical species. The BGM allows
us to compute the stellar component of our Galaxy taking into
account errors and the selection function of the observations,
drawing a consistent picture of the Galaxy with the formation
and evolution scenarios of the Milky Way, stellar formation and
evolution theory, models of stellar atmospheres, and dynamical
constraints (Robin et al. 2003; Czekaj et al. 2014). This popula-
tion synthesis model is a powerful tool that can improve current
stellar evolution models and the physics of different transport
processes occurring in stellar interiors using a comparison with
observations of field stars at different stellar masses, metallic-
ities, ages, evolutionary stages, or at different locations in the
Galaxy. [α/Fe] =
0.014 + 0.0140675 × [Fe/H] + 0.101262 × [Fe/H]2
for the thin disc stars,
0.320 −exp(1.19375 × [Fe/H] −1.6)
for the thick disc stars,
0.3
for halo stars. (1) [α/Fe] =
0.014 + 0.0140675 × [Fe/H] + 0.101262 × [Fe/H]2
for the thin disc stars,
0.320 −exp(1.19375 × [Fe/H] −1.6)
for the thick disc stars,
0.3
for halo stars. (1) for halo stars. for halo stars. (1) For [Fe/H] > 0.1, [α/Fe] is assumed solar. To these relations
an intrinsic Gaussian dispersion of 0.02 dex is added. y
In this paper, we study the effects of thermohaline mixing
with metallicity and mass of giant stars, using the C and N abun-
dances derived for the giants in the Gaia-ESO survey. To this
end, we perform simulations using the BGM with and without
the effects of thermohaline instability. The simulations and data
used for this study are described in Sect. 2, while the theoretical
effects of thermohaline instability with stellar mass and metal-
licity are discussed in Sect. 2. Data set and simulations All data used in this paper are included in
the second, fourth, and fifth internal GES data releases (iDR2,
iDR4, and iDR5) to have a sample that is as large as possible. The main atmospheric parameters of the stars were determined
as described by Smiljanic et al. (2014), the carbon and nitrogen
abundances were determined as described by Tautvaišien˙e et al. (2015). We separated the stars into two groups: Thermohaline instability driven by
3He-burning through
the
pp-chain
has
been
proposed
to
govern
the
photo-
spheric compositions of bright low-mass red giant stars (e.g. Charbonnel & Zahn 2007; Charbonnel & Lagarde 2010). This
double diffusive instability, induced by a mean molecular
weight inversion due to the 3He(3He,2p)4He reaction in the
thin radiative layer between the convective envelope and the
hydrogen-burning shell (Eggleton et al. 2008; Lattanzio et al. 2015), is a physical mechanism that best reproduces the
observational abundances (e.g. of C and N) in giant stars
(e.g. Charbonnel & Lagarde 2010; Angelou et al. 2011, 2012;
Henkel et al. 2017). A few recent papers have suggested that
magnetic fields might play a role in stellar mixing, alone
(Busso et al. 2007) or in combination with thermohaline mixing
(Denissenkov & Merryfield 2011; Palmerini et al. 2011). Since
thermohaline also provides a physical solution of the
3He
problem, well-known in Galactic chemical evolution models
(Lagarde et al. 2011, 2012), we focus on this mechanism in this
paper. p
g
p
– 324 giant field stars with 173 stars located in the Galactic
bulge; – Giants belonging to open and globular clusters (see Table 1). The simulations were made using the revised version of BGM
(Paper I of this series, Lagarde et al. 2017), where a new grid of
stellar evolution models computed with the code STAREVOL
(e.g. Lagarde et al. 2012; Amard et al. 2016) has been imple-
mented. This new grid provides the global properties (e.g. sur-
face gravity, effective temperature) and chemical abundances
(for 54 stable and unstable species). These models also take into
account the effects of thermohaline instability during the red
giant branch (e.g. Charbonnel & Lagarde 2010). As discussed
in Paper I, thermohaline instability changes the photospheric
composition of low-mass brighter giant stars, with a decrease
in carbon and an increase in nitrogen. In addition to the sim-
ulations discussed in Lagarde et al. 2. Data set and simulations surface abundances of giant stars, they proposed a deep diffusive
mixing (e.g. Denissenkov & Weiss 1996; Weiss et al. 1996;
Denissenkov et al. 1998). Nevertheless, these two proposi-
tions of extra mixing are then not related to any physi-
cal mechanism to explain changes in surface abundances,
and depend on free parameters. On the other hand, rota-
tion has been investigated as a possible source of mixing
in RGB stars by several authors (Sweigart & Mengel 1979;
Charbonnel 1995; Denissenkov & Tout 2000; Palacios et al. 2006; Chanamé et al. 2005), showing that the total trans-
port coefficient of rotation at this phase is too low to
imply abundance variation on the first ascent giant branch as
requested by observations of RGB stars brighter than the RGB
bump. surface abundances of giant stars, they proposed a deep diffusive
mixing (e.g. Denissenkov & Weiss 1996; Weiss et al. 1996;
Denissenkov et al. 1998). Nevertheless, these two proposi-
tions of extra mixing are then not related to any physi-
cal mechanism to explain changes in surface abundances,
and depend on free parameters. On the other hand, rota-
tion has been investigated as a possible source of mixing
in RGB stars by several authors (Sweigart & Mengel 1979;
Charbonnel 1995; Denissenkov & Tout 2000; Palacios et al. 2006; Chanamé et al. 2005), showing that the total trans-
port coefficient of rotation at this phase is too low to
imply abundance variation on the first ascent giant branch as
requested by observations of RGB stars brighter than the RGB
bump. The Gaia-ESO survey (Gilmore et al. 2012; Randich et al. 2013,
hereafter GES) uses the Fibre Large Array Multi Element Spec-
trograph (FLAMES) multifibre facility (Pasquini et al. 2002) of
the Very Large Telescope (VLT) to obtain a better understand-
ing of the kinematic and chemical evolution of our Galaxy. Giraffe, the medium-resolution spectrograph (R ∼20000), and
UVES (Dekker et al. 2000), the high-resolution spectrograph
(R ∼47000), are used to observe up to 105 stars in the Milky
Way. y
For our study, we used the observations of giant stars made
with UVES, and the carbon and nitrogen abundances derived
from their spectra. These giant stars lie in different Galactic
regions (see Fig. 1). 1. Introduction After this episode, numerous spectroscopic observa-
tions show that an extra mixing occurs after the bump lumi-
nosity on the red giant branch changing the abundances of
elements lighter than oxygen at the surface of bright red giant A24, page 1 of 11 A&A 621, A24 (2019) 2. Data set and simulations 3. We start with field stars observed
with UVES in Sect. 4, and then enlarge our study to the open and
globular clusters observed by the Gaia-ESO survey and com-
piled from the literature (see Sect. 4.2). We draw our conclusions
in Sect. 5. As discussed in Czekaj et al. (2014), the BGM also simulates
the Poisson noise in the Monte Carlo generation of the simulated
stars. We performed simulations in every GES field referred to in
the iDR5 using this new version of the BGM. As shown in Fig. 2,
all stars in the sample have 0.5 < J −K < 1.0 and 5.0 < J < 14,
so we restricted our simulations to these colour and magnitude
ranges. We did two sets of simulations, with and without the
effects of thermohaline instability. The selection bias introduced
by the additional requirement of measurable carbon and nitrogen
abundances cannot be taken into account in our sample, which
is why simulations produce more stars than are present in the A24, page 2 of 11 N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars Fig. 1. All-sky view centred on the Milky Way Plane in an Aitoffprojection from Mellinger (2009) with a Galactic coordinate grid. Giants observed
by the Gaia-ESO survey for which C and N abundances are determined are colour-coded: clusters members (red) and field stars (yellow). We also
represent nine open clusters (Collinder 261, Melotte 66, NGC 6253, NGC 3960, NGC 2324, NGC 2477, NGC 2506, IC4651, NGC 6134) for which
12C/13C is derived (cyan diamond, see Sect. 4.3). Fig. 1. All-sky view centred on the Milky Way Plane in an Aitoffprojection from Mellinger (2009) with a Galactic coordinate grid. Giants observed
by the Gaia-ESO survey for which C and N abundances are determined are colour-coded: clusters members (red) and field stars (yellow). We also
represent nine open clusters (Collinder 261, Melotte 66, NGC 6253, NGC 3960, NGC 2324, NGC 2477, NGC 2506, IC4651, NGC 6134) for which
12C/13C is derived (cyan diamond, see Sect. 4.3). sample. This difference does not affect the conclusions of this
paper. predict that thermohaline instability is not efficient enough to
explain surface chemical abundances (Wachlin et al. 2014) with
the thermohaline fingers becoming more like blobs. Future
hydrodynamical simulations closer to the conditions met in stel-
lar interiors (Prat et al. 3.1. Physics Thermohaline mixing, as discussed in Paper I, is a double
diffusive process conducted in RGB stars by an inversion of
mean molecular weight induced by 3He(3He,2p)4He reaction
(Charbonnel & Zahn 2007; Siess 2009; Charbonnel & Lagarde
2010; Henkel et al. 2017) and a temperature gradient. For this
study we use the prescriptions advocated by Charbonnel & Zahn
(2007) given by Ulrich (1972) with an aspect ratio of instabil-
ity fingers α ∼6 as referred by Krishnamurti (2003). The ther-
mohaline diffusion coefficient used in stellar evolution models
includes the correction for non-perfect gas and is given by 2. Data set and simulations 2015) or including the effects of other
hydrodynamical processes as recently by Sengupta & Garaud
(2018) would shed light on this discrepancy. Hence, we choose
to use the prescriptions described above to compare theoreti-
cal predictions with current observations at different masses and
metallicities. 1 [X/Y] = A(X)−A(X)⊙−A(Y) + A(Y)⊙, with A(X) = log(N(X)/
N(H)) + 12. 3.2. Impact along the evolution This double diffusive instability develops starting from the lumi-
nosity of the bump during the RGB (e.g. Charbonnel & Lagarde
2010, and Paper I). Indeed, the steady increase in the stellar
luminosity along the RGB momentarily stops when the hydro-
gen burning shell (HBS) crosses the molecular weight barrier
left behind by the first dredge-up. At that moment the mean
molecular weight of the HBS becomes smaller, which implies
a decrease in the total stellar luminosity. This is called a bump in
the luminosity function. When the region of nuclear energy pro-
duction has passed this discontinuity, the mean molecular weight
slightly increases and the stellar luminosity increases again. This
variation in the luminosity causes an accumulation of stars in
the colour-magnitude diagram leading to a bump in the lumi-
nosity distribution (e.g. Iben 1967, 1968; Fusi Pecci et al. 1990;
Charbonnel 1994; Christensen-Dalsgaard 2015). The impact of
thermohaline instability on the theoretical [C/N]1 distributions
at different evolutionary states are shown in Fig. 3. With stel-
lar models, giant stars are divided into three groups: (1) low-
RGB stars: stars ascending the red giant branch before the Dt = Ct K
ϕ
δ
−∇µ
(∇ad −∇)
for ∇µ < 0,
(2) (2) where K is the thermal diffusivity; ϕ = (∂ln ρ/∂ln µ)P,T; δ =
−(∂ln ρ/∂ln ν)P,µ; ∇= (∂ln T/∂ln P); ∇µ and ∇ad are respec-
tively the molecular weight gradient and the adiabatic gradient;
and with the non-dimensional coefficient Ct = 8
3π2α2·
(3) Ct = 8
3π2α2· (3) The value of α is still discussed in the literature by
hydrodynamic simulations in 2D or 3D (Denissenkov et al. 2009; Denissenkov 2010; Denissenkov & Merryfield 2011;
Rosenblum et al. 2011; Traxler et al. 2011; Brown et al. 2013;
Garaud & Brummell 2015). Although these simulations are still
far from stellar conditions and do not take into account the
coupling of this instability with other hydrodynamic processes
occurring in red giant stars (e.g. rotation, magnetic field), they 1 [X/Y] = A(X)−A(X)⊙−A(Y) + A(Y)⊙, with A(X) = log(N(X)/
N(H)) + 12. A24, page 3 of 11 A&A 621, A24 (2019) nces for the chemical properties of globular and open clusters used in the comparison with model predictions. Table 1. References for the chemical properties of globular and open clusters used in the comparison with model predictions. Cluster
MTO
Age
Ref. Mass & Age
Ref. 3.3. Impact as a function of stellar mass RGB bump2. These stars have not yet undergone thermoha-
line mixing; (2) upper-RGB: RGB stars brighter than the RGB-
bump (with logg ≲2.2); (3) clump stars selected according
to their asymptotic period spacing of g-modes ∆Πℓ=1. As dis-
cussed by Charbonnel & Lagarde (2010) and Paper I, thermoha-
line instability occurring at the RGB-bump luminosity changes
the surface abundances of C and N for giant stars brighter than
the RGB-bump and along the red giant branch, resulting in a
decrease in [C/N] (see middle panel of Fig. 3). While thermo-
haline mixing is no longer happening in red clump stars, they
have the lowest [C/N] (as shown in Fig. 3) because they have
undergone a full RGB phase of extra mixing. RGB bump2. These stars have not yet undergone thermoha-
line mixing; (2) upper-RGB: RGB stars brighter than the RGB-
bump (with logg ≲2.2); (3) clump stars selected according
to their asymptotic period spacing of g-modes ∆Πℓ=1. As dis-
cussed by Charbonnel & Lagarde (2010) and Paper I, thermoha-
line instability occurring at the RGB-bump luminosity changes
the surface abundances of C and N for giant stars brighter than
the RGB-bump and along the red giant branch, resulting in a
decrease in [C/N] (see middle panel of Fig. 3). While thermo-
haline mixing is no longer happening in red clump stars, they
have the lowest [C/N] (as shown in Fig. 3) because they have
undergone a full RGB phase of extra mixing. As discussed and explained by Charbonnel & Lagarde (2010;
see Sect. 3.1.2), the global efficiency of thermohaline mixing
increases when considering less massive stars at a given metal-
licity or more metal-poor stars at a given stellar mass. This
results from the combination of several factors like the thermo-
haline diffusion timescale compared to the secular timescale, the
compactness of the hydrogen burning shell and of the thermoha-
line unstable region, and the amount of 3He available to power
the thermohaline instability. Figure 4 compares the effect of thermohaline mixing on
the surface abundances of red clump stars with (blue) and
without (grey) extra mixing, for 1 M⊙(upper panel) and 2 M⊙
(lower panel). On the main sequence, low-mass stars (LMS,
M ≲1.7 M⊙) burn hydrogen mainly through pp-chains rather
than CNO cycle as in intermediate-mass stars (IMS, 1.7 ≲
M
≲
2.2 M⊙). 2 Since the gravity (and thus luminosity) of the RGB-bump changes
with the metallicity of stars, we establish a simple empirical relation
based on stellar evolution models (without the effects of rotation on the
evolutionary path) allowing the distinction of low-RGB and upper-RGB
stars: log gRGBbump = 0.32·[Fe/H]+2.44. 3.2. Impact along the evolution 2012) with the
corresponding ages and metallicities. For globular clusters see the text; for clusters already published, we use the turn-offmass and age indicated
in the corresponding article. Notes. For GES clusters, the approximate turn-offmasses were evaluated using the theoretical PARSEC isochrones (Bressan et al. 2012) with the
corresponding ages and metallicities. For globular clusters see the text; for clusters already published, we use the turn-offmass and age indicated
in the corresponding article. 3.2. Impact along the evolution [C/N]
Symbols
Tr 20
1.9
1.4
Carraro et al. (2010), Donati et al. (2014)
GES
Black plus sign
NGC 104
0.89
12
Charbonnel & Chantereau (2016)
GES
Dark cyan circle
according to VandenBerg et al. (2013) and Parada et al. (2016)
NGC 1851
0.87
11
Charbonnel & Chantereau (2016)
GES
Magenta square
according to VandenBerg et al. (2013)
NGC 5927
0.94
11
Charbonnel & Chantereau (2016)
GES
Blue diamond
according to VandenBerg et al. (2013)
NGC 6705
3.30
0.3
Santos et al. (2005)
GES
Yellow square
Cantat-Gaudin et al. (2014)
NGC 2243
1.19
4.5
WEBDA database
GES
Red square
Br 81
2.06
1.0
Sagar & Griffiths (1998)
GES
Blue open diamond
NGC 6005
2.05
1.2
Piatti et al. (1998)
GES
Black down triangle
NGC 6802
1.90
0.9
Tang et al. (2017)
GES
Purple circle
Tr 23
2.05
0.8
Overbeek et al. (2017)
GES
Green square
Br 31
1.52
2.9
Cignoni et al. (2011)
GES
Yellow open square
Br 36
1.39
7
Donati et al. (2012)
GES
Magenta plus sign
Melotte 71
3.65
0.2
WEBDA database
GES
Pink open circle
NGC 6067
4.75
0.1
WEBDA database
GES
Magenta circle
according to Alonso-Santiago et al. (2017)
NGC 6253
1.32
3-5
WEBDA database
GES
Purple plus sign
M67
1.6
2.6
WEBDA database
GES
Green circle
NGC 6259
3.73
0.2
WEBDA database
GES
Dark blue circle
Dias et al. (2002)
Rup 134
2.18
1.0
Carraro et al. (2006)
GES
Red circle
NGC 2324
2.7
0.44
Tautvaišien˙e et al. (2016)
Red cross
NGC 3960
2.2
0.9
Tautvaišien˙e et al. (2016)
Blue plus sign
NGC 6253
1.4
0.6
Mikolaitis et al. (2012)
Black open circle
NGC 2477
2.3
0.82
Tautvaišien˙e et al. (2016)
Magenta up triangle
Melotte 66
1.2
4
Drazdauskas et al. (2016)
Black cross
Collinder 261
1.1
6.0
Mikolaitis et al. (2012)
Yellow circle
Drazdauskas et al. (2016)
NGC 6134
2.34
0.7
Mikolaitis et al. (2010)
Blue open square
NGC 2506
1.69
1.7
Mikolaitis et al. (2011a)
Green up triangle
IC 4651
1.69
1.7
Mikolaitis et al. (2011b)
Red down triangle
Notes. For GES clusters, the approximate turn-offmasses were evaluated using the theoretical PARSEC isochrones (Bressan et al. 2012) with the
corresponding ages and metallicities. For globular clusters see the text; for clusters already published, we use the turn-offmass and age indicated
in the corresponding article. Notes. For GES clusters, the approximate turn-offmasses were evaluated using the theoretical PARSEC isochrones (Bressan et al. N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars Fig. 2. Colour-magnitude diagram for stars observed by the GES survey
(grey dots), and for which C and N abundances have been determined
(magenta dots). The colour and magnitude values are from the 2MASS
Catalog. Low-RGB
¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2
0
[C=N]
0
2
4
6
8
N?;norm
Up-RGB
¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2
0
[C=N]
0
2
4
6
8
N?;norm Low-RGB
¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2
0
[C=N]
0
2
4
6
8
N?;norm
Up-RGB
¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2
0
[C=N]
0
2
4
6
8
N?;norm
Clump/early-AGB
¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2
0
[C=N]
0
2
4
6
8
N?;norm
Fig. 3. [C/N] distributions normalised to the histogram area of the
synthetic population simulated with the BGM with the effects of ther-
mohaline instability (blue solid histograms) and without (grey shaded
histograms) during the red giant branch. Giant stars are divided into
three groups: low-RGB stars (before the RGB-bump luminosity, top
panel), upper-RGB stars (after the RGB-bump luminosity, middle
panel), and clump/early-AGB stars (bottom panel). Low-RGB
¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2
0
[C=N]
0
2
4
6
8
N?;norm Up-RGB
¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2
0
[C=N]
0
2
4
6
8
N?;norm Up-RGB Fig. 2. Colour-magnitude diagram for stars observed by the GES survey
(grey dots), and for which C and N abundances have been determined
(magenta dots). The colour and magnitude values are from the 2MASS
Catalog. [C/N] distribution is larger for 1.0 M⊙stars than for 2.0 M⊙
stars. Clump/early-AGB
¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2
0
[C=N]
0
2
4
6
8
N?;norm Clump/early-AGB On the other hand, the RGB-bump occurs on the evolution of
low- and intermediate-mass stars only, and depends on the metal-
licity of these stars. More massive stars (HMS, M ≳2.2 M⊙)
ignite central helium burning in a non-degenerate core at rel-
atively low luminosity on the RGB, well before the hydrogen
burning shell reaches the mean molecular weight discontinuity
caused by the first dredge-up. Consequently, these objects do
not go through the bump on their short ascent of the RGB, and
thus thermohaline instability does not develop in this kind of
star. [C=N] Fig. 3. 3.4. Impact as a function of metallicity In low-mass, low-metallicity giants the thermohaline unstable
region is more compact and has a steeper temperature gradient,
resulting in a higher diffusion coefficient and then a more effi-
cient transport process (see Fig. 6 of Lagarde et al. 2011). p
p
g
g
The effect of thermohaline mixing at two metallicities on the
[C/N] value at the surface of clump stars simulated by the BGM
is shown on Fig. 5 (top and middle panels). The mass distribu-
tions for each metallicity range are also shown in Fig. 5 (bot-
tom panel). Although thermohaline mixing has a larger impact
on [C/N] when the metallicity decreases, the figure also clearly
shows a non-negligible impact at solar metallicity. panel, respectively). Low-RGB, up-RGB, and clump stars are
included in this figure (as described above). This figure clearly
shows a stronger impact of thermohaline instability on the sur-
face abundances of giants (here, [C/N]) with [Fe/H] < −0.5. Lower metallicity populations are composed essentially of low-
mass stars, implying thick disc and halo as key populations to
test the efficiency of this extra mixing (see Figs. 5 and 6). g g
p
y
Because of their large mass and metallicity ranges, field
stars provide key data to constrain the efficiency of thermohaline
instability occurring in giant stars. In the next section, we study
the effects of thermohaline instability with mass and metallicity,
comparing the population synthesis and the GES observations. In the same figure, the [C/N] value derived from the observed
field stars are also shown (red dots). Since UVES is centred on
solar neighbourhood MSTO stars (plus bulge, see Fig. 1), where
approximately solar metallicity is expected, the metallicity range
of observations is around the solar metallicity (Stonkut˙e et al. (2016), −0.5 ≲[Fe/H] ≲0.5). Figure 7 presents a com-
parison between the [C/N] distribution predicted by the BGM
with and without the effects of themohaline instability and the
observed distribution of [C/N] in the GES field stars sample. Since the [C/N] range in the simulation including the effects
of thermohaline instability or following the standard theory are
the same, Fig. 6 does not allow us to discriminate between both N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars [C/N] distributions normalised to the histogram area of the
synthetic population simulated with the BGM with the effects of ther-
mohaline instability (blue solid histograms) and without (grey shaded
histograms) during the red giant branch. Giant stars are divided into
three groups: low-RGB stars (before the RGB-bump luminosity, top
panel), upper-RGB stars (after the RGB-bump luminosity, middle
panel), and clump/early-AGB stars (bottom panel). 3.3. Impact as a function of stellar mass Consequently, a large production of
3He
occurs in LMS, favouring the development of thermohaline
instability during the red giant branch. Then the impact on A24, page 4 of 11 N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars 4.1. C and N abundances in field stars 4.1. C and N abundances in field stars Figure 6 displays the surface [C/N] of giant stars simulated with
the BGM (grey dots) as a function of stellar metallicity, with
and without the effects of thermohaline mixing (right and left A24, page 5 of 11 A24, page 5 of 11 A&A 621, A24 (2019) M = 1.0 M⊙
¡1:2
¡1:0
¡0:8
¡0:6
¡0:4
¡0:2
[C=N]
0
5
10
15
N?;norm
M = 2.0 M⊙
¡1:2
¡1:0
¡0:8
¡0:6
¡0:4
¡0:2
[C=N]
0
5
10
15
N?;norm
¡1:5
¡1:0
¡0:5
0
0:5
[Fe=H]
0
200
400
600
800
N? M » 1:0M¯
M » 2:0M¯
Fig. 4. [C/N] distributions for two stellar masses ranges, 0.95
≤
M/M⊙≤1.05 and 1.95 ≤M/M⊙≤2.05 (top and bottom panels,
respectively), for clump stars simulated with the BGM with the effects
of thermohaline instability (blue solid histograms) and without (grey
shaded histograms). Bottom panel: mass distributions for clump stars
simulated with the BGM at both mass ranges. [Fe/H] ∼0
¡1:2
¡1:0
¡0:8
¡0:6
¡0:4
¡0:2
0
[C=N]
0
5
10
15
N?;norm
[Fe/H] ∼−0.8
¡1:2
¡1:0
¡0:8
¡0:6
¡0:4
¡0:2
0
[C=N]
0
5
10
15
N?;norm
1:0
1:5
2:0
2:5
3:0
3:5
4:0
4:5
5:0
5:5
M=M¯
0
1000
2000
3000
N? [Fe=H] » ¡0:8
[Fe=H] » 0
Fig. 5. [C/N] distributions at two metallicities (top and middle panels,
respectively) for clump stars simulated with the BGM with the effects
of thermohaline instability (blue solid histograms) and without (grey
shaded histograms). Bottom panel: mass distributions for clump stars
simulated with the BGM at −0.85 ≤[Fe/H] ≤−0.75 and −0.05 ≤
[Fe/H] ≤0.05. M = 1.0 M⊙
¡1:2
¡1:0
¡0:8
¡0:6
¡0:4
¡0:2
[C=N]
0
5
10
15
N?;norm [Fe/H] ∼0
¡1:2
¡1:0
¡0:8
¡0:6
¡0:4
¡0:2
0
[C=N]
0
5
10
15
N?;norm M = 1.0 M⊙ M = 2.0 M⊙
¡1:2
¡1:0
¡0:8
¡0:6
¡0:4
¡0:2
[C=N]
0
5
10
15
N?;norm [Fe/H] ∼−0.8
¡1:2
¡1:0
¡0:8
¡0:6
¡0:4
¡0:2
0
[C=N]
0
5
10
15
N?;norm M = 2.0 M⊙ ¡1:5
¡1:0
¡0:5
0
0:5
[Fe=H]
0
200
400
600
800
N? M » 1:0M¯
M » 2:0M¯ 1:0
1:5
2:0
2:5
3:0
3:5
4:0
4:5
5:0
5:5
M=M¯
0
1000
2000
3000
N? [Fe=H] » ¡0:8
[Fe=H] » 0 Fig. 5. 4.1. C and N abundances in field stars [C/N] distributions at two metallicities (top and middle panels,
respectively) for clump stars simulated with the BGM with the effects
of thermohaline instability (blue solid histograms) and without (grey
shaded histograms). Bottom panel: mass distributions for clump stars
simulated with the BGM at −0.85 ≤[Fe/H] ≤−0.75 and −0.05 ≤
[Fe/H] ≤0.05. Fig. 4. [C/N] distributions for two stellar masses ranges, 0.95
≤
M/M⊙≤1.05 and 1.95 ≤M/M⊙≤2.05 (top and bottom panels,
respectively), for clump stars simulated with the BGM with the effects
of thermohaline instability (blue solid histograms) and without (grey
shaded histograms). Bottom panel: mass distributions for clump stars
simulated with the BGM at both mass ranges. prescriptions directly. However, Fig. 7 allows a quantitative com-
parison and shows the necessity of extra mixing to reproduce
the observations even at higher metallicities. In this case, infor-
mation on stellar masses and evolutionary states of field stars
are required to add more constraints on the efficiency of extra
mixing with stellar mass. This is now possible with asteroseis-
mology, which can be combined with astrometry from Gaia
(Gaia Collaboration 2016), and will be discussed in a forthcom-
ing paper. 4.2. C and N abundances in clusters [C/N] as a function of [Fe/H] for synthetic populations computed with the BGM with the effects of thermohaline instability (right panel)
and without (left panel). The [C/N] values for our sample of UVES giant field stars are also shown (red dots). Field stars
NGC 104
¡0:8
¡0:6
¡0:4
¡0:2
0
[C=N]
0
1
2
3
4
N?;norm
¡0:8
¡0:6
¡0:4
¡0:2
0
[C=N]
0
1
2
3
4
N?;norm
¡0:9
¡0:8
¡0:7
¡0:6
¡0:5
¡0:4
¡0:3
¡0:2
[C=N]
0
1
2
3
4
5
6
7
8
N?;norm
Fig. 7. Left and middle panels: [C/N] distributions for the synthetic populations in Fig. 6 with the effects of thermohaline instability (middle panel)
and without (left panel). [C/N] for our sample of UVES giant field stars are also shown (red histogram). Right panel: [C/N] distributions for a
synthetic populations computed with the BGM for the globular cluster NGC 104 with the effect of thermohaline instability (blue histogram) and
without (black histogram). The observed [C/N] derived by GES survey is shown (red histogram). NGC 104
¡0:9
¡0:8
¡0:7
¡0:6
¡0:5
¡0:4
¡0:3
¡0:2
[C=N]
0
1
2
3
4
5
6
7
8
N?;norm eld stars
¡0:8
¡0:6
¡0:4
¡0:2
0
[C=N]
0
1
2
3
4
N?;norm Fie
¡0:8
¡0:6
¡0:4
¡0:2
0
[C=N]
0
1
2
3
4
N?;norm Fig. 7. Left and middle panels: [C/N] distributions for the synthetic populations in Fig. 6 with the effects of thermohaline instability (middle panel)
and without (left panel). [C/N] for our sample of UVES giant field stars are also shown (red histogram). Right panel: [C/N] distributions for a
synthetic populations computed with the BGM for the globular cluster NGC 104 with the effect of thermohaline instability (blue histogram) and
without (black histogram). The observed [C/N] derived by GES survey is shown (red histogram). Fig. 8. [C/N] as a function of [Fe/H] for synthetic populations computed with the BGM with the effects of thermohaline instability (right panel)
and without (left panel). [C/N] for our sample of UVES giant stars members of open and globular clusters are also shown (each symbol represents
a cluster, see Table 1). A typical error bar is indicated. Fig. 8. [C/N] as a function of [Fe/H] for synthetic populations computed with the BGM with the effects of thermohaline instability (right panel)
and without (left panel). 4.2. C and N abundances in clusters The GES has observed many different clusters in different
regions of our Galaxy (see red circles in Fig. 1), providing the
homogeneous observational data needed to constrain stellar and
Galactic evolution. We investigate the [C/N] value derived in
giant members of those open and globular clusters. We do not
want to study each cluster in detail; instead, clusters are used
here as tracers of extra mixing. Since stars belonging to a cluster
were formed together, we can assume that they have the same
age, distance, and metallicity, resulting in stronger constraints of
thermohaline efficiency. In addition, the right panel of Fig. 7 shows the simula-
tion performed specifically for NGC 104 (with and without
extra mixing), focussing on cluster region in the sky and the
specific metallicity range. The mean [C/N] and the standard
deviation predicted by the simulations including extra mix-
ing (⟨[C/N]⟩= −0.48 ± 0.19) are in better agreement with
observations in NGC 104 (⟨[C/N]⟩
=
−0.53 ± 0.20), than
for simulations following the standard stellar evolution model
(⟨[C/N]⟩= −0.28 ± 0.05). With this very promising result,
we focus on clusters observed by the GES survey in the next
section. Figures 8–10 show the [C/N] value derived by GES for
evolved stars belonging to different globular and open clus-
ters as a function of metallicity, turn-offmass, and age. We
also add clusters for which [C/N] and 12C/13C determinations
are available from the literature (not from GES): Collinder
261, Melotte 66, NGC 6253, NGC 3960, NGC 2324, NGC 2477,
NGC 2506, IC 4651, NGC 6134 (Mikolaitis et al. 2012, 2011a,b,
2010; Drazdauskas et al. 2016; Tautvaišien˙e et al. 2016). These A24, page 6 of 11 N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars
Fig. 6. [C/N] as a function of [Fe/H] for synthetic populations computed with the BGM with the effects of thermohaline instability (right panel)
and without (left panel). The [C/N] values for our sample of UVES giant field stars are also shown (red dots). N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant sta Fig. 6. 4.2. C and N abundances in clusters [C/N] for our sample of UVES giant stars members of open and globular clusters are also shown (each symbol represents
a cluster, see Table 1). A typical error bar is indicated. A24, page 7 of 11 A&A 621, A24 (2019) Fig. 9. [C/N] as a function of stellar mass for synthetic populations computed with the BGM with the effects of thermohaline instability (right
panel) and without (left panel). [C/N] for our sample of UVES giant stars members of open and globular clusters are also shown (each symbol
represents a cluster, see Table 1). A typical error bar is indicated. Fig. 9. [C/N] as a function of stellar mass for synthetic populations computed with the BGM with the effects of thermohaline instability (right
panel) and without (left panel). [C/N] for our sample of UVES giant stars members of open and globular clusters are also shown (each symbol
represents a cluster, see Table 1). A typical error bar is indicated. Fig. 10. [C/N] as a function of stellar ages for synthetic populations computed with the BGM with the effects of thermohaline instability (right
panel) and without (left panel). [C/N] for our sample of UVES giant stars members of open and globular clusters are also shown (each symbol
represents a cluster, see Table 1). Fig. 10. [C/N] as a function of stellar ages for synthetic populations computed with the BGM with the effects of thermohaline instability (right
panel) and without (left panel). [C/N] for our sample of UVES giant stars members of open and globular clusters are also shown (each symbol
represents a cluster, see Table 1). As
discussed
by
Charbonnel & Lagarde
(2010)
and
described in Sect. 3, three mass ranges can be evoked to
quantify the efficiency of thermohaline instability: clusters were acquired by two complementary programmes,
and were analysed in a homogeneous way by the same
group that produces C and N determinations in GES, result-
ing in a robust comparison with our models. Individual stars
are attributed the turn-offmass and age of their host clus-
ters (e.g. VandenBerg et al. 2013, Chantereau’s priv. comm. using stellar models discussed in Charbonnel & Chantereau
2016, and Drazdauskas et al., in prep.). Synthetic popula-
tions with and without the effects of thermohaline instability
are shown (right and left panels, respectively) for low-RGB,
upper-RGB, and clump stars (as defined in Sect. 4). 4.2. C and N abundances in clusters Simu-
lations and observations in each cluster are a mix of giant
stars at different evolutionary states (i.e. at different luminosi-
ties or gravities on the RGB), implying a wide [C/N] range
for the synthetic populations and for the determination in each
cluster. clusters were acquired by two complementary programmes,
and were analysed in a homogeneous way by the same
group that produces C and N determinations in GES, result-
ing in a robust comparison with our models. Individual stars
are attributed the turn-offmass and age of their host clus-
ters (e.g. VandenBerg et al. 2013, Chantereau’s priv. comm. using stellar models discussed in Charbonnel & Chantereau
2016, and Drazdauskas et al., in prep.). Synthetic popula-
tions with and without the effects of thermohaline instability
are shown (right and left panels, respectively) for low-RGB,
upper-RGB, and clump stars (as defined in Sect. 4). Simu-
lations and observations in each cluster are a mix of giant
stars at different evolutionary states (i.e. at different luminosi-
ties or gravities on the RGB), implying a wide [C/N] range
for the synthetic populations and for the determination in each
cluster. – For low-mass and low-metallicity stars, thermohaline insta-
bility is the most efficient transport process that can change
the C and N surface abundances, which is why the simula-
tions presented here (which take into account thermohaline
mixing only) reproduce very well the observed [C/N] in this
mass range (see Fig. 9) including a very good fit for stars
older than ∼1 Gyr (see Fig. 10). – For intermediate-mass stars (1.7 ≲M ≲2.2 M⊙), our simu-
lations present a slightly higher [C/N] than observations (see
right panel of Fig. 9). Charbonnel & Lagarde (2010) show
that in addition to thermohaline mixing, rotation-induced
mixing plays an equivalent role in changing the surface abun-
dances of the stars in this mass range, resulting in a slightly A24, page 8 of 11 N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars lower [C/N] at the surface of intermediate-mass stars (see
Fig. 17 of Charbonnel & Lagarde 2010). lower [C/N] at the surface of intermediate-mass stars (see
Fig. 17 of Charbonnel & Lagarde 2010). ¡0:8
¡0:7
¡0:6
¡0:5
¡0:4
¡0:3
¡0:2
¡0:1
0
[C=N]
0
10
20
30
40
50
60
70
12C=13C
Fig. 11. 4.2. C and N abundances in clusters 12C/13C as a function of [C/N] for synthetic populations com-
puted with the BGM with the effects of thermohaline instability (grey
dots) and without (green dots) for upper-RGB and clump stars. A sam-
ple of clump field stars are represented by black dots, and nine clus-
ters (Collinder 261, Melotte 66, NGC 6253, NGC 3960, NGC 2324,
NGC 2477, NGC 2506, IC4651, NGC 6134) using different colours and
symbols (see Table 1). ¡0:8
¡0:7
¡0:6
¡0:5
¡0:4
¡0:3
¡0:2
¡0:1
0
[C=N]
0
10
20
30
40
50
60
70
12C=13C – For high-mass stars, thermohaline mixing plays no role
because these stars do not go through the RGB-bump on
their short first ascent of the red giant branch, and thus
thermohaline instability does not occur (see Sect. 3). This
explains why our simulations do not reproduce the spread of
[C/N] observed in clusters more massive than 2.2 M⊙(see
Fig. 9) and younger than 0.5 Gyr (see Fig. 10). As known
for a long time (e.g. Meynet & Maeder 2002; Palacios et al. 2006; Charbonnel & Lagarde 2010), rotation-induced mix-
ing has an impact on the internal chemical structure of main
sequence stars, although its signatures are revealed later, at
the beginning of the RGB. This results in a decrease in the
surface abundances of C while N increases. We plan to focus
on the effects of rotation on stellar ages and chemical prop-
erties in a forthcoming paper. g p p
We note that using the Data Release 12 of the APOGEE sur-
vey, Masseron & Gilmore (2015) showed that extra mixing has
occurred in thin disc stars, but indicated that thick disc stars do
not show any evidence of this extra mixing process. They pro-
posed that the thick disc stars could be formed with a differ-
ent initial abundances than thin disc stars. We also note that our
simulations reproduce very well observations in clusters having
metallicity corresponding to the thick disc or halo population
assuming the same initial abundances for all populations. How-
ever, our comparison is based on clusters members, and com-
plementary to this study it is crucial to consider a larger sample
of thick disc field stars such as those observed by the APOGEE
survey. 4.3. 12C/13C in field stars and clusters The GES cannot derive the carbon isotopic ratio due to the
wavelength regions observed with no good 13CN features, thus
in this part we use data from other studies to investigate the
importance of 12C/13C to constrain extra mixing on the red giant
branch. We compared data from the Gaia-ESO survey with predic-
tions computed using the Besançon Galaxy model in which we
included stellar evolution models taking into account (and not)
the effects of thermohaline instability (Paper I). To date, this
mixing is the only physical process proposed in the literature to
explain the photospheric composition of evolved red giant stars. We focus in the first part of this paper on field stars because of
their wide-coverage properties (e.g. mass, metallicity, and ages)
to deduce an observational trend between [C/N] and stellar mass,
metallicity, or age. Due to the lack of C and N determinations in
field stars at low metallicity, we cannot investigate further the
observational constraints in the metallicity domain where ther-
mohaline instability is more efficient. Nevertheless, the theoret-
ical distribution of [C/N] predicted by the BGM including the
effects of thermohaline instability is in better agreement than the
distribution predicted by the standard model at metallicity close
to solar. Figures 11 and 12 present the carbon isotopic ratio as a func-
tion of C/N and as function of stellar masses and ages, for syn-
thetic populations computed with the BGM taking into account
the effects of thermohaline mixing (grey dots) or not (green
dots). The carbon isotopic ratio decreases abruptly when the
thermohaline mixing develops in RGB stars, as already shown
by Charbonnel & Lagarde (2010). This is in agreement with
the observed abundance ratios found in field stars and open
clusters, shown with symbols in the figures (Mikolaitis et al. 2012; Drazdauskas et al. 2016; Tautvaišien˙e et al. 2016). Even
though the range of [C/N] values agrees with both models in the
observed range, the low values of 12C/13C cannot be reproduced
without extra mixing process. As discussed below in the [C/N]
case, thermohaline mixing explains very well the low-mass (and
older) giants stars, but not the higher mass stars. This proves
that 12C/13C is a more powerful parameter for constraining extra
mixing on the RGB than [C/N], including for solar metallicity
stars. We also investigate the [C/N] derived in giant members of
open and globular clusters by the Gaia-ESO survey and litera-
ture. 4.2. C and N abundances in clusters In addition, the chemical evolution model and the popu-
lation synthesis model should be combined to study the effects of
different initial abundances for the different populations before
drawing any conclusions. Fig. 11. 12C/13C as a function of [C/N] for synthetic populations com-
puted with the BGM with the effects of thermohaline instability (grey
dots) and without (green dots) for upper-RGB and clump stars. A sam-
ple of clump field stars are represented by black dots, and nine clus-
ters (Collinder 261, Melotte 66, NGC 6253, NGC 3960, NGC 2324,
NGC 2477, NGC 2506, IC4651, NGC 6134) using different colours and
symbols (see Table 1). the C and N abundances derived by the Gaia-ESO survey in
field stars and in different clusters, both globular and open. We
conclude from this work that it is crucial to take into account
thermohaline mixing to understand the C and N observed at the
surface of low-mass stars, and in the determination of the stellar
mass and age from their chemical properties (Martig et al. 2015;
Ness et al. 2016). 4.3. 12C/13C in field stars and clusters This comparison shows a very good agreement with stellar
evolution models including thermohaline mixing over the whole
scrutinized metallicity range, explaining the [C/N] observed in
lower mass and older giant stars. This confirms that thermoha-
line instability is crucial to understand the chemical properties of
giant stars. The next step is to use a larger sample of field stars
to strengthen this encouraging result. 5. Conclusions l’Univers (INSU). G.T., A.D., Š.M., R.M., E.S., Y.Ch., and V.B acknowledge
support from the Research Council of Lithuania (MIP-082/2015). R.S. acknowl-
edges support from the Polish Ministry of Science and Higher Education. F.J.E. acknowledges financial support from ASTERICS project (ID:653477, H2020-
EU.1.4.1.1. - Developing new world-class research infrastructures). T.B. was
funded by the project grant “The New Milky Way?” from the Knut and Alice
Wallenberg Foundation. A.J.K acknowledges support from the Swedish National
Space Board (SNSB). T.M. acknowledges support provided by the Spanish
Ministry of Economy and Competitiveness (MINECO) under grant AYA-2017-
88254-P. Additionally, and independently of spectroscopy, asteroseis-
mology paves the way to a better understanding of stellar
interiors, providing valuable and independent constraints on cur-
rent stellar evolution models and on the physics of different
transport processes. The space missions CoRoT (Baglin et al. 2006), Kepler, and K2 (Borucki et al. 2010) observed a large
number of giant stars in different regions in our Galaxy, allow-
ing a unique opportunity to derive some fundamental properties
(e.g. stellar mass, radius, age and gravity, and evolutionary stage
of giants) by observation of mixed modes in red giants (e.g. Chaplin & Miglio 2013). To obtain the most information pos-
sible from the data sample, the asteroseismic properties must be
combined with the observations of the surface chemical abun-
dances and especially the surface 12C/13C. Future studies of
CoRoT inner-field stars (Valentini et al., in prep.) and K2 giants
(campaign 3) already observed by GES, will provide comple-
mentary results for the development of stellar evolution models. References Alonso-Santiago, J., Negueruela, I., Marco, A., et al. 2017, MNRAS, 469, 1330
Amard, L., Palacios, A., Charbonnel, C., Gallet, F., & Bouvier, J. 2016, A&A,
587, A105 Angelou, G. C., Church, R. P., Stancliffe, R. J., Lattanzio, J. C., & Smith, G. H. 2011, ApJ, 728, 79 Angelou, G. C., Stancliffe, R. J., Church, R. P., Lattanzio, J. C., & Smith, G. H. 2012, ApJ, 749, 128 Baglin, A., Auvergne, M., Boisnard, L., et al. 2006, in 36th COSPAR Scientific
Assembly, 36, 3749 Blanton, M. R., Bershady, M. A., Abolfathi, B., et al. 2017, AJ, 154, 28
Boothroyd, A. I., & Sackmann, I.-J. 1999, ApJ, 510, 232 Boothroyd, A. I., Sackmann, I.-J., & Wasserburg, G. J. 1995, ApJ, 442, L21
Borucki, W. J., Koch, D., Basri, G., et al. 2010, Science, 327, 977 Acknowledgements. Based on data products from observations made with ESO
Telescopes at the La Silla Paranal Observatory under programme ID 188.B-
3002. These data products have been processed by the Cambridge Astronomy
Survey Unit (CASU) at the Institute of Astronomy, University of Cambridge,
and by the FLAMES/UVES reduction team at INAF/Osservatorio Astrofisico
di Arcetri. These data have been obtained from the Gaia-ESO Survey Data
Archive, prepared and hosted by the Wide Field Astronomy Unit, Institute for
Astronomy, University of Edinburgh, which is funded by the UK Science and
Technology Facilities Council. This work was partly supported by the European
Union FP7 programme through ERC grant number 320360 and by the Lever-
hulme Trust through grant RPG-2012-541. We acknowledge the support from
INAF and Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) in
the form of the grant “Premiale VLT 2012”. The results presented here bene-
fit from discussions held during the Gaia-ESO workshops and conferences sup-
ported by the ESF (European Science Foundation) through the GREAT Research
Network Programme. N.L., C.R., A.R., and G.N. acknowledge financial support
from the “Programme National de Physique Stellaire” (PNPS) and the “Pro-
gramme National de cosmologie et Galaxie” (PNCG) of CNRS/INSU, France. N.L. acknowledges financial support from the CNES. Simulations have been
executed on computers from the Utinam Institute of the Université de Franche-
Comté, supported by the Région de Franche-Comté and Institut des Sciences de Bressan, A., Marigo, P., Girardi, L., et al. 2012, MNRAS, 427, 127 Brown, J. M., Garaud, P., & Stellmach, S. 2013, ApJ, 768, 34 Busso, M., Wasserburg, G. J., Nollett, K. M., & Calandra, A. Acknowledgements.
Based on data products from observations made with ESO
Telescopes at the La Silla Paranal Observatory under programme ID 188.B-
3002. These data products have been processed by the Cambridge Astronomy
Survey Unit (CASU) at the Institute of Astronomy, University of Cambridge,
and by the FLAMES/UVES reduction team at INAF/Osservatorio Astrofisico
di Arcetri. These data have been obtained from the Gaia-ESO Survey Data
Archive, prepared and hosted by the Wide Field Astronomy Unit, Institute for
Astronomy, University of Edinburgh, which is funded by the UK Science and
Technology Facilities Council. This work was partly supported by the European
Union FP7 programme through ERC grant number 320360 and by the Lever-
hulme Trust through grant RPG-2012-541. We acknowledge the support from
INAF and Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) in
the form of the grant “Premiale VLT 2012”. The results presented here bene-
fit from discussions held during the Gaia-ESO workshops and conferences sup-
ported by the ESF (European Science Foundation) through the GREAT Research
Network Programme. N.L., C.R., A.R., and G.N. acknowledge financial support
from the “Programme National de Physique Stellaire” (PNPS) and the “Pro-
gramme National de cosmologie et Galaxie” (PNCG) of CNRS/INSU, France.
N.L. acknowledges financial support from the CNES. Simulations have been
executed on computers from the Utinam Institute of the Université de Franche-
Comté, supported by the Région de Franche-Comté and Institut des Sciences de 5. Conclusions In this paper, we present the first comparison between synthetic
populations computed with the Besançon Galaxy model and On the other hand, we show that the observed behaviour of
12C/13C with stellar ages is clearly reproduced by models which A24, page 9 of 11 A24, page 9 of 11 A&A 621, A24 (2019) 1:0
1:5
2:0
2:5
3:0
3:5
4:0
4:5
5:0
5:5
M=M¯
0
5
10
15
20
25
30
35
40
12C=13C
0:1
0:2
0:5
1
2
5
10
Age [Gyr]
0
5
10
15
20
25
30
35
40
12C=13C
Fig. 12. 12C/13C as a function of stellar mass and ages for synthetic populations computed with the BGM with the effects of thermohaline instability
(grey dots) and without (green dots). A sample of clump field stars are represented by black dots in the right panel, and nine clusters (Collinder 261,
Melotte 66, NGC 6253, NGC 3960, NGC 2324, NGC 2477, NGC 2506, IC4651, NGC 6134) using different colours and symbols (see Table 1). 1:0
1:5
2:0
2:5
3:0
3:5
4:0
4:5
5:0
5:5
M=M¯
0
5
10
15
20
25
30
35
40
12C=13C 0:1
0:2
0:5
1
2
5
10
Age [Gyr]
0
5
10
15
20
25
30
35
40
12C=13C Fig. 12. 12C/13C as a function of stellar mass and ages for synthetic populations computed with the BGM with the effects of thermohaline instability
(grey dots) and without (green dots). A sample of clump field stars are represented by black dots in the right panel, and nine clusters (Collinder 261,
Melotte 66, NGC 6253, NGC 3960, NGC 2324, NGC 2477, NGC 2506, IC4651, NGC 6134) using different colours and symbols (see Table 1). include the effect of thermohaline mixing. This confirms the
importance, amongst others, of extra mixing when deducing stel-
lar ages from the chemical properties of giant stars. l’Univers (INSU). G.T., A.D., Š.M., R.M., E.S., Y.Ch., and V.B acknowledge
support from the Research Council of Lithuania (MIP-082/2015). R.S. acknowl-
edges support from the Polish Ministry of Science and Higher Education. F.J.E. acknowledges financial support from ASTERICS project (ID:653477, H2020-
EU.1.4.1.1. - Developing new world-class research infrastructures). T.B. was
funded by the project grant “The New Milky Way?” from the Knut and Alice
Wallenberg Foundation. A.J.K acknowledges support from the Swedish National
Space Board (SNSB). T.M. acknowledges support provided by the Spanish
Ministry of Economy and Competitiveness (MINECO) under grant AYA-2017-
88254-P. Luck, R. E. 1994, ApJS, 91, 309 Luck, R. E. 1994, ApJS, 91, 309 p
Majewski, S. R., Schiavon, R. P., Frinchaboy, P. M., et al. 2017, AJ, 154, 94
Martig, M., Rix, H.-W., Aguirre, V. S., et al. 2015, MNRAS, 451, 2230 p
Majewski, S. R., Schiavon, R. P., Frinchaboy, P. M., et al. 2017, AJ, 154, 94
M
i
M Ri
H W A
i
V S
l 2015 MNRAS 451 2230 y
9 GEPI, Observatoire de Paris, CNRS, Université Paris Diderot, 5
Place Jules Janssen, 92190 Meudon, France Martig, M., Rix, H.-W., Aguirre, V. S., et al. 2015, MNRAS, Masseron, T., & Gilmore, G. 2015, MNRAS, 453, 1855 10 Department of Physics and Astronomy, Uppsala University, Box
516, 751 20 Uppsala, Sweden Mellinger, A. 2009, PASP, 121, 1180 Meynet, G., & Maeder, A. 2002, A&A, 390, 561
Š pp
11 Space Science Data Center - Agenzia Spaziale Italiana, via del
Politecnico, s.n.c., 00133 Roma, Italy Mikolaitis, Š., Tautvaišien˙e, G., Gratton, R., Bragaglia, A., & Carretta, E. 2010,
MNRAS, 407, 1866 12 Nicolaus Copernicus Astronomical Center, Polish Academy of Sci-
ences, ul. Bartycka 18, 00-716 Warsaw, Poland Mikolaitis, Š., Tautvaišien˙e, G., Gratton, R., Bragaglia, A., & Carretta, E. 2011a,
MNRAS, 416, 1092
Š ences, ul. Bartycka 18, 00-716 Warsaw, Poland 13 Instituto de Física y Astronomía, Universidad de Valparaíso, Chile
14 Mikolaitis, Š., Tautvaišien˙e, G., Gratton, R., Bragaglia, A., & Carretta, E. 2011b,
MNRAS, 413, 2199
Š 13 Instituto de Física y Astronomía, Universidad de V
14 14 Dipartimento di Fisica e Astronomia, Università di Padova, Vicolo
dell’Osservatorio 3, 35122 Padova, Italy Mikolaitis, Š., Tautvaišien˙e, G., Gratton, R., Bragaglia, A., & Carretta, E. 2012,
A&A, 541, A137 y
15 Departamento de Didáctica, Universidad de Cádiz, 11519 Puerto
Real, Cádiz, Spain Ness, M., Hogg, D. W., Rix, H.-W., et al. 2016, ApJ, 823, 114 Overbeek, J. C., Friel, E. D., Donati, P., et al. 2017, A&A, 598, A 16 Departmento de Astrofísica, Centro de Astrobiología (INTA-CSIC),
ESAC Campus, Camino Bajo del Castillo s/n, 28692 Villanueva de
la Cañada, Madrid, Spain Palacios, A., Charbonnel, C., Talon, S., & Siess, L. 2006, A&A, 453, 261 Palmerini, S., La Cognata, M., Cristallo, S., & Busso, M. 2011, ApJ, 729, 3 arada, J., Richer, H., Heyl, J., Kalirai, J., & Goldsbury, R. 2016, Ap 17 Suffolk University, Madrid Campus, C/ de la Viña 3, 28003 Madrid,
Spain Pasquini, L., Avila, G., Blecha, A., et al. N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars Tautvaišien˙e, G., Edvardsson, B., Puzeras, E., & Ilyin, I. 2005, A&A, 431,
933 De Silva, G. M., Freeman, K. C., Bland-Hawthorn, J., et al. 2015, MNRAS, 449,
2604 Tautvaišien˙e, G., Drazdauskas, A., Mikolaitis, Š., et al. 2015, A&A, 573, A55
Ž Dekker, H., D’Odorico, S., Kaufer, A., Delabre, B., & Kotzlowski, H. 2000, in
Optical and IR Telescope Instrumentation and Detectors, eds. M. Iye, & A. F. Moorwood, Proc. SPIE, 4008, 534 Tautvaišien˙e, G., Drazdauskas, A., Bragaglia, A., Randich, S., & Ženovien˙e, R. 2016, A&A, 595, A16 Denissenkov, P. A. 2010, ApJ, 723, 563 Traxler, A., Garaud, P., & Stellmach, S. 2011, Ap
Ul i h R K 1972 A J 172 165 Traxler, A., Garaud, P., & Stellmach, S. 2011, ApJ, 728, L29 Traxler, A., Garaud, P., & Stellmach, S. 2011, ApJ, 728, L29
Ulrich, R. K. 1972, ApJ, 172, 165 p
Denissenkov, P. A., & Merryfield, W. J. 2011, ApJ, 727, L8 Denissenkov, P. A., & Tout, C. A. 2000, MNRAS, 316, 395 VandenBerg, D. A., Brogaard, K., Leaman, R., & Casagrande, L. 2013, ApJ, 775,
134 Denissenkov, P. A., & Weiss, A. 1996, A&A, 308, 773 Wachlin, F. C., Vauclair, S., & Althaus, L. G. 2014, A&A, 570, A58
Wasserburg, G. J., Boothroyd, A. I., & Sackmann, I. 1995, ApJ, 447, L37
Weiss, A., Wagenhuber, J., & Denissenkov, P. A. 1996, A&A, 313, 581
Yanny, B., Rockosi, C., Newberg, H. J., et al. 2009, AJ, 137, 4377 Denissenkov, P. A., Da Costa, G. S., Norris, J. E., & Weiss, A. 1998, A&A, 333,
926 Denissenkov, P. A., Pinsonneault, M., & MacGregor, K. B. 2009, ApJ, 696, 1823 Dias, W. S., Alessi, B. S., Moitinho, A., & Lépine, J. R. D. 2002, A Donati, P., Bragaglia, A., Cignoni, M., Cocozza, G., & Tosi, M. 2012, MNRAS,
424, 1132 Donati, P., Cantat Gaudin, T., Bragaglia, A., et al. 2014, A&A, 561, A94 1 Institut UTINAM, CNRS UMR6213, Univ. Bourgogne Franche-
Comté, OSU THETA Franche-Comté-Bourgogne, Observatoire de
Besançon, BP 1615, 25010 Besançon Cedex, France
e-mail: nadege.lagarde@utinam.cnrs.fr Drazdauskas, A., Tautvaišiene, G., Randich, S., et al. 2016, A&A, 589, A50 Eggleton, P. P., Dearborn, D. S. P., & Lattanzio, J. C. 2008, ApJ, 677, 581 Fusi Pecci, F., Ferraro, F. R., Crocker, D. A., Rood, R., & Buonanno, D. A. N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars 1990, Fusi Pecci, F., Ferraro, F. R., Crocker, D. A., Rood, R., & Buonanno, D. A. 1990,
A&A, 238, 95 References 2007, ApJ, 671,
802 Cameron, A. G. W., & Fowler, W. A. 1971, ApJ, 164, 111 Cameron, A. G. W., & Fowler, W. A. 1971, ApJ, 164, 111
Cantat-Gaudin, T., Vallenari, A., Zaggia, S., et al. 2014, A&A, 569, A17 p
Cantat-Gaudin, T., Vallenari, A., Zaggia, S., et al. 2014, A&A, 569, A17 Carraro, G., Janes, K. A., Costa, E., & Méndez, R. A. 2006, MNRAS, 368, Carraro, G., Janes, K. A., Costa, E., & Méndez, R. A. 2006, MNRAS, 368, 1078
Carraro, G., Costa, E., & Ahumada, J. A. 2010, AJ, 140, 954 Carraro, G., Costa, E., & Ahumada, J. A. 2010, AJ, 140, 954 Chanamé, J., Pinsonneault, M., & Terndrup, D. M. 2005, ApJ, 631, 540 Chaplin, W. J., & Miglio, A. 2013, ARA&A, 51, 353 Chaplin, W. J., & Miglio, A. 2013, ARA&A, 51, 353 Charbonnel, C. 1994, A&A, 282, 811 Charbonnel, C. 1995, ApJ, 453, L41 Charbonnel, C., & Chantereau, W. 2016, A&A, 586, A21 Charbonnel, C., & Lagarde, N. 2010, A&A, 522, A10 Charbonnel, C., & Zahn, J.-P. 2007, A&A, 467, L15 Christensen-Dalsgaard, J. 2015, MNRAS, 453, 666 Christensen-Dalsgaard, J. 2015, MNRAS, 453, 666 Cignoni, M., Beccari, G., Bragaglia, A., & Tosi, M. 2011, MNRAS, 416, 1077 Cignoni, M., Beccari, G., Bragaglia, A., & Tosi, M. Cui, X.-Q., Zhao, Y.-H., Chu, Y.-Q., et al. 2012, Res. Astron. Astrophy., 12, 1197 ,
Q ,
,
,
,
Q ,
,
p y ,
,
Czekaj, M. A., Robin, A. C., Figueras, F., Luri, X., & Haywood, M. 2014, A&A,
564 A102 Czekaj, M. A., Robin, A. C., Figueras, F., Luri, X., & Haywood, M. 2014, A&A,
564, A102 Czekaj, M. A., Robin, A. C., Figueras, F., Luri, X., & Haywood, M A24, page 10 of 11 A24, page 10 of 11 ,
,
A&A, 238, 95 Gaia Collaboration (Brown, A. G. A., et al.) 2016, A&A, 595, A2 2 Institute of Theoretical Physics and Astronomy, Vilnius University,
Saul˙etekio av. 3, 10257 Vilnius, Lithuania Garaud, P., & Brummell, N. 2015, ApJ, 815, 42 Gilmore, G., Randich, S., Asplund, M., et al. 2012, The Messenger, 147, 25 3 School of Physics and Astronomy, University of Birmingham, Edg-
baston, Birmingham B15 2TT, UK Gilroy, K. K. 1989, ApJ, 347, 835 Gilroy, K. K. 1989, ApJ, 347, 835 Gilroy, K. K., & Brown, J. A. 1991, ApJ, 371, 578 g
4 Institute of Astronomy, University of Cambridge, Madingley Road,
Cambridge CB3 0HA, UK Gratton, R. G., Sneden, C., Carretta, E., & Bragaglia, A. 2000, A&A, 354, 169
Henkel, K., Karakas, A. I., & Lattanzio, J. C. 2017, MNRAS, 469, 4600 ,
,
,
,
,
,
g g
,
,
,
,
Henkel, K., Karakas, A. I., & Lattanzio, J. C. 2017, MNRAS, 469, 4600 Luck, R. E. 1994, ApJS, 91, 309 2002, The Messenger, 110, 1 Piatti, A. E., Clariá, J. J., Bica, E., Geisler, D., & Minniti, D. 1998, AJ, 116, 801 p
18 Núcleo de Astronomía, Facultad de Ingeniería, Universidad Diego
Portales, Av. Ejército 441, Santiago, Chile Prat, V., Lignières, F., & Lagarde, N. 2015, in SF2A-2015: Proceedings of the
Annual meeting of the French Society of Astronomy and Astrophysics, eds. F. Martins, & S. Boissier, V. Buat, L. Cambrésy, & P. Petit, Proc. SPIE, 4008,
534 j
g
19 School of Physics, University of New South Wales, 2052 Sydney,
Australia Randich, S., et al. (Gaia-ESO Consortium) 2013, The Messenger, 154, 47
Robin, A. C., Reylé, C., Derrière, S., & Picaud, S. 2003, A&A, 409, 523 Randich, S., et al. (Gaia-ESO Consortium) 2013, The Messenger, 154, 47
bi
C
lé C
iè
S &
i
d S 2003
&
409 523 20 Instituto de Astrofísica de Canarias, 38205 La Laguna, Tenerife,
Spain Randich, S., et al. (Gaia-ESO Consortium) 2013, The Messenger, 154, 47
Robin, A. C., Reylé, C., Derrière, S., & Picaud, S. 2003, A&A, 409, 523 ,
,
(
)
,
g ,
,
Robin, A. C., Reylé, C., Derrière, S., & Picaud, S. 2003, A&A, 409, 523 ,
,
y ,
,
,
,
,
,
,
,
Rosenblum, E., Garaud, P., Traxler, A., & Stellmach, S. 2011, ApJ, 731, 66
Sackmann I & Boothroyd A I 1999 ApJ 510 217 y
Rosenblum, E., Garaud, P., Traxler, A., & Stellmach, S. 2011, ApJ, 731, 66 Rosenblum, E., Garaud, P., Traxler, A., & Stellmach, S. 2011, ApJ, 731, 66
Sackmann, I., & Boothroyd, A. I. 1999, ApJ, 510, 217 p
21 Universidad de La Laguna, Dept. Astrofísica, 38206 La Laguna,
Tenerife, Spain Rosenblum, E., Garaud, P., Traxler, A., & Stellmach, S. 2011, ApJ, 731, 66
Sackmann, I., & Boothroyd, A. I. 1999, ApJ, 510, 217 Sackmann, I., & Boothroyd, A. I. 1999, ApJ, 510, 217 y
p
Sagar, R., & Griffiths, W. K. 1998, MNRAS, 299, 1 Sagar, R., & Griffiths, W. K. 1998, MNRAS, 299, 1 p
22 Departamento de Ciencias Físicas, Universidad Andres Bello, Fer-
nández Concha 700, Las Condes, Santiago, Chile g
Santos Jr, J. F. C., Bonatto, C., & Bica, E. 2005, A&A, 442, 201 Sengupta, S., & Garaud, P. 2018, ApJ, 862, 136 Iben, Jr, I. 1967, ApJ, 147, 624 Iben, Jr, I. 1967, ApJ, 147, 624 g
5 INAF – Osservatorio Astrofisico di Arcetri, Largo E. Fermi 5, 50125
Florence, Italy p
Iben, Jr, I. 1968, ApJ, 154, 581 p
Krishnamurti, R. 2003, J. Fluid Mech., 483, 287 6 Lund Observatory, Department of Astronomy and Theoretical
Physics, Box 43, 221 00 Lund, Sweden Lagarde, N., Charbonnel, C., Decressin, T., & Hagelberg, J. 2011, A&A, 536,
A28 y
7 INAF – Osservatorio Astronomico di Bologna, via Gobetti 93/3,
40129 Bologna, Italy Lagarde, N., Romano, D., Charbonnel, C., et al. 2012, A&A, 542, A62 g
Lagarde, N., Robin, A. C., Reylé, C., & Nasello, G. 2017, A&A, 601, A27 8 INAF – Osservatorio Astronomico di Palermo, Piazza del Parla-
mento 1, 90134 Palermo, Italy Lattanzio, J. C., Siess, L., Church, R. P., et al. 2015, MN g p
Siess, L. 2009, A&A, 497, 463 Siess, L. 2009, A&A, 497, 463 Siess, L. 2009, A&A, 497, 463 g
23 INAF – Osservatorio Astrofisico di Arcetri, Largo E. Fermi 5, 50125
Florence, Italy
24 Smiljanic, R., Gauderon, R., North, P., et al. 2009, A&A, 502, 267 Smiljanic, R., Korn, A. J., Bergemann, M., et al. 2014, A&A, 570, A122 24 European Southern Observatory, Alonso de Córdova 3107 Vitacura,
Santiago de Chile, Chile Smiljanic, R., Korn, A. J., Bergemann, M., et al. 2014, A&A, 570, A122
Steinmetz, M., Zwitter, T., Siebert, A., et al. 2006, AJ, 132, 1645
Stonkut˙e E Koposov S E Howes L M et al 2016 MNRAS 460 1131 j
,
,
,
,
g
,
,
,
,
,
Steinmetz, M., Zwitter, T., Siebert, A., et al. 2006, AJ, 132, 1645 teinmetz, M., Zwitter, T., Siebert, A., et al. 2006, AJ, 132, 1645 Stonkut˙e, E., Koposov, S. E., Howes, L. M., et al. 2016, MNRAS, 460, 1131
Sweigart, A. V., & Mengel, J. G. 1979, ApJ, 229, 624 25 Instituto de Astrofísica e Ciências do Espaço, Universidade do
Porto, CAUP, Rua das Estrelas, 4150-762 Porto, Portugal
6 Sweigart, A. V., & Mengel, J. G. 1979, ApJ, 229, 624 g
g
p
Tang, B., Geisler, D., Friel, E., et al. 2017, A&A, 601, A56 Tang, B., Geisler, D., Friel, E., et al. 2017, A&A, 601, A56 26 INAF – Padova Observatory, Vicolo dell’Osservatorio 5, 35122
Padova, Italy Tautvaišien˙e, G., Edvardsson, B., Tuominen, I., & Ilyin, I. 2000, A&A, 360, 499
Tautvaišien˙e G Edvardsson B Tuominen I & Ilyin I 2001 A&A 380 578 Tautvaišien˙e, G., Edvardsson, B., Tuominen, I., & Ilyin, I. 2000, A&A, 360, 499 Tautvaišien˙e, G., Edvardsson, B., Tuominen, I., & Ilyin, I. 2001, A Tautvaišien˙e, G., Edvardsson, B., Tuominen, I., & Ilyin, I. 2001, A&A, 380, 578 A24, page 11 of 11
|
https://openalex.org/W2134059007
|
https://jneuroengrehab.biomedcentral.com/counter/pdf/10.1186/1743-0003-10-15
|
English
| null |
The effects of virtual reality game training on trunk to pelvis coupling in a child with cerebral palsy
|
Journal of neuroengineering and rehabilitation
| 2,013
|
cc-by
| 4,692
|
Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15
http://www.jneuroengrehab.com/content/10/1/15 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15
http://www.jneuroengrehab.com/content/10/1/15 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15
http://www.jneuroengrehab.com/content/10/1/15 JNER
JOURNAL OF NEUROENGINEERING
AND REHABILITATION RESEARCH Open Access © 2013 Barton 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: Good control of trunk and pelvic movements is necessary for well controlled leg movements
required to perform activities of daily living. The nature of movement coupling between the trunk and pelvis varies
and depends on the type of activity. Children with cerebral palsy often have reduced ability to modulate coupling
between the trunk and pelvis but movement patterns of the pelvis can be improved by training. The aim of this
study was to examine how pelvis to trunk coupling changed while playing a computer game driven by pelvic
rotations. Methods: One boy with cerebral palsy diplegia played the Goblin Post Office game on the CAREN virtual
rehabilitation system for six weeks. He navigated a flying dragon in a virtual cave towards randomly appearing
targets by rotating the pelvis around a vertical axis. Motion of the pelvis and trunk was captured in real-time by a
Vicon 612 optoelectronic system tracking two clusters of three markers attached to the sacrum and thoracic spine. Results: Convex hull areas calculated from angle-angle plots of pelvic and trunk rotations showed that coupling
increased over game training (F1,11 = 7.482, p = 0.019). Reaching to targets far from the midline required tighter
coupling than reaching near targets (F1,12 = 10.619, p = 0.007). Conclusions: Increasing coupling appears to be an initial compensation mechanism using the better controlled
trunk to drive rotation of the pelvis. Co-contractions causing increased coupling are expected to reduce over longer
exposure to training. The control scheme of the training game can be set to facilitate de-coupling of pelvic
movements from the trunk. Using large ranges of pelvic rotation required more coupling suggesting that training
of selective pelvic movements is likely to be more effective close to a neutral pelvic posture. Keywords: Virtual rehabilitation, Cerebral palsy, Core control, Pelvis and trunk coupling The effects of virtual reality game training on
trunk to pelvis coupling in a child with
cerebral palsy Gabor J Barton1*, Malcolm B Hawken1, Richard J Foster1, Gill Holmes2 and Penny B Butler3 * Correspondence: g.j.barton@ljmu.ac.uk
1Research Institute for Sport and Exercise Sciences, Liverpool John Moores
University, Byrom Street, Liverpool L3 3AF, UK
Full list of author information is available at the end of the article Background [6,7], reduced strength and impaired proprioception
around the core (pelvis and trunk) were found to be
associated with increased risk of injuries, specifically
around the knee. This suggests that good control of the
movement of the core is a prerequisite for well con-
trolled use of the legs. There is evidence to support the hypothesis that the pel-
vis and trunk play an active role in gait, as opposed to
the traditional view [1] which regards the core of the
body as a “passenger unit” carried by the lower extrem-
ities, which are termed the “locomotor unit”. Responses
triggered by balance perturbations involve neck, trunk
and thigh muscles which contract simultaneously, even
before the activation of muscles around the ankle joint
in unimpaired subjects [2-5]. In prospective studies Good control of the interaction between the trunk and
pelvis is required to carry out activities of daily living. During level walking the nature of this interaction
depends on walking speed [8-12]. At slow speeds the
pelvis and trunk are coupled in the transverse plane and
move in-phase, but as walking speed increases there is a
transition from in-phase to anti-phase coupling [9,10]. Retraction of the trunk, together with protraction of the * Correspondence: g.j.barton@ljmu.ac.uk
1Research Institute for Sport and Exercise Sciences, Liverpool John Moores
University, Byrom Street, Liverpool L3 3AF, UK
Full list of author information is available at the end of the article Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15
http://www.jneuroengrehab.com/content/10/1/15 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15
http://www.jneuroengrehab.com/content/10/1/15 Page 2 of 6 Page 2 of 6 pelvis on the swing leg side, increases step length and
therefore improves the efficiency of gait. Ethical approval was obtained from the NHS National Re-
search Ethics Service (07/H1014/83) and local ethics com-
mittees (Royal Liverpool Children’s NHS Trust and
Liverpool John Moores University), confirming that the
research carried out was in compliance with the Helsinki
Declaration. The subject’s parent provided written consent
for the study. Ethical approval was obtained from the NHS National Re-
search Ethics Service (07/H1014/83) and local ethics com-
mittees (Royal Liverpool Children’s NHS Trust and
Liverpool John Moores University), confirming that the
research carried out was in compliance with the Helsinki
Declaration. The subject’s parent provided written consent
for the study. One of the primary problems in cerebral palsy is
reduced selective control of movement [13]. Background Altered se-
lective motor control of the pelvis and trunk hinders
efficient gait and thereby activities of daily living. Improved ability to reduce in-phase coupling between
the trunk and pelvis is expected to improve gait at nor-
mal walking speeds characterised by anti-phase coupling. Activities of daily living are also expected to benefit from
the reduced in-phase coupling. The subject’s task in the game was to use the horn on
a dragon’s head to burst balloons containing letters. After one balloon target was either hit or missed, the
next target appeared at some distance in front of the
dragon in one of four unpredictable locations (Figure 1). Forward speed of the dragon through the virtual cave
was controlled by the game software, the subject con-
trolled left- and rightward movement (speed) of the
dragon by rotating the pelvis about a vertical axis in a
high kneeling posture. This posture was used to simplify
control requirements by eliminating the complexity of
ankle motion. High kneeling ensured that the pelvis and
trunk remained in the same relative posture as standing,
thus giving a more realistic training opportunity. Motion
of the dragon and location of the targets were confined
to a single horizontal plane. Rotations of the pelvis and
trunk about a vertical axis were captured by tracking the
3D orientation of two triangular clusters of three retro- Due to their central location in the body’s linked chain
of segments the trunk and pelvis are mechanically
restricted to a greater degree than the extremities and
the head. In spite of this constraint, movement of the
trunk and pelvis can be altered through training [14,15]. There are numerous reports of successfully using cus-
tom made computer games to both test and train move-
ment control of the upper or lower extremities [16-19]. Applications of serious gaming focused on the pelvis
and trunk, however, are scarce. Our Goblin Post Office
(GPO) training game [20-25] is derived from the con-
ceptual framework of Targeted Training [26] in that it
allows sequential training of body segments from upper
to lower, thereby increasing successively the complexity
of the motor task. We report here some findings (from a
larger training study) in which we observed changes in
coupling between the pelvis and trunk when the game
was controlled by movement of the pelvis. Figure 1 The Goblin Post Office game. Background The dragon flies forwards
through the cave at a speed set by the game software, and
individual targets appear at some distance ahead unpredictably in
one of the four horizontal locations shown. The subject directs the
dragon towards the target by left- and rightward pelvic rotation,
registered by a triangular array of reflective markers. A second set of
markers registers trunk motion, enabling evaluation of trunk to
pelvis coupling. Methods
b One boy with cerebral palsy spastic diplegia (age:
10 years; height: 1.34 m; mass: 36 kg) trained for 6 weeks,
twice a week for 30 minutes (13 sessions in total) on our
custom-made GPO computer game, developed in the
CAREN
system
(Motek
Medical,
Amsterdam,
The
Netherlands). The child had no history of surgical inter-
vention, and was receiving no conventional physiotherapy
or core specific rehabilitation which might have influenced
his responses to game training. His Gross Motor Function
Classification System (GMFCS [27]) score of 1 indicates
that he walks indoors and outdoors without limitations
and can run and jump but speed, balance and co-
ordination may be reduced. He attends mainstream edu-
cation and does not have learning difficulties. On physical
examination he had a 10° equinus contracture in the right
ankle and bilaterally tight hamstrings (popliteal angles of
60°). The hips were offset into internal rotation bilaterally
together with external tibial torsion. Muscle strength was
reduced in right dorsi/plantarflexors, right hip extensors
and bilaterally in hip abductors and foot in/evertors. There
were positive signs of spasticity bilaterally in the plantar-
flexors (Tardieu 5 on right and 2 on left side), hamstrings
(Tardieu 2) and quadriceps (positive Duncan Ely signs). Figure 1 The Goblin Post Office game. The dragon flies forwards
through the cave at a speed set by the game software, and
individual targets appear at some distance ahead unpredictably in
one of the four horizontal locations shown. The subject directs the
dragon towards the target by left- and rightward pelvic rotation,
registered by a triangular array of reflective markers. A second set of
markers registers trunk motion, enabling evaluation of trunk to
pelvis coupling. Page 3 of 6 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15
http://www.jneuroengrehab.com/content/10/1/15 reduced Areas of the convex hull indicating a more syn-
chronised rotation of the trunk and pelvis (Figure 3). reflective markers placed over the thoracic spine and on
the sacrum. Neither rotation of the pelvis about the left/
right axis (tilt), nor trunk rotations about either axis,
had any effect on the motion of the dragon. At the be-
ginning of the first training session the control scheme
of the game was explained to the participant and he
played the training level of the game which responded to
rotations of the pelvis but the dragon was not moving
forwards. Methods
b Values of Area were not normally distributed, but a
natural
log
transform
corrected
the
distribution
(logArea). The mean, maximum, minimum and standard
deviation of logArea over all targets (trials) were calcu-
lated for each Session. Regression of mean logArea
against Session showed that mean logArea reduced sig-
nificantly over the training sessions (F1,11 = 7.482, p = To quantify coupling between the trunk and pelvis,
angle-angle plots of trunk and pelvic rotation, normal-
ised to their respective ranges of motion over all ses-
sions, were generated for sections of gameplay between
consecutive targets. The area of the convex hull contain-
ing all data points of the angle-angle plot was calculated
using the CONVHULL function in MATLAB (The
Mathworks, Natick MA, USA). This area was used to
quantify the level of coupling between the trunk and pel-
vis (Figure 2). Low values of Area indicate tight in-phase
or anti-phase coupling and high values indicate a lack of
association between rotation of the trunk and of the
pelvis. -1
-0.5
0
0.5
1
-1
-0.5
0
0.5
1
Left Pelvic rotation
Right
Left Trunk rotation
Right
First assessment
-1
-0.5
0
0.5
1
-1
-0.5
0
0.5
1
Left Pelvic rotation
Right
Left Trunk rotation
Right
Last assessment
a
b
Figure 3 Examples of angle-angle plots (black line) and convex
hulls (bold grey line) representing coupling between the trunk
and pelvis a) in the first assessment and b) in the last (13th)
assessment of the six weeks training. -1
-0.5
0
0.5
1
-1
-0.5
0
0.5
1
Left Pelvic rotation
Right
Left Trunk rotation
Right
First assessment
a a Discussion Over the period of six weeks while targeting movement
training to the pelvis in a patient with cerebral palsy, a
gradual development of increased coupling of the pelvis
to the trunk was found. This strategy improved perform-
ance during gameplay which we have reported elsewhere
[22] but improved selective movement control of pelvic
rotation was not achieved. Previous findings indicate
better movement control of the trunk in comparison to
the pelvis in children with cerebral palsy during com-
puter based game-play [25]. The tighter coupling of the
pelvis to the trunk appears to be a compensatory mech-
anism which enables the child to improve control of the
pelvis indirectly by locking it to the better controlled
trunk. Development of strategies compensating for impaired
movement can be prevented by constraint induced
movement therapy which physically inhibits the com-
pensation thereby forcing use of the targeted movement
[29]. Such a constraint preventing the use of trunk rota-
tion while driving the GPO game with pelvic rotation
can be provided by the game software and may be a
means to facilitate use of the pelvis independent of the
trunk. The GPO game can be set to either give a visual
warning in case of trunk rotation or to be driven by the
difference between pelvic and trunk rotation thereby
preventing coupled motion. Initial tests however showed
that typically developing children found it more difficult
to play the game with such control schemes so they were
not examined in the present study. Co-contraction, that is simultaneous activation of
agonist and antagonist muscles, is a common motor
control strategy used to improve stability and accuracy -2.8
-2.4
-2.0
-1.6
-1.2
-0.8
-0.4
0.0
1
2
3
4
5
6
7
8
9 10 11 12 13
logArea
Session
mean+2SD
mean
mean-2SD
Figure 4 The reducing means of convex hull areas indicate
increased coupling between the trunk and pelvis over the 13
game sessions. An increased level of coupling was found when using
a large range of pelvic rotation needed to reach targets
far from the midline. The child was able to move the
pelvis with less in-phase coupling when small ranges
were required (indicated by higher logArea) but pelvic
movement was aided by in-phase coupled trunk rotation
when reaching targets further away from the midline
(confirmed by lower logArea). Results
A
l Angle-angle plots from the first session showed a low
level of in-phase coupling between the pelvis and trunk,
indicated by higher Areas of the convex hull. In the final
(13th session) at the end of the sixth week the angle-
angle
plots showed tighter
in-phase
coupling
with Trunk rotation
Right -1
-0.5
0
0.5
1
-1
-0.5
0
0.5
1
Left Pelvic rotation
Right
L ft
Trunk rotation
Ri ht
Last assessment
b -1
-0.5
0
0.5
1
-1
-0.5
0
0.5
1
Left
Pelvic rotation
Right
Left
Trunk rotation
Right
Figure 2 Angle-angle plot (black line) visualising the
interaction between rotation of the trunk and pelvis. The area
of the convex hull surrounding the angle-angle plot (bold grey line)
was used to quantify coupling. Four quadrants of the chart
represent the possible combinations of trunk and pelvic rotation. -1
-0.5
0
0.5
1
-1
-0.5
0
0.5
1
Left
Pelvic rotation
Right
Left
Trunk rotation
Right Last assessment Left Trunk rotation
Right Left Figure 2 Angle-angle plot (black line) visualising the
interaction between rotation of the trunk and pelvis. The area
of the convex hull surrounding the angle-angle plot (bold grey line)
was used to quantify coupling. Four quadrants of the chart
represent the possible combinations of trunk and pelvic rotation. Figure 2 Angle-angle plot (black line) visualising the
interaction between rotation of the trunk and pelvis. The area
of the convex hull surrounding the angle-angle plot (bold grey line)
was used to quantify coupling. Four quadrants of the chart
represent the possible combinations of trunk and pelvic rotation. Figure 3 Examples of angle-angle plots (black line) and convex
hulls (bold grey line) representing coupling between the trunk
and pelvis a) in the first assessment and b) in the last (13th)
assessment of the six weeks training. Figure 3 Examples of angle-angle plots (black line) and convex
hulls (bold grey line) representing coupling between the trunk
and pelvis a) in the first assessment and b) in the last (13th)
assessment of the six weeks training. Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15
http://www.jneuroengrehab.com/content/10/1/15 Barton et al. Results
A
l Journal of NeuroEngineering and Rehabilitation 2013, 10:15
http://www.jneuroengrehab.com/content/10/1/15 Page 4 of 6 -1.8
-1.5
-1.2
-0.9
-0.6
1
2
3
4
5
6
7
8
9 10 11 12 13
logArea
Session
Near
Far
Figure 5 The convex hull areas indicate increased coupling
(smaller logArea) between the trunk and pelvis when
navigating to Far targets as compared to Near targets. 0.019), Figure 4. This, however, concealed more complex
behaviour. The maximum logArea did not change over
the training sessions, but the mean and minimum
logArea reduced significantly as training progressed. As
the maximum and minimum values are not unbiased
measures of data spread values of Mean + 2 × STD and
Mean - 2 × STD of logArea were used instead in regres-
sions against Session. The difference in slopes seen with
maximum and minimum values was again present
(Figure
4:
regressions:
Mean + 2 × STD
F1,11 = 0.539,
p = 0.478, Mean - 2 × STD F1,11 = 15.526, p = 0.002). Due to their spatial distribution, reaching half of the
balloon targets required less pelvic rotation (Near tar-
gets) than reaching the other half (Far targets), Figure 1. Separating the means of logArea for Near and Far tar-
gets for all the Sessions, a paired t-test showed that
trunk to pelvis in-phase coupling is stronger for Far tar-
gets
than
for
Near
targets
(Figure
5),
t12 = 3.259,
p = 0.007. Figure 5 The convex hull areas indicate increased coupling
(smaller logArea) between the trunk and pelvis when
navigating to Far targets as compared to Near targets. when performing an unpractised task [28]. Levels of co-
contraction decrease over time with practice. Playing the
training game twice a week for 30 minutes over 6 weeks
appears to have resulted in the interlocking of the trunk
and pelvis, representing the initial response to the new
game task. Intensifying exposure to the game by means
of increased frequency or duration of play, or both, may
lead to reduced coupling between the trunk and pelvis
as selective control improves. Conclusions
d 8. Bruijn S, Lamoth C, Kingma I, Meijer O, van Dieën J: Coordination between
pelvis, thorax and leg movements in gait. Gait Posture 2006,
24(S2):S10–S11. Our case study of a child with cerebral palsy diplegia
demonstrated that control was translated from the trunk
to the pelvis through tighter coupling over six weeks
training consisting of thirteen game sessions. Increased
exposure to the game is expected to lead to reduction of
coupling due to reduced co-contraction in muscles link-
ing the trunk and pelvis as selective control improves. The Goblin Post Office game has means to facilitate se-
lective control of the pelvis and this might reduce the
time needed to achieve improved selective movements. Pelvic rotation is helped more by trunk rotation at the
ends of the pelvic rotation range due to mechanical con-
straints and so training of selective pelvic control is
likely to be more productive near to the neutral
orientation. 9. Bruijn SM, Meijer OG, van Dieën JH, Kingma I, Lamoth CJC: Coordination of
leg swing, thorax rotations, and pelvis rotations during gait: The
organisation of total body angular momentum. Gait Posture 2008,
27:455–462. 10. Lamoth CJC, Beek PJ, Meijer OG: Pelvis-thorax coordination in the
transverse plane during gait. Gait Posture 2002, 16:101–114. 11. Lamoth CJC, Daffertshofer A, Meijer OG, Beek PJ: How do persons with
chronic low back pain speed up and slow down? Trunk-pelvis
coordination and lumbar erector spinae activity during gait. Gait Posture
2006, 23:230–239. 12. Lamoth CJC, Meijer OG, Daffertshofer A, Wuisman PIJM, Beek PJ: Effects of
chronic low back pain on trunk coordination and back muscle activity
during walking: changes in motor control. Eur Spine 2006, 15:23–40. 13. Gage JR, Novacheck TF: An update on the treatment of gait problems in
cerebral palsy. J Pediatr Orthop B 2001, 10:265–274. 14. Aruin AS: Knee position feedback: Its effect on management of pelvic
instability in a stroke patient. Disabil Rehabil 2000, 22:690–692. 15. Janssen LJF, Verhoeff LL, Horlings CGC, Allum JHJ: Directional effects of
biofeedback on trunk sway during gait tasks in healthy young subjects. Gait Posture 2009, 29:575–581. Received: 16 March 2012 Accepted: 5 February 2013
Published: 7 February 2013 23. Foster RJ, Hawken MB, Barton GJ: Movement co-ordination of the pelvis in
a virtual game environment. Gait Posture 2008, 28S:S10–S11. 24. Barton GJ: Virtual rehabilitation in cerebral palsy. J Sports Sci 2009,
27S1:S3. Discussion Over the duration of six
weeks training the level of coupling increased but the
difference of coupling over small and large ranges of pel-
vic rotations remained (Figure 5). Figure 4 The reducing means of convex hull areas indicate
increased coupling between the trunk and pelvis over the 13
game sessions. Functional measures of the patient showed mixed
results when comparing performance before and after
game training. On one hand the Segmental Assessment Page 5 of 6 Page 5 of 6 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15
http://www.jneuroengrehab.com/content/10/1/15 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15
http://www.jneuroengrehab.com/content/10/1/15 of Trunk Control scores increased (SATCo Static:2 →4
Active:1 →1 Reactive:NA →2 [30]), the child’s parents
commented on improved sitting posture and the swim-
ming coach reported “improvements” in swimming. On
the other hand an objective evaluation of the patient’s
deviation from normality using the Gait Deviation Index
[31] showed negligible changes in gait (75.6 →75.0). 4. Allum JH, Honegger F, Acuña H: Differential control of leg and trunk
muscle activity by vestibulo-spinal and proprioceptive signals during
human balance corrections. Acta Otolaryngol 1995, 115:124–129. 5. Forssberg H, Hirschfeld H: Postural adjustments in sitting humans
following external perturbations: muscle activity and kinematics. Exp Brain Res 1994, 97:515–527. 6. Leetun DT, Ireland ML, Willson JD, Ballantyne BT, Davis IM: Core stability
measures as risk factors for lower extremity injury in athletes. Med Sci
Sports Exerc 2004, 36:926–934. 7. Zazulak BT, Hewett TE, Reeves NP, Goldberg B, Cholewicki J: The effects of
core proprioception on knee injury: a prospective biomechanical-
epidemiological study. Am J Sports Med 2007, 35:368–373. Authors’ contribution
GJB
i
d h
d GJB conceived the study and drafted the manuscript. MBH participated in
the design of the study and performed the statistical analysis. RJF carried out
the data collection and analysis and helped to draft the manuscript. GJB,
MBH and RJF developed the Goblin Post Office game with contribution from
GH and PBB on the conceptual and practical aspects of targeted training. All
authors read and approved the final manuscript. 18. Holden MK: Virtual environments for motor rehabilitation: review. Cyberpsychol Behav 2005, 8:187–211. discussion 212–189. 19. You SH, Jang SH, Kim YH, Kwon YH, Barrow I, Hallett M: Cortical
reorganization induced by virtual reality therapy in a child with
hemiparetic cerebral palsy. Dev Med Child Neurol 2005, 47(9):628–635. 20. Hawkins PJR, Hawken MB, Barton GJ: Effect of game speed and
surface perturbations on postural control in a virtual environment,
Proceedings of the 7th ICDVRAT with ArtAbilitation: 8–11 September
2008; Maia & Porto. Reading, United Kingdom: ICDVRAT; http://www. icdvrat.reading.ac.uk/2008/index2008.htm. Competing interests
Th f
h
h The first two authors have received financial support from Motek Medical
(Amsterdam, The Netherlands) to attend user group meetings and scientific
conferences. Their host university would receive a proportion of the income
Motek Medical generates by sale of the Goblin Post Office game. 16. Bryanton C, Bosse J, Brien M, McLean J, McCormick A, Sveistrup H:
Feasibility, motivation, and selective motor control: Virtual reality
compared to conventional home exercise in children with cerebral
palsy. Cyberpsychol Behav 2006, 9:123–128. 17. Deutsch JE, Latonio J, Burdea GC, Boian R: Post-stroke rehabilitation with
the Rutgers ankle system: A case study. Presence-Teleop Virt 2001,
10:416–430. Acknowledgements This project was funded by The WellChild Trust and Alder Hey Children’s
NHS Foundation Trust. Author details
1R
h I
i 21. Barton GJ: Virtual rehabilitation – a focus on movement function. Biomech Hung 2009, 2/2:7–14. 1Research Institute for Sport and Exercise Sciences, Liverpool John Moores
University, Byrom Street, Liverpool L3 3AF, UK. 2North West Movement
Analysis Centre, Alder Hey Children’s NHS Foundation Trust, Eaton Road,
Liverpool L12 2AP, UK. 3The Movement Centre, Robert Jones and Agnes
Hunt Hospital, Oswestry SY10 7AG, UK. 22. Barton GJ, Hawken MB, Foster RJ, Holmes G, Butler PB: Playing the
goblin post office game improves movement control of the core: a
case study, Proceedings of the international conference on virtual
rehabilitation; 27–29 June 2011; Zurich. http://dx.doi.org/10.1109/
ICVR.2011.5971811. Received: 16 March 2012 Accepted: 5 February 2013
Published: 7 February 2013 References 25. Barton GJ, Hawken MB, Butler P, Holmes G, Foster RJ: Movement control of
the trunk and pelvis in cerebral palsy diplegia. Gait Posture 2009,
30(S1):147–148. 1. Perry J: Gait analysis: normal and pathological function. Thorofare, NJ: SLACK-
Inc.; 1992. 2. Allum JH, Bloem BR, Carpenter MG, Hulliger M, Hadders-Algra M:
Proprioceptive control of posture: a review of new concepts. Gait Posture
1998, 8:214–242. 26. Butler PB, Major RE: The learning of motor control. Biomechanical
considerations. Physiother 1992, 78:1–6. 3. Keshner EA, Woollacott MH, Debu B: Neck, trunk and limb muscle
responses during postural perturbations in humans. Exp Brain Res 1988,
71:455–466. 27. Palisano R, Rosenbaum P, Walter S, Russell D, Wood E, Galuppi B:
Development and reliability of a system to classify gross motor function
in children with cerebral palsy. Dev Med Child Neurol 1997, 39(4):214–223. Page 6 of 6 Page 6 of 6 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15
http://www.jneuroengrehab.com/content/10/1/15 28. Gribble PL, Mullin LI, Cothros N, Mattar A: Role of cocontraction in arm
movement accuracy. J Neurophysiol 2003, 89:2396–2405. 29. Gordon AM: To constrain or not to constrain, and other stories of
intensive upper extremity training for children with unilateral cerebral
palsy. Dev Med Child Neurol 2001, 53:56–61. 30. Butler P, Saavedra S, Sofranac M, Jarvis S, Woollacott M: Refinement,
reliability and validity of the segmental assessment of trunk control
(SATCo). Pediatr Phys Ther 2010, 22(3):246–257. 31. Schwartz MH, Rozumalski A: The gait deviation index: a new
comprehensive index of gait pathology. Gait Posture 2008, 28(3):351–357. doi:10.1186/1743-0003-10-15
Cite this article as: Barton et al.: The effects of virtual reality game
training on trunk to pelvis coupling in a child with cerebral palsy. Journal of NeuroEngineering and Rehabilitation 2013 10:15. 28. Gribble PL, Mullin LI, Cothros N, Mattar A: Role of cocontraction in arm
movement accuracy. J Neurophysiol 2003, 89:2396–2405. 29. Gordon AM: To constrain or not to constrain, and other stories of
intensive upper extremity training for children with unilateral cerebral
palsy. Dev Med Child Neurol 2001, 53:56–61. 30. Butler P, Saavedra S, Sofranac M, Jarvis S, Woollacott M: Refinement,
reliability and validity of the segmental assessment of trunk control
(SATCo). Pediatr Phys Ther 2010, 22(3):246–257. 31. Schwartz MH, Rozumalski A: The gait deviation index: a new
comprehensive index of gait pathology. Gait Posture 2008, 28(3):351–357. References doi:10.1186/1743-0003-10-15
Cite this article as: Barton et al.: The effects of virtual reality game
training on trunk to pelvis coupling in a child with cerebral palsy. Journal of NeuroEngineering and Rehabilitation 2013 10:15. doi:10.1186/1743-0003-10-15
Cite this article as: Barton et al.: The effects of virtual reality game
training on trunk to pelvis coupling in a child with cerebral palsy. Journal of NeuroEngineering and Rehabilitation 2013 10:15. 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:
|
https://openalex.org/W4231471960
|
https://www.qeios.com/read/FFHNHO/pdf
|
English
| null |
Adenocarcinoma of the anal canal
|
Definitions
| 2,020
|
cc-by
| 115
|
Qeios · Definition, February 10, 2020 Open Peer Review on Qeios Adenocarcinoma of the anal canal INSERM Qeios ID: FFHNHO · https://doi.org/10.32388/FFHNHO Source INSERM. (1999). Orphanet: an online rare disease and orphan drug data base. Adenocarcinoma of the anal canal. ORPHA:424016 Adenocarcinoma of the anal canal is a very rare tumor of the intestine, originating from
the epithelium of the anal canal (including the mucosal surface, anal glands, and lining of
fistulous tracts), macroscopically appearing as a nodular, often ulcerated, invasive mass
located in the anal canal. Patients often present with rectal bleeding, as well as difficulty
and pain during defecation. Inguinal lymphadenopathy, if present, usually indicates
metastatic spread. Qeios ID: FFHNHO · https://doi.org/10.32388/FFHNHO 1/1
|
https://openalex.org/W4391548062
|
https://www.jenrs.com/?smd_process_download=1&download_id=3938
|
English
| null |
Baggage Cart with Weighing Mechanism for Hotels and Airlines
|
Journal of engineering research and sciences
| 2,024
|
cc-by-sa
| 2,417
|
Baggage Cart with Weighing Mechanism for Hotels and
Airlines Vishal Verma*,1
, Kuldeep Kumar2
, Rashmi Aggarwal1
, Tanvi Verma1
1 Chitkara College of Hospitality Management, Chitkara University, Rajpura, 140401, Punjab India
2 Chitkara Business School, Chitkara University, Rajpura, 140401, Punjab India
*Corresponding author: Vishal Verma, vishal.verma@chitkara.edu.in, +91 9041078993 ABSTRACT: In this article, it has been proposed a functional design of Baggage Cart with Weighing
Mechanism for Hospitality Industry based on empirical observations. This design is expected to
promote and become one of the high demand products which can later be used especially by hotels
and respective airlines. It has been often observed that travelers are always concerned about the
maximum weight of their baggage allowed in order to board a flight. Because as per norms of airlines
one needs to carry a specific amount of baggage in the flight, in case of extra weighed baggage carried
by a guest, he/she is charged extra amount as per KG by the respective airline. Keeping this point in
mind, travelers are always conscious about the weight of their baggage before boarding a flight to
avoid the last-minute hassle of paying huge extra money, and sometimes they need to drop the
necessary items out from the baggage to adjust the weight of the baggage. In hotels, a guest request to
weigh his/ her luggage is dealt with in the following ways: The front office associate/porter first needs
to fetch the baggage from the guest room and bring it up to the bell desk or need to drag it up till time
office / receiving area where it is weighed on a heavy-duty weighing scale. It is the traditional way of
measuring baggage. The traditional way is time-consuming, uncomfortable, and less suitable to
support the need of the traveler. Hence, to minimize the time and efforts, we wanted to invent a
mechanism that will help and reduce the burden of managing the separate weighing machine, and
therefore we came up with a baggage cart with a weighing mechanism in it. KEYWORDS: Baggage Cart, Weighing Mechanism, Customer Satisfaction, Service Quality weighing his baggage. Moreover, Airports’ key customers
are passengers and their initial impressions of airport
facilities and services can significantly impact their
perceptions and evaluations of the airport. Therefore, it is
vital for the airports to provide safe, comfortable and
convenient services in a cost-effective manner [2]. Transactions involving goods and services are not the only
aspects of business. Baggage Cart with Weighing Mechanism for Hotels and
Airlines Its capacity to last is significantly
reliant on a variety of humanitarian aspects related to the
management and upkeep of commercial relationships
with clients, suppliers, and staff, particularly when
conducting business internationally [3]. Keeping these in
mind, Quality of Service as apparent by customers is a
comparison amongst performance and expectations [4]. As per [5], The overall observation of service quality is
determined by the modification between customers’
expectations and their actual experiences. When a Special Issue on Computing, Engineering and Sciences Received: 04 October, 2023, Revised: 06 January, 2024, Accepted: 06 January, 2024, Online: 30 January, 2024 Received: 04 October, 2023, Revised: 06 January, 2024, Accepted: 06 January, 2024, Online: 30 January, 2024 Received: 04 October, 2023, Revised: 06 January, 2024, Accepted: 06 January, 2024, Online: 30 January, 2024 DOI: https://doi.org/10.55708/js0301002 1. Introduction Passenger Satisfaction is a crucial metric in order to
evaluate airport performance. International airports in
various regions or countries usually do not compete with
each other. Passengers generally do not have the choice to
choose between airports, irrespective of the quality and
price of airport services. However, Passenger’s demand
for airport services is likely to be fairly inflexible [1]. To
minimize time, effort, and ease out the task, the design
provides a mechanism to weigh the baggage as per the
guest’s request. While working in the hotel industry, it
was a routine task to deal with guests and their needs and
out of all to weigh their baggage was one of them. One
genuine problem which is paid less attention and
provided with an ordinary, less effective, or non-
hospitable solution is the handling of a traveler’s need of Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 6 V. Verma et al., Baggage Cart with Weighing Mechanism customer’s experience matches their expectation, service
quality is perceived as upright. In other words, Quality of
the service is measured by comparing customer
expectations with their experiences and this comparison is
based on the performance of service delivery. The goal of
providing better service to clients is to meet their needs in
addition to making sure they are completely satisfied with
the service they are utilizing [6]. In the hospitality
industry, it is a routine task to weigh the baggage of
guests. The proposed design provides a mechanism to
weigh the baggage as per the guest's request with minimal
time, effort. Present solutions are not convenient and need
the efforts of the porter to fetch the baggage to and fro
between the guests' rooms and hotel utilities which is a
time-consuming process. weighing scale kept in their bathroom. However, this
system is not suitable as it is meant for weighing human
body weight. In case one holds a bag and stands on the
machine, it gives total weight which is not an appropriate
method. it’s not even feasible to keep a large piece of
baggage on the surface of a weighing scale. 2. Technical Background Figure 2: Traditional way of Measuring Baggage through a weighing
device with a lifting hook (top view) At present, the hospitality industry is using the
following mechanisms to weigh the baggage. Based on
empirical observations and as shown in Figure 1 below,
Earlier In a traditional way, a porter needs to fetch the bag
from the guest room which is taken to the time office /
receiving area where it is weighed on a heavy-duty
weighing scale. The moment it’s being measured, it is
properly tagged and delivered to the respective guest
room. The process needs to be executed immediately on
the guest’s demand as the guest has to check out and take
a flight or train. Even a reasonable delay in the process
upsets the guests and creates a bad impression of the hotel. Figure 3: Traditional way of Measuring Baggage through a weighing
device with a lifting hook (front view) Figure 3: Traditional way of Measuring Baggage through a weighing
device with a lifting hook (front view) Figure 1: Traditional way of Measuring Baggage through the help of
Porter Figure 4: Traditional way of Measuring Baggage through the weighing
scale kept in their bathroom Figure 4: Traditional way of Measuring Baggage through the weighing
scale kept in their bathroom Figure 1: Traditional way of Measuring Baggage through the help of
Porter 1. Introduction Figure 2: Traditional way of Measuring Baggage through a weighing
device with a lifting hook (top view)
Figure 3: Traditional way of Measuring Baggage through a weighing
device with a lifting hook (front view) Figure 2: Traditional way of Measuring Baggage through a weighing
device with a lifting hook (top view) 3. Related Work In another mechanism, as per empirical observations
and as shown in Figures 2 & 3 below, some hotels use a
weighing device with a lifting hook. The porter has to hold
the device and lift the bag hooked to the device to weigh
it. Sometimes the process may result in a muscular sprain
in the porter’s hand or back or lead to dropping the bag,
which could again lead to a bad impression of the hotel. 4. Hardware Implementation As per empirical observations, the surface of the
baggage cart has a mechanism to measure the weight. Upon placing a bag on the cart's surface, it will display the
weight on the digital display mounted on the cart. The
baggage cart will appear like a regular baggage cart when
the digital meter is turned off. KULDEEP KUMAR has a decade of experience in
the hospitality industry, working with top brands
such as Marriott International, The Oberoi Hotels
and Resorts, Kempinski Hotels, and Wyndham
Hotels. For the last five years, he has been sharing
his industry insights with students at the
prestigious
Chitkara
University,
while
also
pursuing
research
and
innovation V. Verma et al., Baggage Cart with Weighing Mechanism by hospitality professionals because of lesser effort, easy
handling, and being time effective. moving towards their flight one needs to finish check-in
formalities including the weighing of their bags. Using
this mechanism would enable them to get the results faster
than the existing ones. (https://creativecommons.org/licenses/by-sa/4.0/). VISHAL VERMA is an Experienced hotel
professional with 8 years of hotel experience with
5-star hotel brands, like Oberoi Hotels & Resorts,
Leela Kempinski, Carlson Rezidor, Wyndham,
Post that it’s been 8 years since he’s working with
Chitkara University as an Assistant Professor. He
has publications in various reputable journals with
exceptional orientation of research. His area of
interest is Revenue Management and research skills in Tourism, Travel &
Hospitality with expertise in Room Division, Sales and Marketing as
well. The authors declare no conflict of interest. Figure 5: Traditional way of Measuring Baggage through the weighing
scale References [1]
R. Doganis,
The
Airline
Business,
Routledge,
2005,
doi:10.4324/9780203596807. [2]
R. Rendeiro Martín-Cejas, “Tourism service quality begins at the
airport,” Tourism Management, vol. 27, no. 5, pp. 874–877, 2006,
doi:10.1016/j.tourman.2005.05.005. [3]
B.K. Das, S. Sharma, “Influence of Relationship & Behavioural
Aspects on Customer Retention & Loyalty in B2B Flexible
Packaging Industry in Bangladesh,” International Journal of Applied
Business and Economic Research, vol. 15, no. 22, pp. 71–81, 2017. Figure 5: Traditional way of Measuring Baggage through the weighing
scale [4]
A. Parasuraman, V.A. Zeithaml, L.L. Berry, “A Conceptual Model
of Service Quality and Its Implications for Future Research,”
Journal
of
Marketing,
vol. 49,
no. 4,
pp. 41–50,
1985,
doi:10.1177/002224298504900403. 3.2. Advantage of the proposed system in comparison with
prior art 3.2. Advantage of the proposed system in comparison with
prior art •
Time Effective: It saves time as it is always available
with Front Office / Bell Desk. [5]
C. Gronroos, Service management and marketing: managing the
moments of truth in service competition, Lexington Books, 1990. [6]
J. Sharma, J. Singh, A. Singh, “Impact of E-Banking Service Quality
on Customer Satisfaction,” International Journal of Recent Technology
and Engineering (IJRTE), vol. 8, no. 5, pp. 2296–2300, 2020,
doi:10.35940/ijrte.E5841.018520. •
Easy Handling: It is always handy and in use by Bell
Desk Staff. •
Reduced Efforts: No more to-and-fro efforts, as a
handy baggage cart with a weighing mechanism, is
available all the time. Copyright: This article is an open access article distributed
under the terms and conditions of the Creative Commons
Attribution
(CC
BY-SA)
license Copyright: This article is an open access article distributed
under the terms and conditions of the Creative Commons
Attribution
(CC
BY-SA)
license 3.3. Proposed Solution The mechanism was designed to minimize the time
and effort required in the traditional weighing method
through a separate weighing machine. The proposed
design allows installing the weighing mechanism on the
baggage cart. The proposed design for Baggage Cart with
weighing mechanism for hotels and airlines are shown in
figure 6. (https://creativecommons.org/licenses/by-sa/4.0/). 3.1. Prior Art 3.1. Prior Art As per empirical observations, figure 5 represents the
traditional way of measuring the baggage. It would be
easy and convenient for the front office associate/porter to
use efficient ways to reduce their efforts and save time. As per empirical observations, the use of the proposed
mechanism in baggage cart is not restricted or limited to
just hotels only, even the respective airlines can use it
during a passenger's arrival inside the airport, before In a few hotels, as per empirical observations and as
shown in Figure 4 below, the guest is prompted to
measure his / her baggage on his own by using the Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 7 V. Verma et al., Baggage Cart with Weighing Mechanism V. Verma et al., Baggage Cart with Weighing Mechanism enrs.com
Journal of Engineering Research and Sciences, 3(1): 06-10, 2024
8
Figure 6: Proposed design for Baggage Cart with Weighing Mechanism for Hotels and Airlines Figure 6: Proposed design for Baggage Cart with Weighing Mechanism for Hotels and Airlines Figure 6: Proposed design for Baggage Cart with Weighing Mechanism for Hotels and Airlines Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 8 5. Conclusions The product is designed to eliminate the existing flaws
used by respective hotels and airlines of guests for
weighing their luggage, especially at the eleventh hour of
checking out of the hotel. As per the enclosed pictures
from 1-4, traditionally these are the methods of fetching
baggage and measuring them too which are not at all
convenient and time-effective. The most significant
advantage of the baggage cart with a weighing mechanism
is that it can be used by both hotels and airlines for their
purposes. This product is expected to find high acceptance RASHMI AGGARWAL is a Professor and Dean
Commerce at Chitkara Business School, Chitkara
University, Punjab. She is PhD, M. Phil, M. Com
and UGC-NET qualified, with a vast experience of
23 years in teaching and administration at various
positions. Her
areas
of
specialization
are
Accounting, Finance and Management. She has
publications in many reputable journals with excellent research
orientation. She has been awarded with the excellence Award for Most
Collaborating Employee. She also got the letters of appreciation for
strenuous and meticulous efforts by her in guiding and mentoring the
students. She is an effective communicator along with the exceptional Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 9 V. Verma et al., Baggage Cart with Weighing Mechanism ability of relationship management. She is always committed to creating
an inclusive learning environment for the students where they can
explore their potential. TANVI VERMA holds a doctorate from University
Business School, Panjab University, in Finance and
Strategic Management. She possesses a decade of
experience
in
the
fields
of
Accountancy,
Performance Management, Financial Reporting,
Strategic Management, and subjects related to the
Association of Chartered Certified Accountants
(ACCA). At present, she holds the esteemed
position of Assistant Professor at Chitkara University in Punjab. She has
a strong focus on research and a solid list of papers to her name. Her
work has been published in a multitude of prestigious journals. In
several of the national and international conferences where she has
presented, Ms. Verma has been honored with the Best Paper Award. Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 10 www.jenrs.com
|
https://openalex.org/W2799455770
|
https://leicester.figshare.com/articles/journal_contribution/Quantifying_Uncertainty_in_Satellite-Retrieved_Land_Surface_Temperature_from_Cloud_Detection_Errors/10226807/1/files/18448589.pdf
|
English
| null |
Quantifying Uncertainty in Satellite-Retrieved Land Surface Temperature from Cloud Detection Errors
|
Remote sensing
| 2,018
|
cc-by
| 11,102
|
Received: 9 March 2018; Accepted: 6 April 2018; Published: 17 April 2018 Abstract: Clouds remain one of the largest sources of uncertainty in remote sensing of surface
temperature in the infrared, but this uncertainty has not generally been quantified. We present a new
approach to do so, applied here to the Advanced Along-Track Scanning Radiometer (AATSR). We use
an ensemble of cloud masks based on independent methodologies to investigate the magnitude of
cloud detection uncertainties in area-average Land Surface Temperature (LST) retrieval. We find that
at a grid resolution of 625 km2 (commensurate with a 0.25◦grid size at the tropics), cloud detection
uncertainties are positively correlated with cloud-cover fraction in the cell and are larger during the
day than at night. Daytime cloud detection uncertainties range between 2.5 K for clear-sky fractions
of 10–20% and 1.03 K for clear-sky fractions of 90–100%. Corresponding night-time uncertainties
are 1.6 K and 0.38 K, respectively. Cloud detection uncertainty shows a weaker positive correlation
with the number of biomes present within a grid cell, used as a measure of heterogeneity in the
background against which the cloud detection must operate (e.g., surface temperature, emissivity and
reflectance). Uncertainty due to cloud detection errors is strongly dependent on the dominant land
cover classification. We find cloud detection uncertainties of a magnitude of 1.95 K over permanent
snow and ice, 1.2 K over open forest, 0.9–1 K over bare soils and 0.09 K over mosaic cropland,
for a standardised clear-sky fraction of 74.2%. As the uncertainties arising from cloud detection
errors are of a significant magnitude for many surface types and spatially heterogeneous where
land classification varies rapidly, LST data producers are encouraged to quantify cloud-related
uncertainties in gridded products. Keywords: uncertainties; land surface temperature; cloud detection errors Quantifying Uncertainty in Satellite-Retrieved Land
Surface Temperature from Cloud Detection Errors s
Meteo o og ca
st tute ( M ),
00560
e s
k ,
a d
*
Correspondence: c.e.bulgin@reading.ac.uk (C.E.B.); c.j.merchant@reading.ac.uk (C.J.M.);
djg20@leicester.ac.uk (D.G.); thomas.popp@dlr.de (T.P.); Larisa.Sogacheva@fmi.fi(L.S.);
Tel.: +44-118-378-6732 (C.E.B.) remote sensing remote sensing remote sensing www.mdpi.com/journal/remotesensing remote sensing
Article
Quantifying Uncertainty in Satellite-Retrieved Land
Surface Temperature from Cloud Detection Errors
Claire E. Bulgin 1,2,* ID , Christopher J. Merchant 1,2,* ID , Darren Ghent 2,3,* ID , Lars Klüser 4,5,
Thomas Popp 4,* ID , Caroline Poulsen 6 ID and Larisa Sogacheva 7,* ID
1
Department of Meteorology, University of Reading, Reading, RG6 6AL, UK
2
National Centre for Earth Observation, Leicester, UK
3
Space Research Centre, University of Leicester, Leicester, LE1 7RH, UK
4
Deutsches Zentrum für Luft- und Raumfahrt (DLR), 82234 Oberpfaffenhofen, Germany
5
Now at: Adesso Insurance Solutions GmbH, 81541 Munich, Germany; lars.klueser@imail.de
6
Rutherford Appleton Laboratory (RAL), Harwell Campus, Didcot, OX11 0QX, UK;
caroline.poulsen@stfc.ac.uk
7
Finnish Meteorological Institute (FMI), F1-00560 Helsinki, Finland
*
Correspondence: c.e.bulgin@reading.ac.uk (C.E.B.); c.j.merchant@reading.ac.uk (C.J.M.);
djg20@leicester.ac.uk (D.G.); thomas.popp@dlr.de (T.P.); Larisa.Sogacheva@fmi.fi(L.S.);
Tel.: +44-118-378-6732 (C.E.B.)
Received: 9 March 2018; Accepted: 6 April 2018; Published: 17 April 2018
Ab t
t Cl
d
i
f th
l
t
f
t i t
i
t
i
f
f remote sensing
Article
Quantifying Uncertainty in Satellite-Retrieved Land
Surface Temperature from Cloud Detection Errors
Claire E. Bulgin 1,2,* ID , Christopher J. Merchant 1,2,* ID , Darren Ghent 2,3,* ID , Lars Klüser 4,5,
Thomas Popp 4,* ID , Caroline Poulsen 6 ID and Larisa Sogacheva 7,* ID
1
Department of Meteorology, University of Reading, Reading, RG6 6AL, UK
2
National Centre for Earth Observation, Leicester, UK
3
Space Research Centre, University of Leicester, Leicester, LE1 7RH, UK
4
Deutsches Zentrum für Luft- und Raumfahrt (DLR), 82234 Oberpfaffenhofen, Germany
5
Now at: Adesso Insurance Solutions GmbH, 81541 Munich, Germany; lars.klueser@imail.de
6
Rutherford Appleton Laboratory (RAL), Harwell Campus, Didcot, OX11 0QX, UK;
caroline.poulsen@stfc.ac.uk
7
Finnish Meteorological Institute (FMI), F1-00560 Helsinki, Finland
*
Correspondence: c.e.bulgin@reading.ac.uk (C.E.B.); c.j.merchant@reading.ac.uk (C.J.M.);
djg20@leicester.ac.uk (D.G.); thomas.popp@dlr.de (T.P.); Larisa.Sogacheva@fmi.fi(L.S.);
Tel.: +44-118-378-6732 (C.E.B.)
Received: 9 March 2018; Accepted: 6 April 2018; Published: 17 April 2018
Ab t
t Cl
d
i
f th
l
t
f
t i t
i
t
i
f
f 1. Introduction All geophysical variables are retrieved with a level of uncertainty, which is a function of both the
data used and the retrieval methods. Taking account of uncertainty is fundamental to the appropriate
scientific application of these data, and uncertainty information should be provided in the most
complete state possible by data producers, preferably for each datum where uncertainty varies Remote Sens. 2018, 10, 616; doi:10.3390/rs10040616 www.mdpi.com/journal/remotesensing 2 of 20 Remote Sens. 2018, 10, 616 sufficiently to justify this [1]. Within the surface temperature remote sensing community, there have
been recent efforts to quantify measurement uncertainties on a per-pixel basis. This has been achieved
either by developing a model of how uncertainties vary under different retrieval conditions [2,3] or by
estimating uncertainties within the retrieval process itself [4,5]. In the latter case, the uncertainties can
be validated in addition to the measurement data, using independent reference datasets [6]. A method for estimating per-pixel uncertainty in sea surface temperature retrieval was developed
within the context of the European Space Agency (ESA) Climate Change Initiative (CCI) programme [4]. It has since been adopted within the lake surface temperature community via the European
Commission EU Surface Temperature of All Corners of Earth (EUSTACE) project, and within the
land surface temperature (LST) community via the ESA Data User Element (DUE) GlobTemperature
project [5,7]. These data producers consider the propagation of uncertainties arising from random
and systematic errors from Level 1b data into Level 2 and Level 3 products. The investment of these
different data producers in using the same approach to characterise uncertainty in surface temperature
retrievals is an important step in providing comparable products across different domains (land, ocean,
lake, ice). The uncertainties currently characterised by these data producers include both random and
systematic components. Sources of uncorrelated (random) errors in land surface temperature data
include instrument noise and sampling uncertainties. Systematic uncertainties arise due to retrieving
surface temperature through the atmosphere, sub-pixel variability in emissivity and from calibration
and instrument characteristics that change over time. Surface temperature retrievals are predominantly
made using measurements in the infrared region of the electromagnetic spectrum and are therefore
affected by the presence of clouds. Sampling uncertainties due to the presence of cloud limiting the
observations available in gridded data have been considered in sea surface temperature products [8],
but uncertainties due to imperfect cloud screening impacting the retrieved surface temperature have
not previously been quantified. 1. Introduction Cloud detection is an essential pre-processing step in the retrieval of Earth surface temperatures,
as cloud will affect the observed brightness temperatures. Clouds are the source of two different
types of uncertainty in surface temperature retrieval: (1) uncertainty due to ‘missed clouds’ (errors of
omission), which typically result in a cold bias in the retrieved surface temperature; and (2) uncertainty
due to ‘missed measurements’ (errors of commission), resulting from clear-sky pixels falsely classified
as cloud. At Level 2, (2) simply leads to omission of valid data from a given dataset, but at Level 3,
this has an impact on the averaged surface temperatures within a given gridded domain, not explicitly
accounted for within the sampling uncertainty. Where clouds are missed (1), uncertainties may
also include errors arising from the application of a clear-sky infrared LST retrieval algorithm to
cloud-affected observations. Cloud detection algorithms predominantly work on the concept that clouds are ‘bright’ in the
visible part of the spectrum and ‘cold’ in the infrared part of the spectrum. Cloud screening techniques
range from threshold-based testing [9–11], to neural networks [12–15], probabilistic approaches [16,17]
and full Bayesian probability calculations [18]. Problems typically occur where the clouds are of
a similar temperature to the underlying surface (e.g., low-level fog), along clear-cloud transition
zones and over bright and/or cold surfaces [19–22]. At night, visual detection efficiency is reduced,
amplifying the challenge of detecting low-level warm clouds. Detection difficulties can also be
magnified over land due to rapid spatial variations in land surface emissivity and surface reflectance,
making retrievals more difficult over pixels containing multiple land surface types and varying surface
topography [17,23,24]. Cloud detection errors contribute to structural uncertainty in surface temperature retrieval,
an uncertainty arising due to the methodological approaches adopted in the retrieval (e.g., which type
of cloud detection algorithm is used) [25]. Quantifying structural uncertainties in remotely-sensed
LST due to cloud detection failures is challenging without a ‘truth’ mask against which to make
comparisons, and will of course be dependent on the cloud masking technique chosen in the creation 3 of 20 Remote Sens. 2018, 10, 616 of any given data product. There is an awareness within the remote sensing community that cloud mask
failures do impact retrieval uncertainty [2,26], but the magnitude of cloud detection uncertainties in LST
retrieval have yet to be fully quantified. 2. Cloud Clearing Inter-Comparison The cloud clearing inter-comparison within the ESA DUE GlobTemperature project was designed
to assess a range of different cloud detection techniques for their suitability in land surface temperature
(LST) retrieval from the Advanced Along-Track Scanning Radiometer (AATSR). AATSR, the third in a
series of such instruments (following ATSR-1 and ATSR-2), flew aboard Envisat and was the precursor
to the now operational Sea and Land Surface Radiometer (SLSTR) aboard Sentinel-3. This instrument
series was designed specifically for the retrieval of surface temperature and is therefore the optimal
choice for the production of a long-term LST climate data record (CDR) [27]. AASTR is a dual-view radiometer, making observations at 1-km resolution at the sub-satellite
point in the nadir view (with viewing angles of 0–22◦) and a second observation in the forward view
(with viewing angles of 52–56◦). AATSR has seven channels covering the visible and infrared spectrum,
centred at 0.55, 0.66, 0.87, 1.6, 3.7, 10.8 and 12 µm. It flew between 2002 and 2012 in Sun-synchronous
orbit with an Equator overpass time of 10.30 a.m. For LST retrieval, only observations in the nadir
view are used. The cloud-clearing inter-comparison exercise had three phases designed to give participants an
opportunity to develop their algorithms prior to the final comparison: training, testing and submission. At each stage, data for ten AATSR sub-scenes were provided, with a size of 512 × 512 pixels. These scenes were chosen to cover a wide range of conditions, specifically focussing on those where
cloud detection is known to be more challenging. Within each set of ten scenes, as many as possible of
the following conditions were represented: cumulus, stratus and cirrus cloud types; desert, vegetated,
forest, ice and coast land surface types; a range of atmospheric total column water vapour (TCWV)
and scenes where some dust or smoke aerosol was present. The training phase was designed for
algorithm development, and the test dataset was available to ensure that developers had not tuned
their algorithm too closely to a particular scene selection. At these stages, a ‘truth’ cloud mask was
created manually by expert inspection and provided to algorithm developers to assess their cloud
mask performance. The analysis in this paper uses the final submission dataset where participants
provided their data ‘blind’ without reference to a truth cloud mask (although this was available at the
point of analysing the ensemble). 1. Introduction In this paper, we use data from multiple cloud masks collated
within a cloud detection inter-comparison exercise for the Advanced Along-Track Scanning Radiometer
(AATSR), within the European Space Agency (ESA) Data User Element (DUE) GlobTemperature
project. Collating a range of leading cloud masks over a consistent set of images enables us to evaluate
structural uncertainties due to clouds using an ensemble approach. Multiple independent datasets help
to resolve the state-space of the structural uncertainty [25] and provide an estimate of its magnitude. The paper proceeds as follows: we describe the cloud clearing inter-comparison (Section 2), our
approach to deriving LST uncertainties within the retrieval process (Section 3) and our understanding
of cloud masking uncertainties using the ensemble data (Section 4). We present the discussion and
conclusions in Section 5. 5 of 20 Figure 1. The 10.8-µm brightness temperatures for the 10 submission scenes in the cloud clearing
inter-comparison (Table 1) at native resolution (∼1 km). Individual plot headings give the scene
number, date and orbit start time. Figure 1. The 10.8-µm brightness temperatures for the 10 submission scenes in the cloud clearing
inter-comparison (Table 1) at native resolution (∼1 km). Individual plot headings give the scene
number, date and orbit start time. The data provided to algorithm developers with each scene were designed to be comprehensive
enough to implement any type of cloud detection scheme. Observations were provided for collocated
views (forward and nadir) in all channels, in addition to the land-sea mask, and confidence flags from
the L1b data. Numerical weather prediction (NWP) data were provided for those algorithms dependent
on radiative transfer simulation, including skin temperature and TCWV from ERA-Interim data [28],
and surface emissivity and reflectance from the UVIREMIS and BRDF atlases [29,30]. These are used as
inputs into the RTTOV 11 fast radiative transfer model [31] to simulate top of atmosphere reflectance
and brightness temperatures. We also supplied land surface cover data, which is a variant of the
GlobCover dataset [5] and all-sky LST (i.e., with no cloud detection pre-processing). y
p
p
g
Expert cloud masks were generated for each of the 30 scenes across the three phases of the
inter-comparison exercise. These were constructed using a bespoke tool developed for this purpose
by the Institute for Environmental Analytics at the University of Reading [32]. It enables the user
to view a given scene from a number of different instrument channels concurrently and to easily
construct channel inter-comparisons (such as differences, RGB images and local standard deviations). The majority of cloud in a given scene can generally be masked using thresholds on the channel data
specific to the image (set using sliders), and in challenging regions, the mask can be edited by hand. This allows the user to refine the masking in areas such as cloud edges and sunglint where thresholds
appropriate for the majority of the image may not apply locally. Figure 2 shows this manual cloud
mask for each of the scenes in the submission dataset for land only (ocean and inland water bodies
are masked out and shown in blue). The classification tool allowed pixels to be classified as ‘clear’,
‘probably clear’, ‘probably cloud’ and ‘cloud’. 2. Cloud Clearing Inter-Comparison A full description of the submission scenes is provided in Table 1. Figure 1 shows the 10.8-µm brightness temperatures for each of the ten scenes at native resolution
(∼1 km). Particular features to note are that Scene 3 contains a fire with some smoke aerosol in the top
right quadrant of the image, but the spatial extent of high aerosol loading is limited. Scene 5 has a
significant dust aerosol loading across the bottom-right of the image, which will affect the LST where
a retrieval is made. Scene 7 is a composite of ice over land and sea-ice with some cloud cover over
the left side of the peninsula ridge and towards the top of the image. LST is only retrieved over the
Antarctic Peninsula and not over the sea-ice region (GT_ATS_2P v1.0 dataset). Remote Sens. 2018, 10, 616 4 of 20 Table 1. Key characteristics of the ten submission scenes used within the cloud clearing inter-comparison. Scene
Location
Date
Orbit No. Start Time
Lines
Time of Day
Land Cover
Cloud Type
Aerosol
Elevation
1
China
21/05/2007
27304
14:34:28
3,600–4,111
Night
Cropland/Shrubland/Forest
Stratus, Cumulus
None
0–4000 m
2
Russia
22/05/2007
27314
07:20:27
13,789–14,300
Day
Forest/Snow/Ice
Stratus/Cumulus
None
0–300 m
3
Florida
23/05/2007
27333
15:11:50
17,450–17,961
Day
Forest/Vegetation
Cumulus/Cirrus
Smoke
0–250 m
4
UK
23/08/2007
28647
10:18:40
14,675–15,186
Day
Grassland/Shrubland/Cropland
Stratus/Cirrus
None
0–600 m
5
Algeria
11/06/2010
43290
09:26:40
18,140–18,651
Day
Bare Soil
Cirrostratus/Altostratus
Dust
300–600 m
6
Ukraine
05/08/2010
44083
19:01:32
6,250–6,761
Night
Vegetation/Cropland/River
Cumulostratus
None
0–400 m
7
Antarctica
08/08/2010
44121
10:44:17
29,320–29,831
Night
Snow/Ice
Stratus
None
0–2250 m
8
Mauritania
08/80/2010
44128
22:28:29
3,050–3,561
Night
Bare Soil
Stratus
None
100–700 m
9
Canada
07/06/2011
48474
17:22:31
14,500–15,011
Day
Evergreen Forest
Stratus/Cirrus
None
300–800 m
10
Uruguay
11/11/2009
40250
00:26:21
37,570–38,081
Night
Vegetation/Cropland/Shrubland
Stratus
None
0–600 m 5 of 20 Remote Sens. 2018, 10, 616 5 of 20 ‘Clear’ and ‘cloud’ were the most commonly-used
categories, with ‘probably clear’ and ‘probably cloud’ used infrequently, typically in the proximity
of aerosol. For analysis purposes, the few ‘probably clear’ pixels are interpreted as clear-sky and
‘probably cloud’ pixels interpreted as cloud. The dust aerosol in Scene 5 is set as clear-sky by the
manual mask for the purpose of considering cloud masking uncertainties, but aerosol with a high
optical depth will also affect the retrieved LST. Remote Sens. 2018, 10, 616 6 of 20 6 of 20 6 of 20 Figure 2. Manual cloud mask for the 10 submission scenes in the cloud clearing inter-comparison
(Table 1) at native resolution (∼1 km). Individual plot headings give the scene number, date and orbit
start time. Ocean and inland water bodies are plotted in blue. Figure 2. Manual cloud mask for the 10 submission scenes in the cloud clearing inter-comparison
(Table 1) at native resolution (∼1 km). Individual plot headings give the scene number, date and orbit
start time. Ocean and inland water bodies are plotted in blue. There were eight participants in the cloud clearing inter-comparison from across Europe, including
the AATSR operational cloud mask from the Synthesis of ATSR Data Into Sea-Surface Temperature
(SADIST) [9], the AATSR Dual View V2 (ADV V2) [11,33], the Avhrr Processing scheme Over Land,
cLouds and Ocean- Next Generation (APOLLO_NG) [16], Optimal Retrieval of Aerosol and Cloud
(ORAC) [10], Generalised Bayesian Cloud Screening (Bayes Min and Bayes Max) [17,18], Community
Cloud Retrieval for Climate (CC4CL) [14,15] and the University of Leicester Version 3 (UoL_V3) [17,34]
algorithms. This selection of algorithms represents a wide range of cloud detection techniques,
including threshold-based testing, neural networks, probabilistic approaches and full Bayesian
calculations. With the exception of SADIST, the algorithms have all been developed within the
following European Space Agency-funded projects: GlobTemperature Data User Element, Long-Term
Land Surface Temperature Validation, Aerosol Climate Change Initiative (Aerosol_cci) and Cloud
CCI; and have a wider use within the Earth Observation community beyond the extensive datasets
produced within these projects. Full details on each of the contributing algorithms are provided
in Table 2. Subsequent to the time of submission to the cloud clearing inter-comparison exercise,
the ORAC algorithm has been replaced by the CC4CL algorithm within Aerosol_cci and is not therefore
included in the ensemble analyses presented in this paper. 7 of 20 7 of 20 Remote Sens. 2018, 10, 616 Table 2. Key characteristics of the cloud detection algorithms submitted to the GlobTemperature Cloud Clearing Inter-Comparison exercise. Expansion of algorithm
acronyms are as follows: Synthesis of ATSR Data Into Sea-Surface Temperature (SADIST); AATSR Dual View V2 (ADV 2); the Avhrr Processing scheme Over Land,
cLouds and Ocean-Next Generation (APOLLO_NG); Optimal Retrieval of Aerosol and Cloud (ORAC); Bayesian Cloud Detection (Bayes Min and Bayes Max);
Community Cloud Retrieval for Climate (CC4CL); University of Leicester Version 3 (UoL_V3). 3. Uncertainties in LST Retrieval LST from AATSR is retrieved using the 10.8 and 12 µm channels in a nadir-only split window
algorithm [5,35]. LST from AATSR is retrieved using the 10.8 and 12 µm channels in a nadir-only split window
algorithm [5,35]. LST = a f,i,pw + bf,i(T11 −T12)p(θ) + (bf,i + c f,i)T12
(1) (1) where a, b and c are retrieval coefficients, dependent on biome (i), fractional vegetation cover (f),
precipitable water (pw) and satellite viewing angle (p(θ)). T11 and T12 denote the 10.8 and 12 µm
brightness temperatures, respectively. The retrieval coefficients vary as a function of time of day
(day or night), and the dependent parameters vary on differing length scales: the biome or land
cover classification is fixed for a given location throughout time, whilst the precipitable water and
fractional vegetation cover change seasonally. The land cover classification is based on the GlobCover
dataset [36], regridded at 1/120◦resolution. The bare soil category is sub-divided into six different
classes, giving a total of twenty-seven distinct land cover classifications. Fractional vegetation cover
is calculated from the Copernicus Global Land Services dataset [37] at ten-day temporal resolution,
whilst the precipitable water is derived from the six-hourly ERA-Interim dataset [28]. Uncertainties in the LST retrieval arise from instrument noise, instrument calibration, specification
of surface parameters (e.g., surface emissivity, land cover classification) and because the retrieval
is made through the Earth’s atmosphere. Radiative transfer is simulated in the retrieval process
using a fast forward model (RTTOV 11) constrained by Numerical Weather Prediction (NWP) data. Uncertainties are characterised using an approach consistent with the production of sea surface
temperature data within the ESA CCI project [4,8], where an uncertainty budget is constructed from
a number of distinct components: uncorrelated, locally correlated and systematic uncertainties [5]. Random errors are those that have no correlation between observations and include instrument noise
and sub-pixel variability in surface emissivity not captured by the auxiliary data. Locally systematic
uncertainties arise from errors in the surface emissivity for different land cover classifications (captured
implicitly in the fractional vegetation cover), errors in the atmospheric state (via the precipitable water
and radiative transfer parametrisations), errors in the selection of retrieval coefficients, errors in
geolocation and uncertainty in the knowledge of the underlying biome. Within the retrieval, these
locally systematic uncertainties are considered as two separate components relating to the surface (sfc)
and atmosphere (atm). 6 of 20 Name
Project
Type
Institution
Day/Twilight (µm)
Night (µm)
Views
RT 1
Current Application
References
SADIST
Operational
Threshold
RAL a
10.8, 12
3.7, 10.8, 12
Nadir, Forward
No
ATSR Series
[9]
ADV V2
Aerosol_cci
Threshold
FMI b
0.6, 0.8, 10.8, 12
NA
Nadir, Forward
No
AATSR
[11,33]
APOLLO_NG
Aerosol_cci
Probabilistic
DLR c
0.6, 0.8, 10.8, 12
NA
Nadir Only
No
AVHRR, AATSR
[16]
ORAC
Aerosol_cci
Threshold
RAL a
0.5, 0.6, 0.8, 1.6, 10.8, 12
NA
Nadir, Forward
No
ATSR Series
[10]
Bayes Min
GlobTemperature
Bayesian
UoR d
10.8, 12
10.8, 12
Nadir Only
Yes
ATSR Series, AVHRR, SEVIRI, GOES
[17,18]
Bayes Max
GlobTemperature
Bayesian
UoR d
0.6, 1.6, 10.8, 12
3.7, 10.8, 12
Nadir Only
Yes
ATSR Series, AVHRR, SEVIRI, GOES
[17,18]
CC4CL
Cloud CCI
Neural Network
RAL a
0.6, 0.8, 1.6, 10.8, 12
3.7, 10.8, 12
Nadir Only
No
AATSR
[14,15]
UoL_V3
GlobTemperature
Probabilistic
UoL e
3.7, 10.8, 12
3.7, 10.8, 12
Nadir Only
Yes
ATSR Series
[17,34]
a Rutherford Appleton Laboratory; b Finnish Meteorological Institute; c Deutsches Zentrum für Luft- und Raumfahrt; d University of Reading; e University of Leicester; 1 Radiative
Transfer. Remote Sens. 2018, 10, 616 8 of 20 3. Uncertainties in LST Retrieval Finally, the large-scale systematic uncertainty arises from calibration errors
and the bias in the satellite surface temperatures relative to other sources of temperature data, once
all known residual biases are corrected for [38]. This is currently set to a fixed value of 0.03 K [5]. Although the magnitude of individual uncertainty components can be algorithm dependent, the total
uncertainty budget tends to be similar across different LST retrieval schemes. Within this paper, we consider uncertainties in the LST data when gridded at a 25 × 25 pixel
resolution corresponding to a surface area of 625 km2. This is commensurate with a gridded resolution
of 0.25◦at the tropics, a typical grid size for global surface temperature products. At Level 3,
an additional source of uncertainty is introduced via the gridding process as LST can only be retrieved
for clear-sky pixels. The sampling uncertainty (σl3
samp) is calculated as follows: σl3
samp =
s
ncld × σ2
LST,clr
nland −1
(2) (2) where ncld is the number of cloudy pixels in the grid cell, nland is the number of land pixels in the
grid cell and σLST is the standard deviation of the LST across the clear-sky pixels in the grid cell. Where there is only a single clear-sky pixel in the grid cell (and the LST standard deviation cannot
be calculated), the variance is set to the maximum value across the scene. The sampling uncertainty
is uncorrelated between Level 3 grid cells and is added to the upscaled Level 2 random uncertainty,
where σl2
ran and σl3
ran denote the Level 2 and Level 3 random uncertainty components, respectively. Remote Sens. 2018, 10, 616 9 of 20 σl3
ran =
v
u
u
t∑σ2(l2)
ran,clr
n2
land
+ σ2samp
(3) (3) The subscript ‘clr’ denotes clear sky pixels over land within the 625 km2 grid cell. The Level
2 atmospheric uncertainty component (σl2
atm) is upscaled to Level 3 (σl3
atm) as follows, assuming that
atmospheric state effects are correlated over length scales of 25 km: The subscript ‘clr’ denotes clear sky pixels over land within the 625 km2 grid cell. 3. Uncertainties in LST Retrieval Individual plot headings give the scene number, date
and orbit start time. We calculate a cloud masking uncertainty for these ten scenes using the ensemble of contributing
cloud masks. Seven masks are applicable during the day and only five at night (as ADV and
APOLLO_NG were designed for daytime classification only). We apply each cloud mask to the
unfiltered LST field in turn and calculate an average LST in each 625-km2 grid cell. We then calculate
the LST difference between applying the manual mask (MM) in each grid cell and applying each of
the contributed masks (CM) in turn (LSTCM −LSTMM). The cloud masking structural uncertainty
is then the standard deviation of the ensemble of LST differences. We classify any grid cells with
600 or more pixels with an ocean biome (out of 625 pixels in a given grid cell) as water and do not
consider them in this analysis. For any land grid cells, the lower threshold on the number of clear-sky
pixels required to calculate the mean LST is one, as this methodology is applied in the generation
of operational products. Figure 4 shows the cloud masking uncertainties for each of the ten scenes. The magnitude of the cloud masking uncertainty is 0 K where all masks (including the manual mask)
agree that there is clear-sky, and grid cells where all masks agree that it is cloudy are shown in white. Cloud masking uncertainties over largely clear regions range between 0 and 0.5 K. Small variations
in the pixels flagged as cloud within a given grid cell will cause some uncertainty in the retrieved
LST, as this is not homogeneous at 1-km resolution. Cloud masking uncertainty typically increases
around the edges of cloud features and some land features where one or more masks incorrectly
interpret them as cloud, being of the order of 1.5–3 K in magnitude. There is also some evidence of
increased cloud detection uncertainty over broken cloud fields (Scenes 3 and 9). In some regions, the
cloud masking uncertainty increases significantly to >10 K, and this uncertainty has higher spatial
variability than the LST retrieval uncertainty. Figure 5 shows the retrieval (Equation (7)) and cloud
masking uncertainties added in quadrature. The spatial variability from the cloud masking uncertainty
dominates, with minimum total uncertainties typically of the order 1.5 K in clear-sky regions. 3. Uncertainties in LST Retrieval The Level
2 atmospheric uncertainty component (σl2
atm) is upscaled to Level 3 (σl3
atm) as follows, assuming that
atmospheric state effects are correlated over length scales of 25 km: σl3
atm =
v
u
u
t∑σ2(l2)
atm,clr
nland
(4) (4) For the surface component, it is assumed that the uncertainties are correlated with a length scale
of 5 km, consistent with the resolution of the auxiliary surface datasets, but are uncorrelated over larger
spatial scales. The AATSR data are at a native resolution of 1 km, so to calculate the Level 3 surface
uncertainty component (σl3
s f c), we first calculate an intermediate surface uncertainty component at
25 km2 (σs f c_25km) before upscaling this to 625-km2 resolution. σs f c_25km =
v
u
u
t∑σ2(l2)
s f c,clr
nland
(5)
σl3
s f c =
v
u
u
t∑σ2
s f c_25km
n2
land
(6) (5) (6) The total uncertainty in a grid cell (σtotal) is given by adding all of the Level 3 uncertainty
components in quadrature. The total uncertainty in a grid cell (σtotal) is given by adding all of the Level 3 uncertainty
components in quadrature. σtotal =
r
σ2(l3)
ran
+ σ2(l3)
s f c
+ σ2(l3)
atm + σ2(l3)
sys
(7) (7) where the Level 3 large-scale systematic uncertainty (σsys) is equal to 0.03 K as this is fully correlated
across the gridded domain. The retrieval uncertainties (not including cloud detection uncertainties)
for the ten scenes in the cloud clearing inter-comparison are shown in Figure 3. They are calculated
having applied the manual cloud mask to the data, and the uncertainties are set to zero for entirely
cloudy grid cells. We see that typically, the minimum retrieval uncertainty at this resolution is ∼1 K,
increasing in some regions to 2–3 K. This is particularly evident over Florida (Scene 3), across parts of
the U.K. (Scene 4), over Uruguay (Scene 5) and across Mauritania (Scene 9). Remote Sens. 2018, 10, 616 10 of 20 10 of 20 Figure 3. Level 3 LST retrieval uncertainties for the 10 submission scenes in the cloud clearing
inter-comparison (Table 1) at 625-km2 resolution. Individual plot headings give the scene number, date
and orbit start time. Figure 3. Level 3 LST retrieval uncertainties for the 10 submission scenes in the cloud clearing
inter-comparison (Table 1) at 625-km2 resolution. 3. Uncertainties in LST Retrieval Regions
of large cloud masking uncertainty do not always correlate with regions of high retrieval uncertainty,
for example over the U.K. (Scene 4) and Mauritania (Scene 9). Remote Sens. 2018, 10, 616 11 of 20 Figure 4. Cloud detection uncertainties for the 10 submission scenes in the cloud clearing
inter-comparison (Tables 1 and 2) at 625-km2 resolution. Individual plot headings give the scene
number, date and orbit start time. White grid cells denote agreement between all masks on cloudy
conditions. Black grid cells denote regions classified as entirely cloudy by the manual mask, but
clear-sky in some of the contributing masks. Figure 4. Cloud detection uncertainties for the 10 submission scenes in the cloud clearing
inter-comparison (Tables 1 and 2) at 625-km2 resolution. Individual plot headings give the scene
number, date and orbit start time. White grid cells denote agreement between all masks on cloudy
conditions. Black grid cells denote regions classified as entirely cloudy by the manual mask, but
clear-sky in some of the contributing masks. Figure 5. Combined Level 3 LST retrieval and cloud detection uncertainties for the 10 submission
scenes in the cloud clearing inter-comparison (Figures 3 and 4) at 625-km2 resolution. Individual plot
headings give the scene number, date and orbit start time. White grid cells denote agreement between
all masks on cloudy conditions. Black grid cells denote regions classified as entirely cloudy by the
manual mask, but clear-sky in some of the contributing masks. Figure 5. Combined Level 3 LST retrieval and cloud detection uncertainties for the 10 submission
scenes in the cloud clearing inter-comparison (Figures 3 and 4) at 625-km2 resolution. Individual plot
headings give the scene number, date and orbit start time. White grid cells denote agreement between
all masks on cloudy conditions. Black grid cells denote regions classified as entirely cloudy by the
manual mask, but clear-sky in some of the contributing masks. 4. Understanding Cloud Detection Uncertainties Cloud detection uncertainty shows some spatial correlation with the location of cloudy features
(Figure 2), but also has a spatial variability related to the land surface cover. We would expect cloud
detection uncertainty to be positively correlated with the cloud fraction. Where fewer clear-sky pixels
are available, these are less likely to be representative of the gridded mean LST, as the per-pixel values
generally show a degree of heterogeneity. The spatial variability related to land surface cover is
driven by several factors affecting the individual cloud masks within the ensemble: (1) Some cloud
masks, particularly the threshold based methods, are sensitive to land features such as riverbeds. It is very difficult to select globally-applicable thresholds for cloud detection tests, and inevitably,
those chosen work better in some regions than in others. Erroneous flagging of surface features
will generate discrepancies in the mean LST between different cloud masks, increasing the cloud
detection uncertainty. (2) The variability in the land cover classification within a given grid cell is also 12 of 20 Remote Sens. 2018, 10, 616 important. Where the land surface biome has a large variability, the LST is likely to vary more rapidly
spatially, and the difference in sub-sampling of the cell introduced by the different cloud detection
schemes (i.e., variations in the definition of the cloud edge) will have a more significant impact. In
this situation, the spread of differences in LST with respect to the manual cloud mask will increase. Under these conditions, the distribution of different biomes within a grid cell will also be important,
e.g., with a steady gradient in LST across the grid cell potentially giving a different uncertainty to
a more heterogeneous LST field. Assuming a perfect cloud mask under these conditions, sampling
uncertainties would also be higher here, than in more homogeneous regions [8]. Given these factors, it may be expected that we can parametrise Level 3 uncertainties as:
(1) a function of the percentage of clear-sky pixels within a grid cell and (2) as a function of the
underlying variability in LST. We examine both, using the manual mask as ‘truth’ for the percentage of
clear-sky pixels, and the number of land biomes within a given grid cell as a proxy for LST variability,
as we do not have representative LST’s for cloudy pixels. Figure 6 shows the number of clear-sky pixels in each 625-km2 grid cell. 4. Understanding Cloud Detection Uncertainties Figure 8a shows the relationship between the percentage of clear-sky pixels over land and the
cloud detection uncertainty at 625-km2 resolution. The percentage of clear-sky pixel data on the x-axis
is binned in 10% increments, and panel (b) shows the number of grid cells in each bin. The cloud
detection uncertainty is the mean value across all grid cells in a given bin, and the error bars represent
the standard error. The solid line shows all of the data from the eight scenes (excluding Scenes 5 and
7), indicating a negative correlation between the percentage of clear-sky pixels and cloud detection
uncertainty. For a 10–20% clear-sky fraction, we see average cloud detection uncertainties of 2 K, which
fall to 0.65 K for a clear-sky fraction between 90 and 100%. Of the scenes included in the analysis, four are daytime observations and four night-time
observations, and we calculate the cloud detection uncertainty under these conditions independently. We see from the number of grid cells that this still gives ≥50 observations within each bin under
different solar illuminations. The cloud detection uncertainties during the day are of larger magnitude
than at night, but the slope of the relationship between uncertainty and clear-sky fraction is consistent
with that shown for all of the data. At clear-sky fractions of 10–20%, cloud detection uncertainties are
on average 2.5 K during the day and 1.6 K at night, falling to 1 K and 0.38 K respectively for clear-sky
fractions of 90–100%. The magnitude of the day-night difference in cloud detection uncertainties
for any given clear-sky fraction is of the order 0.6–0.8 K. We would not expect the cloud detection
uncertainties to be 0 K at 100% clear-sky as this definition is applicable to the manual mask only. We also consider the cloud detection uncertainty as a function of the number of biomes present
in the Level 3 grid cell, as a measure of the heterogeneity in the background against which the cloud
detection must operate, e.g., surface temperature, emissivity and reflectance (Figure 9). We present
results for grid cells with between four and twelve different biomes as this is the typical range
for the majority of observations. Again, we plot all data and then day and night-time observations
independently. 4. Understanding Cloud Detection Uncertainties Comparing this with
Figure 4, we see some spatial correlations between lower percentages of clear-sky pixels and high
cloud detection uncertainties, notably around the cloud features over China (Scene 1), Russia (Scene 2),
Algeria (Scene 6) and in the top half of the Mauritania image (Scene 9). We also see regions where
larger cloud detection uncertainties do not correspond to lower clear-sky percentages, for example
over Canada (Scene 10, to the left of the image) and over the Antarctic Peninsula (Scene 7). Figure 7
shows the number of biomes in each 625-km2 grid cell for each scene. Here, we see that the number of
biomes correlates positively with cloud detection uncertainty over China (Scene 1, to the right of the
image) and over Florida (Scene 3). Figure 6. Clear-sky fraction for the 10 submission scenes in the cloud clearing inter-comparison (Table 1)
at 625-km2 resolution. Individual plot headings give the scene number, date and orbit start time. Figure 6. Clear-sky fraction for the 10 submission scenes in the cloud clearing inter-comparison (Table 1)
at 625-km2 resolution. Individual plot headings give the scene number, date and orbit start time. We examine the relationship between cloud detection uncertainty and the percentage of clear-sky
pixels as defined by the manual mask across the ten scenes, excluding Scene 5 (high dust aerosol
loading) and Scene 7 (Antarctic Peninsula). These data are excluded for the following reasons: (1) firstly
because aerosol affects the retrieved LST, and the most appropriate treatment of aerosol affected pixels
is dependent on the data application (see the discussion section for further details); (2) secondly
because most cloud detection algorithms show very poor skill over ice, tending to flag all ice as cloud,
or on occasion all ice as clear-sky, but failing to discriminate well between snow or ice surfaces and
cloud. The cloud detection uncertainties in these particularly challenging regions are considered
further at the end of this section. Remote Sens. 2018, 10, 616 13 of 20 13 of 20 Figure 7. Number of land biomes for the 10 submission scenes in the cloud clearing inter-comparison
(Table 1) at 625-km2 resolution. Individual plot headings give the scene number, date and orbit
start time. Figure 7. Number of land biomes for the 10 submission scenes in the cloud clearing inter-comparison
(Table 1) at 625-km2 resolution. Individual plot headings give the scene number, date and orbit
start time. 4. Understanding Cloud Detection Uncertainties We find a general increase in the cloud detection uncertainty with the number of biomes,
although the relationship is not as consistent as that found with the clear-sky fraction. There will
be a dependence on the biome type that folds into this analysis. For example, if all the biomes are
different types of forest vegetation, the LST variability will be much lower than where the biome set
includes more diverse biome types, e.g., bare soil and forest. At night, there seems to be a stronger
relationship between the cloud detection uncertainty and the number of biomes and a much weaker
one during the day. There is a time of day-dependent difference in cloud detection uncertainty where
the number of biomes is few: 0.5 K at night and 1.5 K during the day for four biomes. For 11–12 biomes
in a given grid cell, we see convergence in the uncertainties, which are typically of order 1.7 K under
all conditions (for 11 biomes per grid cell). Remote Sens. 2018, 10, 616 14 of 20 14 of 20 0
20
40
60
80
100
Clear-Sky Pixels / %
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Cloud Detection Uncertainty / K
(a)
All
Day
Night
0
20
40
60
80
100
Clear-Sky Pixels / %
0
100
200
300
400
500
Number of L3 Grid Cells
(b)
All
Day
Night
Figure 8. Cloud detection uncertainties as a function of clear-sky fraction (a) at 625-km2 resolution. The
following are shown: all data (solid line), night-time only (dot-dash line) and daytime only (dashed
line). The linear regression for cloud detection uncertainty as a function of clear-sky percentages is
shown for all data. Panel (b) shows the number of observations in each bin. 0
20
40
60
80
100
Clear-Sky Pixels / %
0
100
200
300
400
500
Number of L3 Grid Cells
(b)
All
Day
Night 0
20
40
60
80
100
Clear-Sky Pixels / %
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Cloud Detection Uncertainty / K
(a)
All
Day
Night Figure 8. Cloud detection uncertainties as a function of clear-sky fraction (a) at 625-km2 resolution. The
following are shown: all data (solid line), night-time only (dot-dash line) and daytime only (dashed
line). The linear regression for cloud detection uncertainty as a function of clear-sky percentages is
shown for all data. Panel (b) shows the number of observations in each bin. 4. Understanding Cloud Detection Uncertainties 4
5
6
7
8
9
10
11
12
Number of Biomes in L3 Grid Cell
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Cloud Detection Uncertainty / K
(a)
All
Day
Night
4
5
6
7
8
9
10
11
12
Number of Biomes in L3 Grid Cell
0
100
200
300
400
500
Number of L3 Grid Cells
(b)
All
Day
Night
Figure 9. Cloud detection uncertainties as a function of number of biomes in each grid cell (a) at
625-km2 resolution. The following are shown: all data (solid line), night-time only (dot-dash line) and
daytime only (dashed line). Panel (b) shows the number of observations in each bin. 4
5
6
7
8
9
10
11
12
Number of Biomes in L3 Grid Cell
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Cloud Detection Uncertainty / K
(a)
All
Day
Night 4
5
6
7
8
9
10
11
12
Number of Biomes in L3 Grid Cell
0
100
200
300
400
500
Number of L3 Grid Cells
(b)
All
Day
Night Figure 9. Cloud detection uncertainties as a function of number of biomes in each grid cell (a) at
625-km2 resolution. The following are shown: all data (solid line), night-time only (dot-dash line) and
daytime only (dashed line). Panel (b) shows the number of observations in each bin. Finally, we examine whether this dataset can reveal any differences in the cloud detection
uncertainties under different land cover classifications. To do this, we consider all ten scenes and look
for grid cells that have a dominant biome, defined as where 80% or more of the land pixels within
the grid cell have a single land cover classification. Within our scenes, we found five biomes for
which we had 40+ grid cells in which they were the dominant land cover, as shown in Table 3. For
each land cover classification considered here, the observations had a range of clear-sky fractions,
upon which cloud detection uncertainty has a significant dependence (as shown in Figure 8). We
therefore standardise all the results to the average percentage of clear-sky pixels across the five biomes
(74.2%). We compare the model slope from a linear regression of all data against the clear-sky fraction
(Figure 8) against the linear regressions of data points falling within each of the five identified biomes. 4. Understanding Cloud Detection Uncertainties We find that the slope of the model is strongly biome dependent (Figure 10a) and that a biome-specific
correction to a clear-sky fraction of 74.2% is required. The linear regression gives each data point
an equal weighting, with the assumption that we have the same confidence in the cloud detection
uncertainty at every location, in the absence of any other information. The uncertainty in the regression 15 of 20 Remote Sens. 2018, 10, 616 model is calculated as the standard deviation of the statistical differences between the model and the
data points (Table 3). The mosaic vegetation has the best constrained fit with a standard deviation in
the model-data differences of 0.11 K. For bare soils, the standard deviation is ∼0.34 K, for forests 0.57
K and for permanent snow and ice 1 K. This means that there is a greater uncertainty in the regression
model for forest and permanent snow and ice classes. Table 3. Land cover classification numbers, descriptions, number of observations and regression model
uncertainties corresponding to the data shown in Figure 10. Land Cover Classification
Description
Number of Observations
Regression Model Uncertainty
4
Mosaic vegetation/cropland
40
0.11 K
9
Open needle-leaved deciduous or evergreen forest
48
0.57 K
21
Bare soil (entisols-orthents)
352
0.33 K
22
Bare soil (shifting sand)
92
0.35 K
27
Permanent snow and ice
178
0.996 K Table 3. Land cover classification numbers, descriptions, number of observations and regression model
uncertainties corresponding to the data shown in Figure 10. Figure 10 shows relative cloud detection uncertainties between the biomes. Panel (b) shows
the mean standardised cloud detection uncertainty at 74.2% clear-sky fraction plotted for each
biome, including the standard error on the mean (note that the absolute magnitude of these will
vary with clear-sky fraction). Panel (c) shows the number of observations available for each land
cover classification. We see here that the magnitude of the cloud detection uncertainty shows a
significant dependence on the underlying biome. The largest cloud detection uncertainties are seen for
permanent snow and ice (biome 27) with a magnitude of 1.95 K. Cloud detection uncertainties over
open needle-leaved deciduous or evergreen forest (Biome 9) had a magnitude of 1.2 K. Two categories
of bare soil were included: entisol-orthents and shifting sand (Biomes 21 and 22), and these give similar
magnitudes of cloud detection uncertainty, 0.9 K and 1.0 K, respectively. 4. Understanding Cloud Detection Uncertainties The lowest cloud detection
uncertainty was found over mosaic cropland/vegetation, with an average value of 0.09 K, suggesting
that the land cover in this sample was very homogeneous in comparison with other vegetated biomes,
e.g., forest (Biome 9). It is also important to note that the sample size available for these analyses is
limited, particularly for the mosaic cropland and forest biomes (Table 3), and with a larger sample size,
these estimates may vary. 0
20
40
60
80
100
Clear-Sky Pixels / %
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Cloud Detection Uncertainty / K
(a)
All data
Veg (4)
Forest (9)
Soil (21)
Sand (22)
Snow (27)
4
9
21
22
27
Land Cover Classification
0.0
0.5
1.0
1.5
2.0
Cloud Detection Uncertainty / K
(b)
0
1
2
3
4
5
6
Land Cover Classification
0
50
100
150
200
250
300
350
400
Number of L3 Grid Cells
(c)
Figure 10. Biome-specific linear regression models for cloud detection uncertainty dependence
on clear-sky fraction (a). Standardised cloud detection uncertainties as a function of land cover
classification (b). Panel (c) shows the number of observations for each land cover classification,
with land cover classifications defined in Table 3. 0
1
2
3
4
5
6
Land Cover Classification
0
50
100
150
200
250
300
350
400
Number of L3 Grid Cells
(c) 0
20
40
60
80
100
Clear-Sky Pixels / %
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Cloud Detection Uncertainty / K
(a)
All data
Veg (4)
Forest (9)
Soil (21)
Sand (22)
Snow (27) 0
20
40
60
80
100
Clear-Sky Pixels / %
0
5
0
5
0
5
0
5
0 (a)
All data
Veg (4)
Forest (9)
Soil (21)
Sand (22)
Snow (27)
4
9
21
22
27
Land Cover Classification
0.0
0.5
1.0
1.5
2.0
Cloud Detection Uncertainty / K
(b) 4
9
21
22
27
Land Cover Classification
0.0
0.5
1.0
1.5
2.0
Cloud Detection Uncertainty / K
(b) Figure 10. Biome-specific linear regression models for cloud detection uncertainty dependence
on clear-sky fraction (a). Standardised cloud detection uncertainties as a function of land cover
classification (b). Panel (c) shows the number of observations for each land cover classification,
with land cover classifications defined in Table 3. 5. Discussion and Conclusions The use of a cloud mask ensemble enables us to gain insight into the magnitude of cloud detection
uncertainties in Level 3 data. There is a strong dependence on the percentage of clear-sky pixels in
a given grid cell, with larger uncertainties in daytime data than at night. This is to be expected, as
diurnal warming of the land surface during the day results in a more heterogeneous LST field, as 16 of 20 Remote Sens. 2018, 10, 616 16 of 20 the local rate of warming depends on the underlying land surface. Cloud shadowing will also affect
LST, so where clouds are more abundant, giving fewer clear-sky pixels, shadowing may increase the
LST variability and increase cloud detection uncertainty due to imperfect masking. At night, the land
surface cools rapidly, and temperatures across neighbouring pixels will generally show less variation. One exception to this is boundaries between forest and grass or bare soil. Forests stay warmer at
night, whilst grass and bare soil cool more quickly. The number of biomes is also a weak indicator of
cloud detection uncertainty, used as a measure of heterogeneity in the background against which the
cloud detection must operate. The correlation between the number of biomes and clear-sky fraction is
stronger at night where it is the more dominant factor in LST variability, as other factors such as cloud
shadowing are less important. As mentioned in Section 4, the appropriate classification of aerosol-affected observations is
dependent on the application of the data, e.g., for retrievals of cloud properties, aerosol should be
classified as ‘not cloud’, whilst for surface retrievals, it is often preferable for optically-thick aerosol
to be classified as ‘not clear’. In the infrared, optically-thick aerosol can obscure the signal from the
land surface, and optically-thin aerosol will do so in part. The relative temperature of the aerosol
with respect to the land surface will depend on the aerosol composition and altitude, and aerosol is
likely to modify the underlying surface temperature (unobserved by infrared satellite measurements)
by modifying the atmospheric radiative transfer relative to clear-sky conditions. For these reasons,
we excluded the large area of dust aerosol affected observations from the analysis of cloud detection
uncertainty as a function of clear-sky fraction and the number of biomes. 5. Discussion and Conclusions These regions were included
in the biome-dependent cloud detection uncertainty (Figure 10), with aerosol affecting a significant
proportion of the observations over entisols-orthents bare soil (Biome 21). The cloud detection
uncertainties were slightly larger here than for shifting sand (Biome 22). We would expect a more
varied response in the cloud detection algorithms in aerosol-affected areas depending on their primary
application, but find that the spread in the data is smaller than over shifting sand. There are however a
number of additional factors that could affect these results, including variations in surface emissivity
and cloud shadowing, which it is not possible to separate out using these data. Within this paper, we have considered only data gridded at 625-km2 resolution, but it could
be expected that cloud detection uncertainty would vary as a function of gridding resolution. To
address this point, we plot the cloud detection uncertainty as a function of both clear-sky fraction
and number of biomes for the same data regridded at 25-km2, commensurate with a resolution of
0.05◦at the tropics. This resolution is commonly used in the provision of Level 3 uncollated data from
polar orbiting satellite instruments. We assume that the locally systematic surface component of the
uncertainty budget is correlated at this resolution, but otherwise follow the methodology described in
Section 3. As the gridded resolution is increased, there is a significant increase in the number of Level
3 grid cells included in the analysis. Figure 11 shows that the overall magnitude of the cloud detection uncertainty is lower at 25-km2
than at 625-km2 resolution. Cloud detection uncertainties are still larger for daytime observations
than for night-time observations, but at this resolution do not show such a strong correlation with
the clear-sky fraction. During the day, cloud detection uncertainties are of a magnitude of ∼1.7
K for clear-sky fractions <80%, falling to ∼1.2 K for >80% clear-sky. Night-time cloud detection
uncertainties fall more consistently with increasing clear-sky fraction, from ∼1 K at 0–40% clear-sky
to ∼0.4 K at >80% clear-sky. Comparing against the 625-km2 data, the cloud detection uncertainties
are similar for large clear-sky fractions, but lower at small clear-sky fractions. This may reflect less
variability in the underlying LST for data gridded at a higher resolution. The difference in magnitude
between day and night-time cloud detection uncertainties is similar, ranging between ∼0.6 and 1 K. 17 of 20 17 of 20 Remote Sens. 5. Discussion and Conclusions 2018, 10, 616 0
20
40
60
80
100
Clear-Sky Pixels / %
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Cloud Detection Uncertainty / K
(a)
All
Day
Night
0
20
40
60
80
100
Clear-Sky Pixels / %
1000
2000
3000
4000
5000
6000
7000
8000
Number of L3 Grid Cells
(b)
All
Day
Night
4
5
6
7
8
Number of Biomes in L3 Grid Cell
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Cloud Detection Uncertainty / K
(c)
All
Day
Night
4
5
6
7
8
Number of Biomes in L3 Grid Cell
0
2000
4000
6000
8000
10000
12000
Number of L3 Grid Cells
(d)
All
Day
Night
Figure 11. Cloud detection uncertainties as a function of clear-sky fraction (a) and number of biomes
(c) at 25-km2 resolution. Right-hand panels show the number of observations in each clear-sky bin (b)
and for each number of biomes represented in a given grid cell (d). The following are shown: all data
(solid line), night-time only (dot-dash line) and daytime only (dashed line). 0
20
40
60
80
100
Clear-Sky Pixels / %
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Cloud Detection Uncertainty / K
(a)
All
Day
Night
4
5
6
7
8
Number of Biomes in L3 Grid Cell
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Cloud Detection Uncertainty / K
(c)
All
Day
Night
N mber of L3 Grid Cells 0
20
40
60
80
100
Clear-Sky Pixels / %
1000
2000
3000
4000
5000
6000
7000
8000
Number of L3 Grid Cells
(b)
All
Day
Night
4
5
6
7
8
Number of Biomes in L3 Grid Cell
0
2000
4000
6000
8000
10000
12000
Number of L3 Grid Cells
(d)
All
Day
Night Number of L3 Grid Cells Figure 11. Cloud detection uncertainties as a function of clear-sky fraction (a) and number of biomes
(c) at 25-km2 resolution. Right-hand panels show the number of observations in each clear-sky bin (b)
and for each number of biomes represented in a given grid cell (d). The following are shown: all data
(solid line), night-time only (dot-dash line) and daytime only (dashed line). Figure 11. Cloud detection uncertainties as a function of clear-sky fraction (a) and number of biomes
(c) at 25-km2 resolution. References 1. Merchant, C.J.; Paul, F.; Popp, T.; Ablain, M.; Bontemps, S.; Defourny, P.; Hollmann, R.; Lavergne, T.;
Laeng, A.; de Leeuw, G.; et al. Uncertainty information in climate data records from Earth Observation. Earth Syst. Sci. Data 2017, 9, 511–527. [CrossRef] 2. Freitas, S.C.; Trigo, I.F.; Bioucas-Dias, J.M.; Göttsche, F.M. Quantifying the Uncertainty of Land Surface
Temperature Retrievals From SEVIRI/Meteosat. EEE Trans. Geosci. Remote Sens. 2010, 48, 523–534. [CrossRef] 2. Freitas, S.C.; Trigo, I.F.; Bioucas-Dias, J.M.; Göttsche, F.M. Quantifying the Uncertainty of Land Surface
Temperature Retrievals From SEVIRI/Meteosat. EEE Trans. Geosci. Remote Sens. 2010, 48, 523–534. [CrossRef]
3. Hulley, G.C.; Hughes, C.G.; Hook, S.J. Quantifying uncertainties in land surface temperature and emissivity
retrievals from ASTER and MODIS thermal infrared data. J. Geophys. Res.-Atmos. 2012, 117, D23113. 3. Hulley, G.C.; Hughes, C.G.; Hook, S.J. Quantifying uncertainties in land surface temperature and emissivity
retrievals from ASTER and MODIS thermal infrared data. J. Geophys. Res.-Atmos. 2012, 117, D23113. [CrossRef] 4. Bulgin, C.E.; Embury, O.; Corlett, G.; Merchant, C.J. Independent uncertainty budget estimates for
coefficient based sea surface temperature retrieval from the Along-Track Scanning Radiometer instruments. Remote Sens. Environ. 2016, 178, 213–222. [CrossRef] 5. Ghent, D.J.; Corlett, G.K.; Göttsche, F.M.; Remedios, J.J. Global Land Surface Temperature from the
along-Track Scanning Radiometers. J. Geophys. Res. Atmos. 2017, 122, 12167–12193. [CrossRef] 6. Corlett, G.; Atkinson, C.; Rayner, N.; Good, S.; Fiedler, E.; McLaren, A.; Hoeyer, J.; Bulgin, C.E. CCI Phase 1 (SST): Product Validation and Intercomparison Report (PVIR); ESA SST CCI Project,
SST_CCI-PVIR-UoL-001; 2015. Available online: www.esa-sst-cci.org/PUG/pdf/SST_CCI-PVIR-UoL-
201-Issue_1-signed.pdf (accessed on 17 April 2018). 7. Merchant, C.J.; Ghent, D.; Kennedy, J.; Good, E.; Hoyer, J. Common Approach to Providing Uncertainty Estimates
Across All Surfaces; EUSTACE (640171) Deliverable 1.1; 2015. Available online: https://www.eustaceproject. eu/eustace/static/media/uploads/Deliverables/eustace_d1-2.pdf (accessed on 17 April 2018). 8. Bulgin, C.E.; Embury, O.; Merchant, C.J. Sampling Uncertainty in Global Area Coverage (GAC) and G
Sea Surface Temperature Products. Remote Sens. Environ. 2016, 177, 287–294. [CrossRef] 9. Zavody, A.M; Mutlow, C.T.; Llewellyn-Jones, D.T. Cloud Clearing over the Ocean in the Processing of Data
from the Along-Track Scanning Radiometer (ATSR). J. Atmos. Ocean. Technol. 2000, 17, 595–615. [CrossRef] 10. Thomas, G.E.; Poulsen, C.A.; Siddans, R.; Carboni, E.; Grainger, R.G.; Povey, A.C. Algorithm Theoretical
Basis Document (ATBD) AATSR Optimal Retrieval of Aerosol and Cloud (ORAC). 2015. Version 2.1, ESA
Climate Change Initiative, Aerosol_cci_ORAC_ATBD_V2.1.pdf. Available online: http://www.esa-aerosol-
cci.org/?q=webfm_send/1022/1 (accessed on 1 November 2017). 11. 5. Discussion and Conclusions at a gridded resolution of 625 km2), LST data producers are encouraged to quantify and provide
cloud-related uncertainties in gridded data products. Acknowledgments: We acknowledge the work of G. H. Griffiths (Institute for Environmental Analytics, University
of Reading) in developing the bespoke manual cloud clearing tool for the cloud clearing inter-comparison exercise. We also acknowledge NCEO funding, supporting the preparation of the cloud clearing inter-comparison datasets,
and ESA Data User Element (DUE) GlobTemperature funding for conducting this research. For further information
and possible access to the datasets used please contact the corresponding author. Author Contributions: The lead author, Claire Bulgin, undertook the majority of this work with input from
Christopher Merchant. Darren Ghent, Lars Klüser, Thomas Popp, Caroline Poulsen and Larissa Sogacheva all
provided data used in the analysis by taking part in the GlobTemperature cloud mask inter-comparison exercise. Conflicts of Interest: The authors declare no conflict of interest. 5. Discussion and Conclusions Right-hand panels show the number of observations in each clear-sky bin (b)
and for each number of biomes represented in a given grid cell (d). The following are shown: all data
(solid line), night-time only (dot-dash line) and daytime only (dashed line). When considering the cloud detection uncertainty as a function of the number of biomes, the range
is smaller at 25-km2 resolution than at 625-km2 resolution. Here, we find that the number of biomes
typically ranges between one and eight, with cloud detection uncertainties remaining fairly constant
between one and four biomes and increasing thereafter. This increase happens at lower numbers
of biomes in a given grid cell than for 625-km2 resolution data, where cloud detection uncertainty
tends to increase for 7+ biomes. As in the comparison against the clear-sky fraction, uncertainties in
the daytime data are of a larger magnitude than at night, with a typical difference of ∼0.6–1 K. For
pixels containing 4–8 biomes, where a direct comparison can be made with the 625-km2 data, the cloud
detection uncertainties are lower in the 25-km2 data, typically by a magnitude of ∼0.5 K. These analyses all provide insight into the magnitude of cloud detection uncertainties for Level
3 data gridded at different resolutions, but do not provide a mathematical model of cloud detection
uncertainty for a single given cloud mask, which is usually all that is available for any given LST
product. The uncertainty in LST arising from any single mask will be dependent on the tendency of
that mask to over-flag or under-flag, and whether this varies as a function of the underlying land cover
classification and vegetation fraction. This study does not separate cloud detection uncertainties due
to omission (missed cloud) from those due to commission (unnecessarily flagged clear-sky pixels),
although it could be hypothesised that the former may typically be larger in magnitude across many
land cover classifications, as clouds are often significantly colder than the underlying land surface. Given that the structural uncertainties arising from cloud detection errors are not insignificant (typically
of order 1.95 K for snow, 1.2 K for forest and 0.9 K for bare soil with a clear-sky fraction of 74.2% 18 of 20 18 of 20 Remote Sens. 2018, 10, 616 at a gridded resolution of 625 km2), LST data producers are encouraged to quantify and provide
cloud-related uncertainties in gridded data products. References Remote Sens. Environ. 2015,
162, 396–407. [CrossRef] 22. Hutchison, K.D.; Jackson, J.M. Cloud detection over desert regions using the 412 nanometer MODIS channel. Geophys. Res. Lett. 2003, 30, 2187–2190. [CrossRef] 23. Simpson,
J.J.;
Gobat,
J.I. Improved cloud detection for daytime AVHRR scenes over land. Remote Sens. Environ. 1996, 55, 21–49. [CrossRef] 24. Bendix, J.; Rollenbeck, R.; Palacios, W.E. Cloud detection in the Tropics—A suitable tool for climate-ecological
studies in the high mountains of Ecuador. Int. J. Remote Sens. 2004, 25, 4521–4540. [CrossRef] 25. Thorne, P.W.; Parker, D.E.; Christy, J.R.; Mears, C.A. Uncertainties in climate trends: Lessons from Upper-Air
Temperature Records. Bull. Am. Meteorol. Soc. 2005, 10, 1437–1442. [CrossRef] 26. Cho, H.-M.; Zhang, Z.; Meyer, K.; Lebsock, M.; Platnick, S.; Ackerman, A.S.; Di Girolamo, L.; Labonnote, L.C.;
Cornet, C.; Riedi, J.; et al. Frequency and causes of failed MODIS cloud property retrievals for liquid phase
clouds over global oceans. J. Geophys. Res.-Atmos. 2015, 120, 4132–4154. [CrossRef] 27. Good, E.J.; Ghent, D.J.; Bulgin, C.E.; Remedios, J.J. A spatiotemporal analysis of the relationship between
near-surface air temperature and satellite land surface temperatures using 17 years of data from the ATSR
series. J. Geophys. Res.-Atmos. 2017, 122, 9185–9210. [CrossRef] 28. Berrisford, P.; Dee, D.; Poli, P.; Brugge, R.; Fielding, K.; Fuentes, M.; Kaliberg, P.; Kobayashi, S.; Uppala,
S.; Simmons, S. The ERA-Interim archive Version 2.0; ERA Report Series; 2011. Available online: https:
//www.ecmwf.int/en/elibrary/8174-era-interim-archive-version-20 (accessed on 17 April 2018). 29. Borbas, E.; Ruston, B. The RTTOV UWiremis IR Land Surface Emissivity Model;
EUMETSAT Satellite
Application Facility on Numerical Weather Prediction; NWPSAF-MO-VS-042; Version 1; 2010. Available
online: https://www.nwpsaf.eu/vs-reports/nwpsaf-mo-vs-042.pdf (accessed on 17 April 2018). 30. Vidot, J.; Borbás, É. Land surface VIS/NIR BRDF atlas for RTTOV-11: Model and validation against SEVIRI
land SAF albedo product. Q. J. R. Meteorol. Soc. 2014, 140, 2186–2196. [CrossRef] 31. Hocking J.; Rayer, P.; Rundle, D.; Saunders, R.; Matricardi, M.; Geer, A.; Brunel, P.; Vidot, J. RTTOV
v11 Users Guide; EUMETSAT Satellite Application Facility on Numerical Weather Prediction (NWP SAF),
NWPSAF-MO-UD-028; 2014. Available online: www.nwpsaf.eu/site/download/documentation/rtm/
docs_rttov11/user_guide_11_v1.4pdf?e6316e&e6316e (accessed on 17 April 2018). 32. Griffiths, G.H. Cloudmask: Release 1.1. 2017. Available online: https://doi.org/10.5281/zenodo.1095557
(accessed on 1 November 2017). 33. Sogacheva, L.; Kolmonen, P.; Virtanen, T.H.; Rodriguez, E.; Sapanaro, G.; de Leeuw, G. Post-processing
to remove residual clouds from aerosol optical depth retrieved using the Advance along Track Scanning
Radiometer. Atmos. Meas. Tech. 2017, 10, 491–505. [CrossRef] 34. Ghent, D. References Kolmonen, P.; Sogacheva, L.; Virtanen, T.H.; de Leeuw, G.; Kulmala, M. The ADV/ASV AATSR aerosol
retrieval algorithm: current status and presentation of a full-mission AOD dataset. Int. J. Digit. Earth 2016,
9, 545–561. [CrossRef] 12. Jang, J.-D.; Viau, A.A.; Anctil, F.; Bartholomé, E. Neural network application for cloud detection in SPOT
VEGETATION images. Int. J. Remote Sens. 2006, 27, 719–736. [CrossRef] 12. Jang, J.-D.; Viau, A.A.; Anctil, F.; Bartholomé, E. Neural network application for cloud detection in SPOT
VEGETATION images. Int. J. Remote Sens. 2006, 27, 719–736. [CrossRef]
13. Yhann, S.R.; Simpson, J.J. Application of neural networks to AVHRR cloud segmentation. IEEE Trans. Geosci. R
t S
1995 33 590 604 [C
R f] Yhann, S.R.; Simpson, J.J. Application of neural networks to AVHRR cloud segmentation. IEEE Trans. Geosci
Remote Sens. 1995, 33, 590–604. [CrossRef] 14. Sus, O.; Stengel, M.; Stapelberg, S.; McGarragh, G.; Poulsen, C.A.; Povey, A.C.; Schlundt, C.; Thomas, G.;
Christensen, M.; Proud, S.; et al. The Community Cloud retrieval for Climate (CC4CL). Part I: A framework
applied to multiple satellite imaging sensors. Atmos. Meas. Tech. Discuss. 2017. [CrossRef] 19 of 20 19 of 20 Remote Sens. 2018, 10, 616 15. McGarragh, G.R.; Poulsen, C.A.; Thomas, G.E.; Povey, A.C.; Sus, O.; Stapelberg, S.; Schlundt, C.; Proud, S.;
Christensen, M.W.; Stengel, M.; et al. The Community Cloud retrieval for CLimate (CC4CL). Part II: The
optimal estimation approach. Atmos. Meas. Tech. Discuss. 2017. [CrossRef] 6. Klüser, L.; Killius, N.; Gesell, G. APOLLO NG—A probabilistic interpretation of the APOLLO legacy
AVHRR heritage channels. Atmos. Meas. Tech. 2015, 8, 4155–4170. [CrossRef] 17. Bulgin, C.E.; Sembhi, H.; Ghent, D.; Remedios, J.J.; Merchant, C.J. Cloud clearing techniques over land for
land surface temperature retrieval from the Advanced along Track Scanning Radiometer. Int. J. Remote Sens. 2014, 35, 3594–3615. [CrossRef] 18. Merchant, C.J.; Harris, A.R.; Maturi, E.; MacCallum, S. Probabilistic physically-based cloud screening of
satellite infra-red imagery for operational sea surface temperature retrieval. Q. J. R. Meteorol. Soc. 2005,
131, 2735–2755. [CrossRef] 19. Ellrod, G.P. Advances in the detection and analysis of fog at night using GOES multispectral infrared imagery. Weather Forecast. 1995, 10, 606–619. [CrossRef] 20. Breon, F.M.; Colzy, S. Cloud detection from the spaceborne POLDER instrument and validation against
surface synoptic observations. J. Appl. Meteorol. 1999, 38, 777–785. [CrossRef] 21. Bulgin, C.E.; Eastwood, S.; Embury, O.; Merchant, C.J. Sea surface temperature climate change initiative:
alternative image classification algorithms for sea-ice affected oceans. References LST Validation and Algorithm Verification; Long-Term Land Surface Temperature Validation
Project; Technical Note 19054/05/NL/FF; 2012. Available online: https://earth.esa.int/documents/700255/
2411932/QC3_D4.1+Validation_Report_Issue_1A_20120416.pdf (accessed on 17 April 2018). 20 of 20 Remote Sens. 2018, 10, 616 20 of 20 35. Prata F. Land Surface Temperature Measurement from Space: AATSR Algorithm Theoretical Basis Document. ESA Tech. Note 2002, 2002, 1–34. 36. Arino, O.; Leroy, M.; Bicheron, P.; Brockman, C.; Defourney, P.; Vancutsem, C.; Achard, C.; Durieux, L.;
Bourg, L.; Latham, J.; et al. GlobCover: ESA service for Global Land Cover from MERIS. In Proceedings
of the IEEE International Geoscience and Remote Sensing Symposium (IGARSS 2007), Barcelona, Spain,
23–28 July 2007; pp. 2412–2415. 37. Baret, F.; Weiss, M.; Lacaze, R.; Camacho, F.; Makhmara, H.; Pacholcyzk, P.; Smets, B. GEOV1: LAI and
FAPAR essential climate variables and FCOVER global time series capitalising over existing products. Part1:
Principles of development and production. Remote Sens. Environ. 2013, 137, 299–309. [CrossRef] 38. Ghent, D.; Trigo, I.; Pires, A.; Sardou, O.; Bruniquel, J.; Göttsche, F.; Martin, M.; Prigent, C.;
Jimenez,
C. Product
User
Guide;
DUE
GlobTemperature
Project;
GlobT-WP3-DEL-11;
2016. Available online: http://www.globtemperature.info/index.php/public-documentation/deliverables-1/
108-globtemperature-product-user-guide/file (accessed on 1 November 2017). c⃝2018 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/).
|
https://openalex.org/W2172840010
|
https://europepmc.org/articles/pmc4646955?pdf=render
|
English
| null |
Potential for Genetic Improvement of Sugarcane as a Source of Biomass for Biofuels
|
Frontiers in bioengineering and biotechnology
| 2,015
|
cc-by
| 17,013
|
Abbreviations: AFLP, amplified fragment length polymorphism; BAC, bacterial artificial chromosome; CAD, cinnamyl
alcohol dehydrogenase (EC 1.1.1.195); cDNA, complementary DNA; COMT, caffeic acid O-methyltransferase (EC 2.1.1.68);
DArT, diversity array technology; EST, expressed sequence tag; Gb/Mb, gigabase/megabase; LD, linkage disequilibrium;
Lignin G, lignin guaiacyl; Lignin H, lignin hydroxyphenyl; Lignin S, lignin syringyl; NGS, next-generation sequencing; QTL,
quantitative trait locus; RFLP, restricted fragment length polymorphism; RNAi, RNA interference; S/G ratio, syringyl/guaiacyl
ratio; SNP, single nucleotide polymorphism; SUCEST, sugarcane EST database; TF, transcription factor; TIGR, the institute
for genome research. Edited by:
P. C. Abhilash,
Banaras Hindu University, India Edited by:
P. C. Abhilash,
Banaras Hindu University, India Reviewed by:
Yu-Shen Cheng,
National Yunlin University of Science
and Technology, Taiwan
Tianju Chen,
Chinese Academy of Sciences, China
*Correspondence:
Nam V. Hoang
hoang.nam@uq.net.au Reviewed by:
Yu-Shen Cheng,
National Yunlin University of Science
and Technology, Taiwan
Tianju Chen,
Chinese Academy of Sciences, China *Correspondence:
Nam V. Hoang
hoang.nam@uq.net.au Specialty section:
This article was submitted to
Bioenergy and Biofuels, a section of
the journal Frontiers in Bioengineering
and Biotechnology Potential for Genetic Improvement
of Sugarcane as a Source
of Biomass for Biofuels 1 Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, QLD, Australia,
2 College of Agriculture and Forestry, Hue University, Hue, Vietnam, 3 Sugar Research Australia, Indooroopilly, QLD, Australia,
4 Joint BioEnergy Institute, Emeryville, CA, USA Sugarcane (Saccharum spp. hybrids) has great potential as a major feedstock for
biofuel production worldwide. It is considered among the best options for producing
biofuels today due to an exceptional biomass production capacity, high carbohydrate
(sugar + fiber) content, and a favorable energy input/output ratio. To maximize the conver-
sion of sugarcane biomass into biofuels, it is imperative to generate improved sugarcane
varieties with better biomass degradability. However, unlike many diploid plants, where
genetic tools are well developed, biotechnological improvement is hindered in sugarcane
by our current limited understanding of the large and complex genome. Therefore,
understanding the genetics of the key biofuel traits in sugarcane and optimization of
sugarcane biomass composition will advance efficient conversion of sugarcane biomass
into fermentable sugars for biofuel production. The large existing phenotypic variation
in Saccharum germplasm and the availability of the current genomics technologies will
allow biofuel traits to be characterized, the genetic basis of critical differences in biomass
composition to be determined, and targets for improvement of sugarcane for biofuels
to be established. Emerging options for genetic improvement of sugarcane for the use
as a bioenergy crop are reviewed. This will better define the targets for potential genetic
manipulation of sugarcane biomass composition for biofuels. REVIEW REVIEW
published: 17 November 2015
doi: 10.3389/fbioe.2015.00182 published: 17 November 2015
doi: 10.3389/fbioe.2015.00182 Keywords: sugarcane, biofuels, biomass for biofuels, biofuel traits, association studies INTRODUCTION Plant biomass from grasses such as sugarcane or woody species contains mostly cellulose, hemicel-
lulose, and lignin (also referred to as lignocellulosic biomass), which can be converted to biofuels as
a source of renewable energy. At the moment, plant biomass-derived biofuels have great potential in
countries that have limited oil resources because they reduce the dependence on fossil fuel, mitigate
air pollution by cutting down greenhouse gas emissions, and can be produced from a wide range Received: 25 July 2015
Accepted: 26 October 2015
Published: 17 November 2015 Biology and Genetics At the moment,
the cost of bagasse pretreatment (to remove or separate its recal-
citrant components before converting to biofuels) and microbial
enzymes contributes mostly to the total production cost, resulting
in reducing the incentive to transition from first generation to
second generation of biofuels in sugarcane (Yuan et al., 2008). To
maximize the efficiency of conversion of sugarcane biomass into
biofuels, it is imperative to generate improved sugarcane cultivars
with not only high biomass yield and fiber content but also better
biomass degradability for conversion to biofuels in addition to
improving the pretreatment and enzyme digestion technologies. Hybrid sugarcane was derived from crosses between a female
S. officinarum (2n = 80) and a male S. spontaneum (2n = 40–128). Due to the female restitution phenomenon, at first, the F1 hybrid
conserves the whole S. officinarum chromosome set and half of
the S. spontaneum which was 2n + n, then a few backcrosses
later, this hybrid breaks down to n + n, establishing the hybrid
chromosome set of modern sugarcane hybrid (Bremer, 1961). For
this reason, current sugarcane cultivars (Saccharum spp. hybrids)
have a combination of a highly aneuploid and interspecific set
of chromosomes. By using genomic in situ hybridization (GISH)
and fluorescent in situ hybridization (FISH), it is revealed that
among chromosomes in the nucleus of modern hybrid sugarcane,
approximately 80% are contributed by S. officinarum, 10–20%
from S. spontaneum, and less than 5–17% from recombination of
chromosomes of the two species (D’Hont et al., 1996; Piperidis
et al., 2001; Cuadrado et al., 2004). Modern sugarcane hybrids
are normally crosses between varieties/clones, which makes the
combination of the chromosomes in each offspring unique and
unpredictable due to the random sorting of the chromosomes in
the genome (Grivet and Arruda, 2002). The first sugarcane breed-
ing program, which started more than one century ago, generated
a few interspecific hybrids and constitutes the basic germplasm
used by sugarcane breeding programs (Ming et al., 2010). Modern
sugarcane cultivars are derived from the basic germplasm, but This review focuses on the potential for the genetic improve-
ment of sugarcane as a source of biomass for biofuels, exploring
the beneficial characteristics of sugarcane, the available genetic
resources and germplasm, the potential of cell wall modifi-
cation by breeding and biotechnology, and the potential of
whole genome/transcriptome sequencing applications in dis-
secting important biofuel traits to improve sugarcane biomass
composition. Biology and Genetics Taxonomically, sugarcane belongs to the genus Saccharum (estab-
lished by Carl Linnaeus in 1753), in the grass family Poaceae
(or Gramineae), subfamily Panicoideae, tribe Andropogoneae, sub-
tribe Sacharinae, under the group Saccharastrae and has a very
close genetic relationship to sorghum and other grass family
members such as Erianthus and Miscanthus (Amalraj and Bala-
sundaram, 2006). Typically, the genus is divided into six different
species namely Saccharum barberi, Saccharum edule, Saccharum
officinarum, Saccharum robustum, Saccharum sinense, and Sac-
charum spontaneum (Daniels and Roach, 1987; Amalraj and Bala-
sundaram, 2006), in which S. spontaneum and S. robustum are wild
species; S. officinarum, S. barberi, and S. sinense are early cultivars
while S. edule is a marginal specialty cultivar. All genotypes of
the Saccharum genus are reported to be polyploid with the ploidy
level ranging from 5× to 16× and are considered as among the
most complex plant genomes (Manners et al., 2004). The cytotype
(2n, the number of chromosomes in the cell) was reported to be
different in each species as follows: S. officinarum (2n = 80), S. spontaneum (2n = 40–128), S. barberi (2n = 111–120), S. sinense
(2n = 81–124), S. edule (2n = 60–80), and S. robustum (2n = 60,
80); hence, the basic chromosome number (x, the monoploid
set of chromosomes in the cell) ranges from 5, 6, 8, 10 to 12
(Sreenivasan et al., 1987). The basic chromosome number of S. spontaneum is 8 (even though a number of very variable cytotype
is observed) and of S. officinarum and S. robustum is 10 [Panje
and Babu, 1960, D’Hont et al. (1998), and Piperidis et al. (2010)]. For the other three species, S. sinense, S. barberi, and S. edule, due
to the fact that these are early interspecific hybrid cultivars, there
have not been a consensus reported, but a study by Ming et al. (1998) suggested that the basic chromosome number for these
three species could also be 10. Conventionally, sugarcane bagasse is usually burned to produce
fertilizer or steam and electricity to fuel the boilers in sugar
mills (Pandey et al., 2000). Recently, it has been used for biofuel
production; however, the production cost of biofuels from ligno-
cellulosic biomass is still considered to be relatively high, which
makes it difficult to be price-competitive and commercialized on
a large scale (Halling and Simms-Borre, 2008). Citation: Hoang NV, Furtado A, Botha FC,
Simmons BA and Henry RJ (2015)
Potential for Genetic Improvement
of Sugarcane as a Source
of Biomass for Biofuels. Front. Bioeng. Biotechnol. 3:182. doi: 10.3389/fbioe.2015.00182 November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 Sugarcane Genetic Improvement for Biofuels Hoang et al. SUGARCANE AT A QUICK GLANCE of abundant sources (Matsuoka et al., 2009). Biofuels generated
from plant lignocellulosic biomass (also known as the second
generation of biofuels) have been shown to be advantageous over
the first generation (from plant starches, sugar, and oil) in terms
of net energy and CO2 balance and, more importantly, they do
not compete with food industries for supplies (Yuan et al., 2008). To date, producing bioethanol from the sugar in sugarcane has
been one of the world’s most commercially successful biofuel pro-
duction systems, with the potential to deliver second-generation
fuels with a high positive energy balance and at a relatively low
production cost (Yuan et al., 2008; Botha, 2009; Matsuoka et al.,
2009). The rapid growth and high yield of sugarcane compared
to other grasses and woody plants makes it a good candidate for
ethanol processing platform and the second generation of biofuels
in general (Pandey et al., 2000). Sugarcane has an exceptional
ability to produce biomass as a C4 plant with the potential of a
perennial grass crop allowing harvest four to five times by using
ratoons without requiring replanting (Verheye, 2010), resulting in
a lower cost of energy production from sugarcane than for most
of the other potential sources of biomass (Botha, 2009). Brazil is
the world’s first country to launch a national fuel alcohol program
(ProAlcooL). This program is based on the use of sugarcane and
substitutes the usage of gasoline by ethanol (Dias De Oliveira
et al., 2005). Approximately, 23.4 billion liters (6.19 billion U.S. liquid gallons) of ethanol was produced in Brazil in the year
2014 (Renewable Fuels Association, 2015). As of 2009, sugarcane
bagasse contributes to about 15% of the total electricity consumed
in Brazil, and it is predicted that energy generated from sugarcane
stalks could supply more than 30% of the country energy needs
by 2020 and will be equal to or more than the electricity produced
from hydropower (Matsuoka et al., 2009). The Nature of a Complex, Polyploid, and
Repetitive Genome Biofuels from sugarcane can be produced extensively not
only from its soluble sugar but also from main residues in sugar-
cane production, bagasse and trash, on the same production area
(Seabra et al., 2010; Alonso Pippo et al., 2011a,b; Macrelli et al.,
2012). The total estimated available lignocellulosic biomass from
sugarcane worldwide was 584 million dry tons per year, with an
average lignocellulosic biomass yield of 22.9 dry tons per hectare
per year (Van Der Weijde et al., 2013). Sugarcane bioethanol yield
from bagasse is estimated at about 3,000 L per hectare in a total
yield of 9,950 L per hectare from sugar and bagasse (Somerville
et al., 2010). Biology and Genetics This will define the targets for potential genetic
manipulation and better exploitation of sugarcane biomass for
biofuels. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org November 2015 | Volume 3 | Article 182 2 Sugarcane Genetic Improvement for Biofuels Hoang et al. TABLE 1 | Average lignocellulosic biomass yield (dry matter) from sugar-
cane compared to other sources. Plant name
Yield
(tons/ha/year)
Reference
Sugarcane
22.9a
Van Der Weijde et al. (2013)
Switch grass
7–35
Reviewed in Hattori and Morita (2010)
Miscanthus
12–40
Reviewed in Hattori and Morita (2010)
Erianthus
40–60
Reviewed in Hattori and Morita (2010)
Eucalyptus
15–40
Reviewed in Johansson and Burnham (1993)
aAverage total cane biomass dry matter is 39 tons/ha/year (Moore, 2009). TABLE 1 | Average lignocellulosic biomass yield (dry matter) from sugar-
cane compared to other sources. there has been only a few generations for chromosome recombi-
nation opportunities (the number of meiosis that chromosomes
have undergone is mainly about 2–7) as the sugarcane breeding
processes normally take between 10 and 15 years (Raboin et al.,
2008; Ming et al., 2010). As a result, the modern sugarcane popu-
lation has a narrow genetic basis and high linkage disequilibrium
(Roach, 1989; Lima et al., 2002; Raboin et al., 2008). The Nature of a Complex, Polyploid, and
Repetitive Genome Globally, sugarcane is
the most important crop in about 100 countries with a produc-
tion area of 26.9 million hectares, total production of ~1.9 billion
tons, and yield of 70.9 tons of fresh cane per hectare (FAOSTAT,
2015). At present, Brazil is the world’s largest sugarcane producer
followed by India, China, Thailand, Pakistan, Mexico, Colombia,
Indonesia, Philippines, U.S., and Australia (FAOSTAT, 2015). In
sugarcane internodal tissue, sucrose concentration ranges from 14
to 42% of the dry weight (Whittaker and Botha, 1997), while the
rest of dry biomass comes from the cell wall lignocellulose, mostly
containing cellulose, hemicellulose, lignin, and ash (Pereira et al.,
2015). Biofuels from sugarcane can be produced extensively not
only from its soluble sugar but also from main residues in sugar-
cane production, bagasse and trash, on the same production area
(Seabra et al., 2010; Alonso Pippo et al., 2011a,b; Macrelli et al.,
2012). The total estimated available lignocellulosic biomass from
sugarcane worldwide was 584 million dry tons per year, with an
average lignocellulosic biomass yield of 22.9 dry tons per hectare
per year (Van Der Weijde et al., 2013). Sugarcane bioethanol yield
from bagasse is estimated at about 3,000 L per hectare in a total
yield of 9,950 L per hectare from sugar and bagasse (Somerville
et al., 2010). et al., 2009; Moore, 2009; Henry, 2010a). At present, the most
suitable energy crop is probably sugarcane because of its high
biomass yield and the potential for production on other than
prime agricultural land avoiding competing with the land used for
food industries (Waclawovsky et al., 2010). Globally, sugarcane is
the most important crop in about 100 countries with a produc-
tion area of 26.9 million hectares, total production of ~1.9 billion
tons, and yield of 70.9 tons of fresh cane per hectare (FAOSTAT,
2015). At present, Brazil is the world’s largest sugarcane producer
followed by India, China, Thailand, Pakistan, Mexico, Colombia,
Indonesia, Philippines, U.S., and Australia (FAOSTAT, 2015). In
sugarcane internodal tissue, sucrose concentration ranges from 14
to 42% of the dry weight (Whittaker and Botha, 1997), while the
rest of dry biomass comes from the cell wall lignocellulose, mostly
containing cellulose, hemicellulose, lignin, and ash (Pereira et al.,
2015). Existing Variations within
Saccharum Germplasm Genetically diverse sugarcane germplasm may play a key role in
improving sugarcane for biofuels through breeding and biotech-
nological approaches. Genetic variation may be found in biomass
yield, fiber content, and sugar composition in the Saccharum
germplasm. This includes the diversity among the cultivars within
one species and also diversity among species within the genus. A relatively high genetic variability within sugarcane hybrid
cultivars was reported thanks to their heterozygosity and high
polyploidy despite their originating from a few clones of a
narrow genetic base (Aitken and McNeil, 2010). There is also
great genetic and morphological diversity within Saccharum
species, Miscanthus species, and Erianthus species to be poten-
tially exploited and incorporated to broaden the genetic base in The Nature of a Complex, Polyploid, and
Repetitive Genome aAverage total cane biomass dry matter is 39 tons/ha/year (Moore, 2009). p
The complex and polyploid genome of sugarcane makes the pro-
cess of analyzing and understanding difficult by normal methods
applied to diploid plants. The size of the sugarcane genome is
about 10 Gb while its genome complexity is due to the mixture of
euploid and aneuploid chromosome sets with homologous genes
present in from 8 to 12 copies (Souza et al., 2011). The estimated
monoploid genome size is about 750–930 Mb (the monoploid
genome size of the two parental species, S. officinarum and S. spon-
taneum, are 930 Mb and 750 Mb, respectively), not much larger
than the sorghum genome (~730 Mb) and about twice the size
of the rice genome (~380 Mb) (D’Hont and Glaszmann, 2001). On the other hand, studies revealed that despite this complex and
polyploid genome, sugarcane showed synteny with other grasses,
especially sorghum (collinear, due to the limited divergence time)
and maize (orthologous but altered loci collinearity) [reviewed
in Grivet and Arruda (2002)]. It was thought that the sugarcane
genome contains roughly the same amount of repetitive DNA as
in the sorghum genome (Jannoo et al., 2007); however, studies
on BAC-end sequences by Wang et al. (2010), Figueira et al. (2012), and Kim et al. (2013) suggested that there is less repet-
itive content in the sugarcane genome (e.g., 45.2% and 42.8%
repetitive sequences observed in large BAC collections in com-
parison to 61% in the sorghum genome). More recently, using
the k-mer approach, Berkman et al. (2014) found that the repet-
itive proportion in three sugarcane hybrid cultivars ranges from
63.74 to 78.37% and higher than that in the sorghum genome
(55.5%) using the same approach. The authors postulated that the
increased proportion could be attributed to ploidy level rather
than repetitive content in the sugarcane genome. A high gene-
copy number, the integration of two chromosome sets from two
different species, and a significant repeat content hinder the
understanding of how the genome functions and obtaining a gen-
uine assembled monoploid genome (Souza et al., 2011; Figueira
et al., 2012). et al., 2009; Moore, 2009; Henry, 2010a). At present, the most
suitable energy crop is probably sugarcane because of its high
biomass yield and the potential for production on other than
prime agricultural land avoiding competing with the land used for
food industries (Waclawovsky et al., 2010). Candidate Crop for Future Biofuels These collections could be potentially selected and utilized
for breeding purpose to improve sugarcane germplasm for new
biofuel traits (Todd et al., 2014). The wild sugarcane species show
wider variability in comparison to the domesticated species. In
the Saccharum genus, S. spontaneum has the widest range of
morphological variability, ratoon yielding, as well as biotic and
abiotic stress tolerance (Tai and Miller, 2001; Aitken and McNeil,
2010; Govindaraj et al., 2014). The coefficient of variation (CV%)
for some of the traits such as internode length, midrib width, leaf
width, plant height, and stalk height studied by Govindaraj et al. (2014) were reported to be between 15 and 30%, which indicates
a very high variability within the collection. It has been shown
that the diversity within modern sugarcane hybrids was mostly
contributed by the introgression from S. spontaneum (D’Hont
et al., 1996). On the other hand, S. robustum also possesses a large
amount of phenotypic variations in many traits studied (Aitken
and McNeil, 2010). Sugarcane parental species (S. officinarum,
S. spontaneum, and S. robustum), Miscanthus species, Erianthus
species, and sorghum species with their diversity in genome con-
tent, structure, and tremendous allelic variation are a valuable
and significant genetic reservoir which could be exploited for
improving sugarcane biomass. Therefore, it is difficult to allocate these markers into linkage
groups or cosegregation groups or sugarcane expected chromo-
some number (Souza et al., 2011). More detailed linkage maps of S. officinarum cultivar IJ76-545 (534 markers in 123 linkage groups)
and cultivar Green German (615 markers in 72 linkage groups); S. spontaneum cultivar IND (536 markers in 69 linkage groups); and
the hybrid cultivars R570 and Q165 (with 2,000 markers placed
in more than 100 linkage groups) have been constructed (Souza
et al., 2011; Aitken et al., 2014). Most recently, using Diversity
Array Technology (DArT), Aitken et al. (2014) integrated DArT
markers, RFLPs, AFLPs, SSRs, and SNPs into the largest marker
collection for sugarcane, which contains 2,467 single-dose mark-
ers for the cross between Q165 and IJ76-514 (a S. officinarum
accession) and 2,267 markers from the cultivar Q165. These were
placed into 160 linkage groups and eight homology groups, with
some uncategorized linkage groups indicating that more markers
are required. There is still a need to develop high-throughput
marker arrays for sugarcane association studies, to generate more
markers, and also to make use of the available markers. Candidate Crop for Future Biofuels These
markers will be a valuable resource in facilitating and unraveling
the complex genome structure of sugarcane. It is worth consider-
ing that information on DNA-based molecular markers of progen-
itor plants can potentially reveal available genetic polymorphism
for the analysis of their progenies (Henry et al., 2012). This could
be a useful strategy in the case of sugarcane, where the genomes
of the parental species are less complex than that of the hybrids. g
p
p
(
g
,
)
There are two world largest collections of germplasm of Sac-
charum species, one is located in Florida (USA) while the other
is in Kerala (India), containing approximately 1,200 accessions
collected from 45 countries (Tai and Miller, 2001; Todd et al.,
2014). These collections could be potentially selected and utilized
for breeding purpose to improve sugarcane germplasm for new
biofuel traits (Todd et al., 2014). The wild sugarcane species show
wider variability in comparison to the domesticated species. In
the Saccharum genus, S. spontaneum has the widest range of
morphological variability, ratoon yielding, as well as biotic and
abiotic stress tolerance (Tai and Miller, 2001; Aitken and McNeil,
2010; Govindaraj et al., 2014). The coefficient of variation (CV%)
for some of the traits such as internode length, midrib width, leaf
width, plant height, and stalk height studied by Govindaraj et al. (2014) were reported to be between 15 and 30%, which indicates
a very high variability within the collection. It has been shown
that the diversity within modern sugarcane hybrids was mostly
contributed by the introgression from S. spontaneum (D’Hont
et al., 1996). On the other hand, S. robustum also possesses a large
amount of phenotypic variations in many traits studied (Aitken
and McNeil, 2010). Sugarcane parental species (S. officinarum,
S. spontaneum, and S. robustum), Miscanthus species, Erianthus
species, and sorghum species with their diversity in genome con-
tent, structure, and tremendous allelic variation are a valuable
and significant genetic reservoir which could be exploited for
improving sugarcane biomass. Transcriptome Sequences and
Transcription Factors p
Expressed sequence tags (ESTs) and complementary DNA
(cDNA) sequences provide direct evidence of the genes present
in the samples, and this sequence information is very useful for
genome exploration, gene prediction/discovery, genome structure
identification, SNP characterization, and transcriptome and pro-
teome analysis (Nagaraj et al., 2007). As of May 2015, the GenBank
EST database (dbEST) was composed of 75,906,308 ESTs from
different organisms of which 284,818 hits were detected under
the search term sugarcane (“S. officinarum” or “Saccharum hybrid
cultivar” or sugarcane). In the last 20 years, sugarcane ESTs have
been used for gene discovery, BAC clone selection, and dissecting
the coding regions of the genome, involving many projects in
South Africa, France, U.S., Australia, and Brazil (Carson and
Botha, 2000, 2002; Vettore et al., 2001; Casu et al., 2003, 2004;
Grivet et al., 2003; Pinto et al., 2004; Bower et al., 2005). The largest
collection of sugarcane ESTs was generated by SUCEST, which is
composed of approximate 238,208 ESTs from 26 diverse cDNA
libraries of different tissues of sugarcane cultivars, e.g., SP80-3280,
SP70-1143, RB845205, RB845298, and RB805028 (Vettore et al.,
2001, 2003; Souza et al., 2011). These sequences were assembled
into 42,982 sugarcane assembled sequences representing more
than 30,000 unique genes (~90% of the estimated genes, about
43,141, of S. officinarum) (Vettore et al., 2003; Hotta et al., 2010;
Grassius: Grass Regulatory Information Server, 2015). There are
other sugarcane EST collections containing less EST entries gen-
erated by Casu et al. (2003, 2004) (8,342 ESTs), Ma et al. (2004)
(7,993 ESTs), Gupta et al. (2010) (~35,000 ESTs) and small number
of ESTs by Carson and Botha (2000, 2002). Frontiers in Bioengineering and Biotechnology | www.frontiersin.org Candidate Crop for Future Biofuels To date, sugarcane is among the most efficient crops in the world
together with other C4 grasses such as switch grass (Panicum
virgatum), Miscanthus species (Miscanthus x giganteus), and Eri-
anthus species (Erianthus arundinaceus Retz.) in terms of con-
verting solar energy into stored chemical energy and biomass
accumulation (Tew and Cobill, 2008; Furtado et al., 2014). In gen-
eral, C4 plants outperform C3 plants in biomass yield, including
grain, stem, and leaf yield (Jakob et al., 2009; Wang and Pater-
son, 2013). Sugarcane and other C4 grasses are the highest yield
potential feedstocks (Table 1), and for sugarcane, the potential
yield can exceed 100 tons dry matter per hectare per year (Jakob Frontiers in Bioengineering and Biotechnology | www.frontiersin.org November 2015 | Volume 3 | Article 182 3 Sugarcane Genetic Improvement for Biofuels Hoang et al. breeding programs (Harvey et al., 1994; Aitken and McNeil, 2010). To date, the genetic diversity of S. officinarum has been exploited
in breeding programs; however, the diversity of S. spontaneum and
other species have not been used much (Aitken and McNeil, 2010). Saccharum species have also been shown to have varied genome
size, S. officinarum genome is about 7.50–8.55 Gb, S. robustum
ranging from 7.56 to 11.78 Gb, and S. spontaneum ranging from
3.36 to 12.64 Gb, whereas the other three species – S. sinense, S. barberi, S. edule – and modern sugarcane are interspecific hybrids
whose genome size depends upon each cross (Zhang et al., 2012). breeding programs (Harvey et al., 1994; Aitken and McNeil, 2010). To date, the genetic diversity of S. officinarum has been exploited
in breeding programs; however, the diversity of S. spontaneum and
other species have not been used much (Aitken and McNeil, 2010). Saccharum species have also been shown to have varied genome
size, S. officinarum genome is about 7.50–8.55 Gb, S. robustum
ranging from 7.56 to 11.78 Gb, and S. spontaneum ranging from
3.36 to 12.64 Gb, whereas the other three species – S. sinense, S. barberi, S. edule – and modern sugarcane are interspecific hybrids
whose genome size depends upon each cross (Zhang et al., 2012). There are two world largest collections of germplasm of Sac-
charum species, one is located in Florida (USA) while the other
is in Kerala (India), containing approximately 1,200 accessions
collected from 45 countries (Tai and Miller, 2001; Todd et al.,
2014). Sorghum bicolor Genome as the Closest
Related Reference Genome Due to the homology between genomes, genome-wide map-
ping of ESTs of one species provides an important framework for
the genome structure of other related species (Sato et al., 2011). However, it is noteworthy that the discovery of the ESTs may
be restricted to specific cultivars, as within sugarcane germplasm
each cultivar has been shown to have different gene expres-
sion level [reviewed in Hotta et al. (2010)]. Moreover, for bio-
fuel trait analysis, the TFs regulating monolignol biosynthesis
in lignin pathway have received attention as understanding this
allows reducing and modifying lignin content and composition
which are essential in addressing the recalcitrant problem in
biomass conversion (Santos Brito et al., 2015). It is shown that the
lignin regulation can be species specific and information on TFs
obtained from model plants such as Arabidopsis may require to
be validated in other species (Santos Brito et al., 2015). A limited
number of TFs in grass and sugarcane have been preliminarily
characterized recently including those involved in monolignol
biosynthesis, for example, in grass (Handakumbura and Hazen,
2012), rice (Yoshida et al., 2013), sorghum (Yan et al., 2013),
and sugarcane (Santos Brito et al., 2015). Gene discovery of
sugarcane has progressed to some extent despite the complex-
ity of the genome. The valuable information of ESTs, TFs, full-
length cDNAs, and BACs provides an understanding of allelic
variations in the genome while a full-genome sequence is not
available. Sorghum is the most closely related species to sugarcane (Grivet
et al., 1994; Dillon et al., 2007). The sorghum genome sequencing
project was initiated and completed in 2007 with the total genome
size of ~730 Mb, and 34,496 protein-coding loci, at the coverage
of 8.5× using whole-genome shotgun sequencing by standard
Sanger methodologies (Paterson et al., 2009). The sequenced
genome is composed of 10 pairs of chromosomes and 3,294
supercontigs (most of these have been placed into chunks on 10
chromosomes), covering 90% of the genome and 99% of protein-
coding regions (including the majority of available non-repetitive
markers, known sorghum protein-coding genes, and the major-
ity of ESTs) (Paterson et al., 2009). The sorghum genome has
approximately 61% repetitive DNA, a low level of gene duplication
compared to other C4 grasses, and a high degree of gene paral-
lelism with sugarcane, even though the sugarcane genome is much
more polyploid (Paterson et al., 2009, 2010). Sorghum bicolor Genome as the Closest
Related Reference Genome Microcollinearity
between sugarcane and sorghum genomes indicated that sorghum
is suitable as the template for sugarcane genome assembly (Ming
et al., 1998; Wang et al., 2010; Figueira et al., 2012). It has been
suggested that the sugarcane genome could be 20–30% smaller
than that of sorghum despite the estimated monoploid genome
size of sugarcane being about 760–930 Mb, at approximately the
size of the sorghum genome (Figueira et al., 2012). BIOMASS-DERIVED BIOFUELS AND THE
CHALLENGING ISSUES IN BIOMASS
CONVERSION TO BIOFUELS g
Sugarcane cultivar R570 and other cultivars including ones from
the parental species S. officinarum and S. spontaneum have been
used for constructing of bacterial artificial chromosome (BAC)
libraries (Hotta et al., 2010). BAC libraries from the sugarcane
include hybrid cultivar R570 (103,296 clones, average insert size
of 130 kb and two other libraries of 100,000 clones) (Tomkins
et al., 1999; Grivet and Arruda, 2002), S. spontaneum cultivar
SES208 (38,400 clones, average insert size of 120 kb), and S. officinarum cultivar LA Purple (74,880 clones, average insert
size of 150 kb) generated from different restriction enzymes, e.g.,
HindIII and BamH1 [reviewed in Souza et al. (2011)]. BAC
sequencing in sugarcane is currently based on the sequencing
of BAC clones anchored to an available physical map. Even
though it requires a higher cost compared to the whole-genome
shotgun sequencing (using high-throughput platforms, Illumina,
for example), it is a reliable approach for reference construc-
tion, especially, for highly repetitive genomes which cannot
be sequenced and resolved by a short-read method (Eversole
et al., 2009; Steuernagel et al., 2009). This BAC sequencing
approach has been used successfully in sequencing of Arabidop-
sis, rice, and maize genomes and producing the barley reference
genome [reviewed in Steuernagel et al. (2009)]. The ongoing
Sugarcane Genome Sequencing Initiative (SUGESI) has selected
5,000 BAC clones for sequencing from a library by Tomkins
et al. (1999) of cultivar R570, the most intensively character-
ized cultivar to date, to help assembly of the monoploid cov-
erage (monoploid tiling path) of the sugarcane genome using
the sorghum sequence as the guide (Souza et al., 2011; Sugesi,
2015). Genetic Markers and Maps To support the effort of understanding the sugarcane genome,
many physical maps, molecular markers, and resources such as
RFLP, RAPD, AFLP, SSR, and ESTs have been developed over
time. These common markers have been applied for genetic stud-
ies such as diversity, mapping, quantitative trait loci (QTL), and
synteny definition; however, these systems have been developed
mostly for well-established diploid species and are less effective
for polyploidy plants (Garcia et al., 2013). Markers like AFLP,
SSR, and RFLP are unable to estimate the number of allelic copies
and level of polyploidy in complicated genomes such as potato,
strawberry, and sugarcane (Garcia et al., 2013). More recently,
the use of SNPs markers, which are distributed at high density
across the genome, for complex genomes can allow estimation of
the number of allelic copies and the ploidy level of genomes (Zhu
et al., 2008; Hall et al., 2010). The currently available genetic maps
and markers have been generated for sugarcane by using low-
throughput methods, providing limited information on genome
organization due to the low density of markers and coverage
(most of them have less than 1,000 markers) (Aitken et al., 2014). Frontiers in Bioengineering and Biotechnology | www.frontiersin.org November 2015 | Volume 3 | Article 182 4 Sugarcane Genetic Improvement for Biofuels Hoang et al. Dealing with the Conversion Issues Dealing with the Conversion Issues
Even though sugarcane biomass is less resistant to enzymatic
digestion compared to that from woody plants, it is reported
that biomass recalcitrant components impede the efficiency of
the conversion to ethanol (Jung, 1989; Anterola and Lewis, 2002;
Chen and Dixon, 2007; Himmel et al., 2007; Balat et al., 2008;
Li et al., 2013). Biomass recalcitrance is caused by many factors
such as the presence of epidermal and sclerenchyma tissues, vas-
cular bundle density and arrangement, degree of lignification,
heterogeneity and complexity of cell wall constituents, insolu-
ble matter, natural inhibitors, and cellulose crystallinity (Him-
mel et al., 2007). Most approaches for producing biofuels from
biomass at the moment rely on the disruption of the biomass,
to separate lignocellulose and remove lignin in the biomass, and
then conversion using microbial enzymes (Sticklen, 2006). In
general, overcoming the recalcitrant issue can be addressed by
physical, chemical, and genetic approaches. Physical and chemical
strategies deal mainly with the pretreatment and involve loosening
the cell wall structure, lowering the biomass heterogeneity, pro-
viding the enzymes access to the cellulose, cleaving the crossing
linkages, and removing enzymatic inhibitors (Balat et al., 2008;
Saathoff et al., 2011). To make the physical and chemical changes
in plant biomass, pretreatment processing conditions must be
tailored to the specific chemical and structural composition of the
various and variable sources of lignocellulosic biomass (Mosier
et al., 2005). Currently available physical and chemical pretreat-
ment methods are varied and can be listed as uncatalyzed steam
explosion, flow-through acid, liquid hot water, pH-controlled hot
water, dilute acid, ammonia, lime and, more recently, the method
using ionic liquids (Mosier et al., 2005; Shi et al., 2013; Sun
et al., 2013). Genetic approaches involve genetic enhancement,
molecular biology, and plant breeding efforts to improve biomass
sources by having crops with less lignin, modified lignin, crops
that self-produced enzymes, and crops with increased cellulose
and biomass overall [reviewed in Sticklen (2006)]. The Second Generation of Biofuels –
Cell Walls for Fuels Due to the depletion of fossil fuel sources, the potential for oil
to become more expensive, and the raising awareness of the
negative impact of fossil fuels on the environment, biomass-
derived biofuels have been investigated and developed recently as
an alternative source of renewable, sufficient, and clean energy
(Botha, 2009). The demand for renewable biofuels is predicted
to be increasing (Fedenko et al., 2013). The first generation of
biofuels from plant biomass involved the process of conversion
of stored polysaccharides, non-structural carbohydrates, and oils
from plants (starchy, sugary, and oily parts of plants such as corn
starch, sugarcane molasses, soybeans, canola seeds, and palm oil)
into fuels like ethanol and diesel (Schubert, 2006; Yuan et al.,
2008). However, these sources are also used as food supplies
and are limited due to the increasing demand from the growing
world’s population (Schubert, 2006). The second generation of
biofuels can be generated by using the non-food parts of plants
such as cell walls, composed of structural polysaccharides, such
as cellulose and hemicellulose (Schubert, 2006; Yuan et al., 2008;
Henry, 2010a). This is considered to be advantageous over the
first generation of biofuels as it has a higher energy production
potential, lower cost, sustainable CO2 balance, no competition
with the food production, and a wide range of plant biomass
sources are available at costs affordable to a biorefinery (Yuan
et al., 2008; Henry, 2010a). As of 2009, sugarcane biomass as November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 5 Sugarcane Genetic Improvement for Biofuels Hoang et al. and the inner part of stem has higher syringyl/guaiacyl (S/G) ratio
than the outer part. Polysaccharides found in sugarcane leaf and
culm walls were similar but different in the proportions of xyloglu-
can and arabinoxylan (Souza et al., 2013). The major monosaccha-
rides released from sugarcane cell walls were glucose, xylose, and
arabinose (Loureiro et al., 2011; Rabemanolontsoa and Saka, 2013;
Souza et al., 2013). Understanding the fine structure and detailed
composition of sugarcane cell wall will assist in optimizing the
tissue pretreatment and cell wall hydrolysis protocol. The Second Generation of Biofuels –
Cell Walls for Fuels At present,
converting sugarcane lignocellulosic biomass to ethanol includes
(1) pretreatment to remove the lignin and other recalcitrant cel-
lular constituents (or hemicellulose) to free cellulose, (2) enzyme-
mediated action to depolymerize carbohydrates to simple sugars,
and (3) fermentation of sugars and distillation of ethanol as the
end product (Canilha et al., 2012). sucrose accounted for about 40% of biofuels feedstock worldwide
for first-generation biofuel production (Lam et al., 2009). Using
sugarcane bagasse as a feedstock for second-generation biofuels
would lead to doubling the current output of biofuel production
from sugarcane (Halling and Simms-Borre, 2008). Frontiers in Bioengineering and Biotechnology | www.frontiersin.org Sugarcane Cell Wall and Biomass
Composition Physically, sugarcane biomass can be divided into four major frac-
tions, whose content depends on the industrial process: fiber (het-
erogeneous organic solid fraction), non-soluble solids (inorganic
substances), soluble solids (sucrose, waxes, and other chemicals),
and water (Canilha et al., 2012; Shi et al., 2013). Second generation
of biofuels focuses on using the fiber fraction especially the cell
wall constituents of the plant to produce biofuels (Schubert, 2006;
Henry, 2010a). This approach may be made more efficient by opti-
mizing the composition of the biomass source for biofuel produc-
tion. This could be achieved by advances in pretreatment methods
or biotechnological modification of cell wall synthesis pathways
to create a biomass that can be more efficiently processed (Sims
et al., 2006; Yuan et al., 2008; Simpson, 2009; Viikari et al., 2012). Three major components make up the fiber fraction of sugarcane,
namely, cellulose, hemicellulose (or non-cellulosic polysaccharide
components), and lignin. Cellulose constitutes around 50% of the
dry weight sugarcane bagasse while hemicellulose and lignin each
account for about 25% (Loureiro et al., 2011). These three com-
ponents are biosynthesized through different complex pathways
(Higuchi, 1981; Whetten and Ron, 1995; Saxena and Brown, 2000;
Mutwil et al., 2008; Harris and DeBolt, 2010; Pauly et al., 2013). Cellulose and hemicellulose molecules form the cell walls which
act as the skeleton of plants and are strengthened by lignin and
phenolic cross-linkages (Carpita, 1996; Henry, 2010b). The com-
plex interlinking between cell wall components plays an important
role in grass defense and yet challenges the biofuel production by
requiring the pretreatment to separate them (De O. Buanafina,
2009). Dealing with the Conversion Issues The costs of the
enzymatic pretreatment of cellulosic biomass (which accounts for
about 25% of total processing expenses), biomass conversion, and
microbial tanks limit the price-competitiveness of biofuel from
lignocellulosic biomass in comparison to fossil fuel (Gnansounou
and Dauriat, 2010; Macrelli et al., 2012, 2014; Van Der Weijde The sugarcane and grass cell wall are categorized as type II
cell wall, which differs from the type I and type III cell walls of
other plants [reviewed in Souza et al. (2013)]. In general, there is
little pectin, less lignin, and less structural proteins in grass cell
walls than that in the non-grasses (Carpita, 1996; Henry, 2010b;
Saathoff et al., 2011). There is similar cellulose content between
grass and non-grass primary and secondary cell walls; however,
hemicellulose composition is different between two groups. Grass
cell walls have four to eight times more xylans, higher mixed
linkage glucans, and lower levels of xyloglucans, mannans, gluco-
mannans, and pectin in primary cell wall, but higher phenolics
and lignin in the secondary cell wall (Loureiro et al., 2011). Grassy lignin is composed of three monolignols (lignin syringyl –
S, lignin guaiacyl – G and lignin hydroxyphenyl – H subunits)
forming various ratios of them and normally has more H subunit
(more coumaryl derivatives) than in non-grasses (Vogel, 2008). A
recent study by Bottcher et al. (2013) showed that sugarcane lignin
content and composition are varied depending on tissue types
and stem positions on the plant. Within one plant, the bottom
internode has higher lignin accumulation than the top internode, November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 6 Sugarcane Genetic Improvement for Biofuels Hoang et al. the genetic diversity of the germplasm (Tabasum et al., 2010;
Berkman et al., 2012). The use of markers associated with the
desired traits in combination with the advances in next-generation
sequencing (NGS) technology, bioinformatics tools, and high-
throughput phenotyping methods will significantly improve the
sugarcane breeding programs (Lam et al., 2009). NGS will allow
a great number of markers such as SNPs to be generated, which
could be used to obtain a high density of marker at high coverage
across the genome, to dissect the important traits they associate
with. Dealing with the Conversion Issues These sources of markers will be essential in breeding
programs for screening of the parental plants from germplasm
collection and of progenies derived from the crosses, selecting
traits where the phenotypic methods are not practical (Berkman
et al., 2012). High-throughput phenotyping methods will collect
data from a large number of samples to overcome the small effects
of genes, especially the QTL, controlling the traits (Lam et al.,
2009). et al., 2013). This emphasizes the value of genetic improvement
of biomass composition to reduce processing costs. POTENTIAL IMPROVEMENT BY
MOLECULAR GENETICS FOR BIOFUELS The competitiveness of biofuels over other options relies on
biotechnology
advancement. Efficient
conversion
of
plant
biomass to biofuels requires the supply of appropriate feedstocks
that can be sustainably produced in large quantities at high yields. The efficient conversion of the biomass in these feedstocks will be
facilitated by having a composition that is optimized for efficient
processing to deliver high yields of the desired end products. Manipulating of the carbohydrates of the cell walls is the key
of improving the biomass composition for biofuels (Harris and
DeBolt, 2010). Powerful tools of biotechnology could aim to
produce genetically modified sugarcane plants with a favorable
ratio of cellulose to non-cellulose content; with in planta enzymes
that can digest the biomass or degrade the lignin prior to its
conversion to ethanol; with pest and disease resistance, flower
inhibition, abiotic resistance; or incorporate them into elite
sugarcane cultivars for better agronomic performance (Sticklen,
2006; Yuan et al., 2008; Matsuoka et al., 2009; Arruda, 2012). To facilitate the second generation of biofuels, sugarcane breed-
ing programs need to be focusing not only on important traits such
as total biomass yield, sugar yield adaptability to local environ-
ment, and resistance to major pathogens but also on biofuel traits
(e.g., less lignin, improve biomass composition for conversion)
as a whole (Matsuoka et al., 2009; Waclawovsky et al., 2010). In
sugarcane breeding, to maximize heterosis, the parents are usually
selected from divergent genotypes of genetic background (Taba-
sum et al., 2010). Increasing sugarcane biomass yield and produc-
tivity is getting more and more difficult to achieve by conventional
methods; hence, broadening the sugarcane genetic basis by intro-
gression of its ancestors or closely related species such as Miscant-
hus and Erianthus is being explored in sugarcane improvement
[reviewed in Dal-Bianco et al. (2012) and De Siqueira Ferreira
et al. (2013)]. This is normally done by crossing S. officinarum
and Erianthus, Miscanthus, or backcrossing the hybrids to S. spon-
taneum (Matsuoka et al., 2009). Dual-purpose cane and energy
cane, sugarcane lines for lignocellulosic biomass production, have
been derived from two sugarcane species, S. spontaneum and S. robustum, by crossing to develop lines with a high ability to accu-
mulate fiber and high biomass content in addition to accumulat-
ing soluble sugars (De Siqueira Ferreira et al., 2013). Another case
is Miscane, which was the result of crossing between Saccharum
x Miscanthus. POTENTIAL IMPROVEMENT
OF SUGARCANE BY BREEDING
FOR BIOFUELS The complex and highly polyploid genome of sugarcane poses
a great challenge in unraveling and studying its functions. Each
cross of modern sugarcane cultivar has a unique set of chromo-
somes due to the random sorting of chromosomes and recombi-
nation of alleles from two progenitor species (Grivet and Arruda,
2002). There are several distinct alleles at each locus in sugar-
cane chromosomes, making the characteristics of the offspring
unpredictable and requiring evaluation of thousands of lines from
many parents to gather sufficient information in breeding pro-
grams (Matsuoka et al., 2009). In conventional breeding, after
crossing and obtaining the F1 generation, hundreds of thousands
of F1 seedlings are used for screening for the desired traits such
as disease resistance, sugar content, agronomic characteristics,
and adaptability (Matsuoka et al., 2009). The process is normally
repeated for some vegetatively propagated generations to obtain
the required stability of the traits. For industrial purpose, after a
long process of selection, from hundreds of thousands seedlings
at the beginning, breeders normally end up at a limited number of
clones for release as commercial lines or cultivars. POTENTIAL IMPROVEMENT BY
MOLECULAR GENETICS FOR BIOFUELS This produces cane varieties with more biomass
(lignocellulose and total fermentable sugars), disease resistance,
and cold tolerance. This effectively adapts Miscanthus to a tropical
climate and expands sugarcane production to temperate, dry, and
cold conditions (Alexander, 1985; Burner et al., 2009; Lam et al.,
2009). Recently, using molecular markers in sugarcane breeding
program (marker-assisted selection) allows the direct comparison
of DNA genetic diversity and provides a precise tool in assessing Among the grasses potentially used for biofuel production such
as sugarcane, switch grass, Miscanthus, and Erianthus, sugarcane
has been used more for gene transformation studies (Falco et al.,
2000; Manickavasagam et al., 2004; Basnayake et al., 2011) and
the first transgenic sugarcane was established by Bower and Birch
(1992). The current status of improving sugarcane biomass by
using the genetic tools is hindered by its genome complexity, low
transformation efficiency, transgene inactivation (gene silencing
and regulation), somaclonal variation, and difficulty in backcross-
ing (Ingelbrecht et al., 1999; Hotta et al., 2010; Arruda, 2012;
Dal-Bianco et al., 2012). Targets tackled so far on sugarcane
include sucrose and biomass yield increase [i.e., in Ma et al. (2000) and Botha et al. (2001)], downregulation of lignin con-
tent or monolignol changes in lignin to lower biomass recalci-
trance (described later), expression and accumulation of microbial
cellulosic enzymes in leaf [i.e., in Harrison et al. (2011)], her-
bicide tolerance [i.e., in Gallo-Meagher and Irvine (1996) and
Enríquez-Obregón et al. (1998)], disease or pest resistance [i.e.,
in Joyce et al. (1998), Arencibia et al. (1999), and Zhang et al. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org November 2015 | Volume 3 | Article 182 7 Sugarcane Genetic Improvement for Biofuels Hoang et al. wall constituent biosynthesis (Jung et al., 2012; Bottcher et al.,
2013). (1999)], flowering inhibition [reviewed in Matsuoka et al. (2009)
and Hotta et al. (2010)], and drought tolerance [i.e., in Zhang et al. (2006)]. Genetically modified sugarcane has great potential to
contribute to biofuel production, with new varieties incorporating
these characteristics (Arruda, 2012). Unexploited genes not only
from the Saccharum germplasm but also in other related species,
such as cold-tolerant genes in S. spontaneum and Miscanthus or
drought-tolerant genes in sorghum, once identified would allow
their integration into the sugarcane genome, facilitating the pro-
duction of more sugarcane biomass in temperate areas or under
dry conditions (Lam et al., 2009). POTENTIAL IMPROVEMENT BY
MOLECULAR GENETICS FOR BIOFUELS Producing enzymes in planta is another way to cut the cost
of biofuel production as it reduces the expense of enzymes and
enzyme treatment. Cellulase has been produced within the plant
(in the apoplast) of Arabidopsis, rice, and maize without effects
on the growth and development of the host plants [reviewed in
Sticklen (2006)]. In planta enzyme expression in sugarcane is
still in its infancy; however, a high-yield biofuel plant such as
sugarcane must be a target for the production of enzymes within
the biomass. Recombinant protein enzymes have been targeted
to organelles such as chloroplasts, vacuoles, and the endoplasmic
reticulum to separate the enzymes produced and their substrates
(Harrison et al., 2011). In sugarcane, thanks to its well-established
transformation methods via Agrobacterium, the expression of
enzymes in leaves and other tissues is feasible (Manickavasagam
et al., 2004; Taylor et al., 2008). Endoglucanases and exoglu-
canases have been overexpressed in sugarcane leaves by using the
maize PepC promoter achieving an accumulation level of 0.05%
of total soluble proteins (endoglucanase, in chloroplast) and less
of exoglucanases without altering the phenotype (Harrison et al.,
2011). In the future, enzymes might be synthesized in specific
energy cane plants that could be coprocessed with other biomass
sources from sugarcane for sugar and biomass production (e.g.,
bagasse from sugar mills) (Arruda, 2012). Increasing plant cellulose and total biomass content may be
achieved by using approaches such as manipulation of growth
regulators or key nutrients, increasing the ability of the plant to
fix carbon by increasing atmospheric CO2 and also manipulat-
ing some key metabolic enzymes in biomass synthesis pathways
[reviewed in Sticklen (2006)]. Reduction of the cross-links of
the maize cell walls (including ferulate and diferulate cross-links;
benzyl ether and ester cross-links) has been shown to increase
the initial hydrolysis of its cell wall polysaccharides by up to
46% (Grabber, 2005). In general, selection of grasses with less
ferulate cross-linking or potent microbial xylanases by breeding
or engineering tools are more attractive than pretreatment of the
cell wall with a feruloyl esterase (Grabber, 2005). y
Lignin content accounts for about 25% of sugarcane total lig-
nocellulosic biomass and is probably the main obstacle affecting
the efficiency of saccharification during conversion to ethanol
(Canilha et al., 2012, 2013). Lignin and other recalcitrant com-
ponents in cell walls prevent cellulase accessing the cellulose
molecules and need to be removed before further processing
(Sticklen, 2006). POTENTIAL IMPROVEMENT BY
MOLECULAR GENETICS FOR BIOFUELS Lignin biosynthesis pathways are complicated
and at least 10 different enzymes have been found involved in
the lignin pathway in sugarcane (Higuchi, 1981; Whetten and
Ron, 1995) and a total of 28 unigenes associated with monolignol
biosynthesis were identified in sugarcane using SUCEST database
and annotated genes from closely related species such as sorghum,
maize, and rice (Bottcher et al., 2013). Tailoring sugarcane biomass
composition for biofuels can be achieved by manipulating some
of the key genes in lignin pathway (downregulation of some
key enzymes), mostly targeting genes which encode the terminal
enzymes such as caffeic acid O-methyltransferase (COMT) and
cinnamyl alcohol dehydrogenase (CAD), to minimize the impact
of the modifications on growth and development of the plant [as
reviewed in Sticklen (2006), Jung et al. (2012), and Furtado et al. (2014)]. Not only lignin content but also the lignin S/G ratio is
a very important aspect to consider in terms of modifying the
lignin content because these two are both associated with biomass
recalcitrance (Chen and Dixon, 2007; Li et al., 2010). Sugarcane
lignin content was reduced by 3.9–13.7% using RNA interference
(RNAi) suppression to downregulate the COMT gene [which has
at least 31 different ESTs involved (Ramos et al., 2001)] by 67–97%
and at the same time, the lignin S/G ratio was reduced from
1.47 to 1.27–0.79 (Jung et al., 2012). This resulted in an increase
of up to 29% in total sugar yield without pretreatment (34%
with dilute acid pretreatment). This study suggests that RNAi-
mediated gene suppression is a promising method for suppres-
sion of target genes not only in lignin pathway but also for cell POTENTIAL OF SUGARCANE WHOLE
GENOME AND TRANSCIPTOME
SEQUENCING FOR BIOFUELS The advent of NGS technology and a sharp reduction in per-base
cost in the past decade [as reviewed in Van Dijk et al. (2014)]
allows us to sequence the whole genome of a species, even a
complex genome such as sugarcane, at a relatively low price within
a relatively short time. At present, the cost of sequencing of a
human genome at 30× coverage using the latest Illumina’s Hiseq
X is around US $1,000. Since the first plant genome was com-
pletely sequenced (Arabidopsis thaliana in 2000) using the tradi-
tional Sanger sequencing platform, the sequencing strategies have
moved to high-throughput and cost-effective approaches (Henry
et al., 2012). High-throughput genome sequencing platforms have
recently advanced and facilitated improved genotyping, allowing
huge data output to be generated for polymorphism detection
(especially SNPs) and marker discovery. Potential Strategies in Dissection of
Biofuel Traits in Sugarcane Due
to the limited number of generations, low recombination rate
between chromosomes, and strong founder effect, it is expected
that sugarcane has an extensive linkage disequilibrium despite the
large number of chromosomes and being an outcrossing species
(Huang et al., 2010). In fact, attaining a F2 population (such
as inbred backcrosses or recombinant inbred lines and double
haploid lines) in sugarcane is not practical due to its clonal prop-
agation, high heterozygosity, and inbreeding depression (Aitken
and McNeil, 2010; Sreedhar and Collins, 2010). Therefore, more
commonly, a segregating F1 population from biparental crosses
or self-pollinated progenies from heterozygous parents (as the
pseudo F2 population) are used, and hence, most of sugarcane
linkage maps (as AFLP, RAPD, isozyme, and SSR) were developed
on this type of F1 population (Sreedhar and Collins, 2010). To
date, most of these maps have low coverage and a limited number
of markers because of the genome complexity and high cost of
marker generation (Aitken et al., 2014). The high redundancy
of the chromosomes in the sugarcane genome implies that with
conventional approaches only the single-dose markers (present
on only one of the homologous/homoelogous haplotype) can be
used to obtain a high-resolution mapping (Hoarau et al., 2002;
Le Cunff et al., 2008). The potential applications of the current genotyping technolo-
gies to sugarcane association studies employ both whole-genome is restricted to genes which are likely thought to be associated with
traits of interest based on prior knowledge (Hirschhorn and Daly,
2005; Ingvarsson and Street, 2011). At present, whole-genome
sequencing based on the random sequencing of fragments of
whole genomic DNA has been successfully applied to medium-
size genomes with limited amount of repetitive elements, genome
resequencing with the guide of a reference sequence, or de novo
assembly of small genomes (Steuernagel et al., 2009; Henry et al.,
2012; Xu et al., 2012; Edwards et al., 2013). The large genome size
of sugarcane is partially attributable to sugarcane being a poly-
ploid and the genome having a significant amount of repetitive
sequences (Berkman et al., 2014). As a result, the current short
reads generated from NGS technologies cannot resolve completely
the challenges in the sugarcane genomes. For highly repetitive
genomes, the genomic complexity will be lost or reduced by
using the de novo assembly approaches of NGS-derived short
reads as the identical repeat sequences in the genome will be col-
lapsed (Green, 2002). Potential Strategies in Dissection of
Biofuel Traits in Sugarcane Therefore, it is required to develop efficient
genotyping strategies using whole-genome sequencing data for
sugarcane system to overcome the challenges. Moreover, whole
transcriptome sequencing gives details of the entire transcript
expressed in the samples across the whole genome and could
be applicable to the sugarcane genome in identifying biological
significant variations (SNPs) between different developmental
stages, between varieties, or for transcripts de novo assembly and
gene discovery (Henry et al., 2012). g
y
y
For large and polyploid genomes, there are still requirements
to enrich the genomic DNA to capture the coding regions to
ensure the depth of coverage, resolve the variable short reads,
and lessen the effect of repetitive sequences in the genome on
discovery of polymorphisms (Bundock et al., 2012; Henry et al.,
2012). Selective sequencing of genomic loci of interest (genes
or exomes) can reduce the cost compared to whole-genome
sequencing and therefore simplify the data interpretation since
non-coding regions are not abundant in the data. The enrich-
ment techniques can be hybrid capture (e.g., Agilent SureSelect,
NimbleGen, FlexGen) or selective circularization (e.g., Selector
probes) or PCR amplification (e.g., Raindance). Hybrid capture
supported by a microarray platform has been applied to sugarcane
and other complex genomes due to its high capacity to enrich
large regions of interest (1–50 Mb), the possibility of multiplexing,
the availability of kits, and a the small amount of input DNA
required (<1–3 μg) (Mertes et al., 2011). This approach uses a
selection library of fragmented DNA or RNA representing the
targets (normally oligonucleotides from 80 to 180 bases produced
from known information such as gene indices, ESTs) to capture
the cDNA fragments from a shotgun DNA library based on the
hybridization and then sequence the captured fragments (Mertes
et al., 2011; Bundock et al., 2012). Bundock et al. (2012) conducted
the solution-based hybridization (Agilent SureSelect) to capture
the exome regions of sugarcane using sorghum and sugarcane
coding probes, enriched the genome 10–11 folds, and detected
270,000–280,000 SNPs in each genotype of the material tested. At
the moment, a great number of SNPs from a genome or haplo-
type can be generated by using high-capacity genome sequencing
instruments or high-density oligonucleotide arrays (Zhu et al.,
2008). The continuous advancement in genotyping technology The potential applications of the current genotyping technolo-
gies to sugarcane association studies employ both whole-genome
sequencing and whole transcriptome sequencing technologies. Potential Strategies in Dissection of
Biofuel Traits in Sugarcane At present, a whole-genome sequence of sugarcane is not avail-
able to support its biofuel trait analysis. However, a strategy to
overcome this using the currently available resources, for dis-
secting biofuel traits, for example, in sugarcane biomass, is to
carry on the association studies, in which a population of genetic
variability is selected, phenotyped, and genotyped. Association
studies use the molecular markers from the genetic variability
to detect the association between markers and traits of inter-
est in order to validate the location of the genes, especially for November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 8 Sugarcane Genetic Improvement for Biofuels Hoang et al. quantitative traits (Huang et al., 2010). This strategy has been
used for human and animal genetic studies since it was first
established and more recently also for plants. To date, associ-
ation studies have been applied successfully to many different
plants including Arabidopsis, wheat, barley, rice, cotton, maize,
potato, soybean, sugar beet, Pinus, Eucalyptus, ryegrass [also Zhu
et al. (2008); for a review, see Hall et al. (2010)], and sugar-
cane (Aitken et al., 2005; Wei et al., 2006) for important traits
like pathogen resistance, flowering time, grain composition, and
quality. Association studies differ from traditional QTL stud-
ies, where in QTL analysis the linkage disequilibrium between
markers and QLTs from a segregating population is established
in a cross of different genotypes, whereas in association stud-
ies a non-structured population is used (Neale and Savolainen,
2004; Ingvarsson and Street, 2011). Therefore, association studies
investigate variations of the whole population not just variations
between parents. Association studies analyze the direct linkage
disequilibrium between genetic markers and traits to overcome
the limitations of the traditional QTL in sample size, low varia-
tion, and recombination in the population (Ingvarsson and Street,
2011). In sugarcane, association studies are a powerful method for
understanding the complex traits which are controlled by many
loci and dosage effects (i.e., Ming et al., 2001; Wei et al., 2006;
Banerjee et al., 2015). In general, association studies involve pop-
ulation selection, phenotyping, genotyping, population structure,
and statistical testing for the association. For these, there is a
requirement to have a population with genetic variability and high
linkage disequilibrium; and for sugarcane, the most important
aspect of doing association studies is having marker data and a
breeding population of elite varieties (Huang et al., 2010). Frontiers in Bioengineering and Biotechnology | www.frontiersin.org CONCLUSION Some of the association studies have been carried out on sugar-
cane recently such as those for QTLs which control the Pachymetra
root rot and brown rust resistance on 154 genotypes (McIntyre
et al., 2005); genetics of root rot, leaf scald, Fiji leaf gall, cane
sugar, and yield using 1,068 AFLP, 141 SRR (on 154 genotypes),
and 1,531 DArT markers (on 480 genotypes) (Wei et al., 2006,
2010); smut and eldana stalk borer using 275 RFLP and 1,056
AFLP markers on 77 genotypes (Butterfield, 2007); resistance
to sugarcane yellow leaf virus using 3,949 polymorphic markers
(DArT and AFLP) on 189 genotypes (Debibakas et al., 2014);
markers agro-morphological traits, sugar yield disease resistance,
and bagasse content using 3,327 DArT, AFLP, and SSR markers
on 183 genotypes (Gouy et al., 2015); and sucrose and yield
contributing traits using 989 SSR markers on 108 genotypes
(Banerjee et al., 2015). Using the Affymetrix GeneChip Sugar-
cane Genome Array, Casu et al. (2007) identified 119 transcripts
associated with enzymes of cell wall metabolism and development
on sugarcane variety Q177. These promising preliminary studies
were carried out on small sample sizes and limited numbers of
markers (even though a small number of significant associations
have been identified) while the polyploid sugarcane genome and
small effect of quantitative traits requires larger sample sizes and
more markers (e.g., genome-wide markers) so that significant
association can be detected (Huang et al., 2010; Gouy et al.,
2015). Sugarcane has been shown to be a good candidate for use as a
lignocellulosic biomass feedstock for second-generation biofuel
production. However, its genome complexity still remains a great
bottleneck restricting the dissection of biofuel traits. The most
significant achievements in improving sugarcane biomass for bio-
fuels so far have been the establishment of the high fiber cane vari-
eties to generate more lignocellulosic biomass, and preliminary
results in modifying biomass with more cellulose, less lignin con-
tent, a preferable lignin S/G ratio, and enzyme expressed in planta
(in leaves) for easy conversion to biofuels. The improvement of
sugarcane biomass has been by traditional breeding, molecu-
lar genetics approaches and, more recently, accelerated with the
use of NGS technology. The future of second-generation biofuel
production using sugarcane lignocellulosic biomass will depend
greatly on advances in understanding of the key biofuel traits
required to deliver more efficient and price-competitive biofuels. This objective will be facilitated once the whole genome of sug-
arcane is fully sequenced. Potential Strategies in Dissection of
Biofuel Traits in Sugarcane Genotyping is normally either by analysis candidate genes or
genome-wide approaches, in which the candidate gene approach November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 9 Sugarcane Genetic Improvement for Biofuels Hoang et al. resources such as the sorghum gene indices (sorghum gene mod-
els), sugarcane gene indices (DFCI Sugarcane Gene Index version
3.0, an integrated collection of sugarcane ESTs, complete cDNA
sequences, non-redundant data of all sugarcane genes and their
related information), transcription factors (TFs), and sugarcane
tentative consensus/assembled sequences. For example, in the
study mentioned earlier, Bundock et al. (2012), based on the gene
sequences in the sorghum genome and sugarcane gene indices,
captured the exomic regions of two sugarcane genotypes Q165
and IJ76-514, detected SNPs present in 13,000–16,000 targeted
genes from Illumina short read data of these samples, and 87–91%
of SNPs were validated and confirmed by 454 sequencing. For
transcript profiling, the reference transcriptome sequence can be
constructed for specific tissues using de novo assembly such as in
Vargas et al. (2014) and Cardoso-Silva et al. (2014) and validated
to find suitable reference gene sets to be used for gene expression
normalization as in Guo et al. (2014). The currently available
resources, on the other hand, are also utilized. Park et al. (2015)
used the Sugarcane Assembled Sequences from SUCEST-FUN
database as reference sequences in a study on cold-responsive gene
expression profiling of sugarcane hybrids and S. spontaneum and
found that more than 600 genes are differentially expressed in each
genotype after applying stress. allows generation of up to 1 million SNPs spanning across the
entire genome in one reaction (e.g., using SNP chip), and the
newest SNP chip can measure the copy number as well as the
allelic variation. Examples of available platforms are Affymetrix
(e.g., Affymetrix Genome-Wide Human SNP Array 6.0) and Illu-
mina (e.g., Illumina’s WGGT Infinium BeadChips). Due to the
multiple chromosomes in the homologous groups of sugarcane
genome and the number of alleles at each locus (and the SNPs
numbers consequently), an allele would likely to be defined by
a combination of SNPs, not just a single SNP (McIntyre et al.,
2006, 2015). SNP genotyping including SNP calling and statistical
methods to estimate the ploidy level and allele dosage within
homologous groups have been developed for sugarcane by Garcia
et al. (2013) to allow in-depth association analysis of the genome. Potential Strategies in Dissection of
Biofuel Traits in Sugarcane In this study, SNPs were developed by SEQUENOM iPLEX Mas-
sARRAY and capture primers and then discovered by QualitySNP
software, mass-based procedures, and the SuperMASSA software. For whole transcriptome sequencing, Cardoso-Silva et al. (2014)
identified 5,106 SSRs and 708,125 SNPs from the unigenes assem-
bled from RNA-seq data of contrasting sugarcane varieties. These
advances in sugarcane genotyping technology, together with well-
developed high-throughput phenotyping methods for biofuel
traits [reviewed in Lupoi et al. (2013) and Lupoi et al. (2015)] and
bioinformatics tools, could accelerate sugarcane analysis while a
reference genome is not available. CONCLUSION Optimizing sugarcane lignocellulosic
bagasse composition may result in biomass with better digestibil-
ity, modified carbohydrates, and reduction of cross-linking or
self-produced enzymes (in planta). Currently available sugarcane
genetic resources include diverse germplasm in the genus Sac-
charum, genetic markers and maps, ESTs, and the sequence of
a closely related species genome. However, novel strategies need
to be developed to overcome the challenges posed by the com-
plex genetics. Traditional approaches using breeding and molec-
ular genetics have potential for wider use improving sugarcane
while the advent of NGS technology and high-throughput phe-
notyping technologies will accelerate the process of dissection
of biofuel traits, genome-wide. By using these approaches, the
loci of interest will be defined for use to improve sugarcane The Reference Sequence Matters REFERENCES Botha, F. (2009). “Energy yield and cost in a sugarcane biomass system,” in:
Proceedings Australian Society Sugarcane Technologists. 1–9. g
y
g
g
Botha, F., Sawyer, B., and Birch, R. (2001). “Sucrose metabolism in the culm of
transgenic sugarcane with reduced soluble acid invertase activity,” in Proceedings
of the International Society of Sugar Cane Technologists, 588–591. Aitken, K., Jackson, P., and McIntyre, C. (2005). A combination of AFLP and SSR
markers provides extensive map coverage and identification of homo(eo)logous
linkage groups in a sugarcane cultivar. Theor. Appl. Genet. 110, 789–801. doi:10. 1007/s00122-004-1813-7 Bottcher, A., Cesarino, I., Santos, A. B. D., Vicentini, R., Mayer, J. L. S., Vanholme,
R., et al. (2013). Lignification in sugarcane: biochemical characterization, gene
discovery, and expression analysis in two genotypes contrasting for lignin con-
tent. Plant Physiol. 163, 1539–1557. doi:10.1104/pp.113.225250 Aitken, K., and McNeil, M. (2010). “Diversity analysis,” in Genetics, Genomics
and Breeding of Sugarcane, eds R. Henry and C. Kole (Enfield, NH: Science
Publishers), 19–42. Bower, N. I., Casu, R. E., Maclean, D. J., Reverter, A., Chapman, S. C., and Manners,
J. M. (2005). Transcriptional response of sugarcane roots to methyl jasmonate. Plant Sci. 168, 761–772. doi:10.1016/j.plantsci.2004.10.006 Aitken, K. S., McNeil, M. D., Hermann, S., Bundock, P. C., Kilian, A., Heller-
Uszynska, K., et al. (2014). A comprehensive genetic map of sugarcane that
provides enhanced map coverage and integrates high-throughput diversity array
technology (DArT) markers. BMC Genomics 15:152. doi:10.1186/1471-2164-
15-152 Bower, R., and Birch, R. G. (1992). Transgenic sugarcane plants via microprojectile
bombardment. Plant J. 2, 409–416. doi:10.1111/j.1365-313X.1992.00409.x Bremer, G. (1961). Problems in breeding and cytology of sugar cane. Euphytica 10,
59–78. doi:10.1007/BF00037206 Alexander, A. G. (1985). The Energy Cane Alternative. Amsterdam: Elsevier Science
Publishers BV. Bundock, P. C., Casu, R. E., and Henry, R. J. (2012). Enrichment of genomic DNA
for polymorphism detection in a non-model highly polyploid crop plant. Plant
Biotechnol. J. 10, 657–667. doi:10.1111/j.1467-7652.2012.00707.x Alonso Pippo, W., Luengo, C. A., Alonsoamador Morales Alberteris, L., Garzone,
P., and Cornacchia, G. (2011a). Energy recovery from sugarcane-trash in the
light of 2nd generation biofuel. Part 2: socio-economic aspects and techno-
economic analysis. Waste Biomass Valorization 2, 257–266. doi:10.1007/s12649-
011-9069-3 Burner, D. M., Tew, T. L., Harvey, J. J., and Belesky, D. P. (2009). Dry matter
partitioning and quality of Miscanthus, Panicum, and Saccharum genotypes in
Arkansas, USA. Biomass Bioenergy 33, 610–619. doi:10.1016/j.biombioe.2008. 10.002 Alonso Pippo, W., Luengo, C. REFERENCES A., Alonsoamador Morales Alberteris, L., Garzone, P.,
and Cornacchia, G. (2011b). Energy recovery from sugarcane-trash in the light
of 2nd generation biofuels. Part 1: current situation and environmental aspects. Waste Biomass Valorization 2, 1–16. doi:10.1007/s12649-010-9048-0 Butterfield, M. K. (2007). Marker Assisted Breeding in Sugarcane: A Complex Poly-
ploidy. PhD Thesis, Univ Stellenbosch, Matieland, Stellenbosch, South Africa. Amalraj, V. A., and Balasundaram, N. (2006). On the taxonomy of the members of
‘Saccharum complex’. Genet. Resour. Crop Evol. 53, 35–41. doi:10.1007/s10722-
004-0581-1 Canilha, L., Kumar Chandel, A., Dos Santos Milessi, T. S., Fernandes Antunes,
F. A., Da Costa Freitas, W. L., Das Gracas Almeida Felipe, M., et al. (2012). Bioconversion of sugarcane biomass into ethanol: an overview about compo-
sition, pretreatment methods, detoxification of hydrolysates, enzymatic sac-
charification, and ethanol fermentation. J. Biomed. Biotechnol. 2012, 989572. doi:10.1155/2012/989572 Anterola, A. M., and Lewis, N. G. (2002). Trends in lignin modification: a com-
prehensive analysis of the effects of genetic manipulations/mutations on ligni-
fication and vascular integrity. Phytochemistry 61, 221–294. doi:10.1016/S0031-
9422(02)00211-X Canilha, L., Rodrigues, R., Antunes, F. A. F., Chandel, A. K., Milessi, T., Felipe, M. D. G. A., et al. (2013). “Bioconversion of hemicellulose from sugarcane biomass
into sustainable products,” in Sustainable Degradation of Lignocellulosic Biomass-
Techniques, Applications and Commercialization, eds A. K. Chandel and S. S. da
Silva (Rijeka: InTech), 15–45. doi:10.5772/53832 Arencibia, A., Carmona, E., Cornide, M., Castiglione, S., O’relly, J., Chinea, A., et al. (1999). Somaclonal variation in insect-resistant transgenic sugarcane (Saccha-
rum hybrid) plants produced by cell electroporation. Transgenic Res. 8, 349–360. doi:10.1023/A:1008900230144 Cardoso-Silva, C. B., Costa, E. A., Mancini, M. C., Balsalobre, T. W. A., Canesin, L. E. C., Pinto, L. R., et al. (2014). De novo assembly and transcriptome analysis of
contrasting sugarcane varieties. PLoS ONE 9:e88462. doi:10.1371/journal.pone. 0088462 Arruda, P. (2012). Genetically modified sugarcane for bioenergy generation. Curr. Opin. Biotechnol. 23, 315–322. doi:10.1016/j.copbio.2011.10.012 Balat, M., Balat, H., and Öz, C. (2008). Progress in bioethanol processing. Prog. Energy Combust. Sci. 34, 551–573. doi:10.1016/j.pecs.2007.11.001 Carpita, N. C. (1996). Structure and biogenesis of the cell walls of grasses. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 445–476. doi:10.1146/annurev.arplant.47. 1.445 Banerjee, N., Siraree, A., Yadav, S., Kumar, S., Singh, J., Kumar, S., et al. (2015). Marker-trait association study for sucrose and yield contributing traits in sug-
arcane (Saccharum spp. hybrid). Euphytica 205, 185–201. doi:10.1007/s10681-
015-1422-3 Carson, D., and Botha, F. (2002). Genes expressed in sugarcane maturing internodal
tissue. ACKNOWLEDGMENTS biomass. Once a better understanding of the genes controlling cell
wall biosynthesis is achieved, breeding programs will be able to
accelerate the selection and development of varieties with opti-
mized biomass composition to generate better sugarcane biomass
sources to meet the demand of biofuel production. We are grateful to the Australian Agency for International Devel-
opment (AusAID) for financial support through an Australian
Development Scholarship (ADS) to NVH and to the Queensland
Government for funding this research. This work was part of the
DOE Joint BioEnergy Institute (http://www.jbei.org) supported
by the U. S. Department of Energy, Office of Science, Office of Bio-
logical and Environmental Research, through contract DE-AC02-
05CH11231 between Lawrence Berkeley National Laboratory and
the U. S. Department of Energy. The Reference Sequence Matters As mentioned earlier, construction of a sugarcane nuclear genome
reference sequence is an important objective, even though it might
take some time to finish. However, in the meantime, sugarcane
genome analysis still can exploit the currently available genetic November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 10 Sugarcane Genetic Improvement for Biofuels Hoang et al. AUTHOR CONTRIBUTIONS NVH wrote the paper. AF, FCB, BAS, and RJH discussed and
edited the manuscript. All authors read and approved the final
manuscript. NVH wrote the paper. AF, FCB, BAS, and RJH discussed and
edited the manuscript. All authors read and approved the final
manuscript. Botha, F. (2009). “Energy yield and cost in a sugarcane biomass system,” in:
Proceedings Australian Society Sugarcane Technologists. 1–9. REFERENCES biortech.2010.02.009 Cuadrado, A., Acevedo, R., Moreno Diaz De La Espina, S., Jouve, N., and De La
Torre, C. (2004). Genome remodelling in three modern S. officinarumx S. spon-
taneum sugarcane cultivars. J. Exp. Bot. 55, 847–854. doi:10.1093/jxb/erh093 Gouy, M., Rousselle, Y., Thong Chane, A., Anglade, A., Royaert, S., Nibouche, S.,
et al. (2015). Genome wide association mapping of agro-morphological and
disease resistance traits in sugarcane. Euphytica 202, 269–284. doi:10.1007/
s10681-014-1294-y Dal-Bianco, M., Carneiro, M. S., Hotta, C. T., Chapola, R. G., Hoffmann, H. P.,
Garcia, A. A. F., et al. (2012). Sugarcane improvement: how far can we go? Curr. Opin. Biotechnol. 23, 265–270. doi:10.1016/j.copbio.2011.09.002 Govindaraj, P., Amalraj, V. A., Mohanraj, K., and Nair, N. V. (2014). Collection,
characterization and phenotypic diversity of Saccharum spontaneum L. from
arid and semi arid zones of northwestern India. Sugar Tech 16, 36–43. doi:10. 1007/s12355-013-0255-4 Daniels, J., and Roach, B. T. (1987). “Chapter 2 – taxonomy and evolution,” in
Developments in Crop Science, ed. J. H. Don (Amsterdam: Elsevier), 7–84. De O. Buanafina, M. M. (2009). Feruloylation in grasses: current and future per-
spectives. Mol. Plant. 2, 861–872. doi:10.1093/mp/ssp067 Grabber, J. H. (2005). How do lignin composition, structure, and cross-linking
affect degradability? A review of cell wall model studies. Crop Sci. 45, 820. doi:10.2135/cropsci2004.0191 De Siqueira Ferreira, S., Nishiyama, M. Y. Jr., Paterson, A. H., and Souza, G. M. (2013). Biofuel and energy crops: high-yield Saccharinae take center stage in the
post-genomics era. Genome Biol. 14, 210. doi:10.1186/gb-2013-14-6-210 Grassius: Grass Regulatory Information Server. (2015). Available at: http://grassius. org/ [accessed May 25, 2015]. Green, P. (2002). Whole-genome disassembly. Proc. Natl. Acad. Sci. U.S.A. 99,
4143–4144. doi:10.1073/pnas.082095999 Debibakas, S., Rocher, S., Garsmeur, O., Toubi, L., Roques, D., D’Hont, A., et al. (2014). Prospecting sugarcane resistance to sugarcane yellow leaf virus by
genome-wide association. Theor. Appl. Genet. 127, 1719–1732. doi:10.1007/
s00122-014-2334-7 Grivet, L., and Arruda, P. (2002). Sugarcane genomics: depicting the complex
genome of an important tropical crop. Curr. Opin. Plant Biol. 5, 122–127. doi:
10.1016/S1369-5266(02)00234-0 D’Hont, A., and Glaszmann, J. C. (2001). “Sugarcane genome analysis with molec-
ular markers, a first decade of research,” in Proceedings of International Society
of Sugar Cane Technologists, 556–559. Grivet, L., Dhont, A., Dufour, P., Hamon, P., Roques, D., and Glaszmann, J. C. (1994). Comparative genome mapping of sugar-cane with other species within
the Andropogoneae tribe. Heredity 73, 500–508. REFERENCES doi:10.1038/hdy.1994.148 D’Hont, A., Grivet, L., Feldmann, P., Rao, S., Berding, N., and Glaszmann, J. C. (1996). Characterisation of the double genome structure of modern sugarcane
cultivars (Saccharum spp.) by molecular cytogenetics. Mol. Gen. Genet. 250,
405–413. doi:10.1007/s004380050092 Grivet, L., Glaszmann, J. C., Vincentz, M., Da Silva, F., and Arruda, P. (2003). ESTs as a source for sequence polymorphism discovery in sugarcane: example
of the Adh genes. Theor. Appl. Genet. 106, 190–197. doi:10.1007/s00122-002-
1075-1 D’Hont, A., Ison, D., Alix, K., Roux, C., and Glaszmann, J. C. (1998). Determination
of basic chromosome numbers in the genus Saccharum by physical mapping of
ribosomal RNA genes. Genome 41, 221–225. doi:10.1139/g98-023 Guo, J., Ling, H., Wu, Q., Xu, L., and Que, Y. (2014). The choice of reference genes
for assessing gene expression in sugarcane under salinity and drought stresses. Sci. Rep. 4, 7042. doi:10.1038/srep07042 Dias De Oliveira, M. E., Vaughan, B. E., and Rykiel, E. J. (2005). Ethanol as
fuel: energy, carbon dioxide balances, and ecological footprint. Bioscience 55,
593–602. doi:10.1641/0006-3568(2005)055[0593:EAFECD]2.0.CO;2 Gupta, V., Raghuvanshi, S., Gupta, A., Saini, N., Gaur, A., Khan, M. S., et al. (2010). The water-deficit stress- and red-rot-related genes in sugarcane. Funct. Integr. Genomics 10, 207–214. doi:10.1007/s10142-009-0144-9 Dillon, S. L., Shapter, F. M., Henry, R. J., Cordeiro, G., Izquierdo, L., and Lee, L. S. (2007). Domestication to crop improvement: genetic resources for Sorghum and
Saccharum (Andropogoneae). Ann. Bot. 100, 975–989. doi:10.1093/aob/mcm192 Hall, D., Tegstrom, C., and Ingvarsson, P. K. (2010). Using association mapping to
dissect the genetic basis of complex traits in plants. Brief. Funct. Genomics 9,
157–165. doi:10.1093/bfgp/elp048 Edwards, D., Batley, J., and Snowdon, R. J. (2013). Accessing complex crop genomes
with next-generation sequencing. Theor. Appl. Genet. 126, 1–11. doi:10.1007/
s00122-012-1964-x gp
p
Halling, P., and Simms-Borre, P. (2008). Overview of lignocellulosic feedstock
conversion into ethanol-focus on sugarcane bagasse. Int. Sugar J. 110, 191. Enríquez-Obregón, G. A., Vázquez-Padrón, R. I., Prieto-Samsonov, D. L., De La
Riva, G. A., and Selman-Housein, G. (1998). Herbicide-resistant sugarcane
(Saccharum officinarum L.) plants by Agrobacterium-mediated transformation. Planta 206, 20–27. doi:10.1007/s004250050369 Handakumbura, P. P., and Hazen, S. P. (2012). Transcriptional regulation of grass
secondary cell wall biosynthesis: playing catch-up with Arabidopsis thaliana. Front. Plant Sci. 3:74. doi:10.3389/fpls.2012.00074 Harris, D., and DeBolt, S. (2010). Synthesis, regulation and utilization of lignocel-
lulosic biomass. Plant Biotechnol. J. 8, 244–262. doi:10.1111/j.1467-7652.2009. 00481.x Eversole, K., Graner, A., and Stein, N. (2009). REFERENCES Plant Cell Rep. 20, 1075–1081. doi:10.1007/s00299-002-0444-1 Basnayake, S. W. V., Moyle, R., and Birch, R. G. (2011). Embryogenic cal-
lus proliferation and regeneration conditions for genetic transformation of
diverse sugarcane cultivars. Plant Cell Rep. 30, 439–448. doi:10.1007/s00299-
010-0927-4 Carson, D. L., and Botha, F. C. (2000). Preliminary analysis of expressed sequence
tags for sugarcane. Crop Sci. 40, 1769–1779. doi:10.2135/cropsci2000.4061769x Casu, R., Dimmock, C., Chapman, S., Grof, C. L., McIntyre, C. L., Bonnett, G.,
et al. (2004). Identification of differentially expressed transcripts from maturing
stem of sugarcane by in silico analysis of stem expressed sequence tags and
gene expression profiling. Plant Mol. Biol. 54, 503–517. doi:10.1023/B:PLAN. 0000038255.96128.41 Berkman, P. J., Bundock, P. C., Casu, R. E., Henry, R. J., Rae, A. L., and Aitken, K. S. (2014). A survey sequence comparison of Saccharum genotypes reveals allelic
diversity differences. Trop. Plant Biol. 7, 71–83. doi:10.1007/s12042-014-9139-3 Berkman, P. J., Lai, K., Lorenc, M. T., and Edwards, D. (2012). Next-generation
sequencing applications for wheat crop improvement. Am. J. Bot. 99, 365–371. doi:10.3732/ajb.1100309 Casu, R., Grof, C. L., Rae, A., McIntyre, C. L., Dimmock, C., and Manners, J. (2003). Identification of a novel sugar transporter homologue strongly expressed November 2015 | Volume 3 | Article 182 11 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org Sugarcane Genetic Improvement for Biofuels Hoang et al. Gallo-Meagher, M., and Irvine, J. E. (1996). Herbicide resistant transgenic sug-
arcane plants containing the bar gene. Crop Sci. 36, 1367–1374. doi:10.2135/
cropsci1996.0011183X003600050047x in maturing stem vascular tissues of sugarcane by expressed sequence tag and
microarray analysis. Plant Mol. Biol. 52, 371–386. doi:10.1023/A:1023957214644 y
y
Casu, R. E., Jarmey, J. M., Bonnett, G. D., and Manners, J. M. (2007). Identification
of transcripts associated with cell wall metabolism and development in the
stem of sugarcane by Affymetrix GeneChip Sugarcane Genome Array expres-
sion profiling. Funct. Integr. Genomics 7, 153–167. doi:10.1007/s10142-006-
0038-z Garcia, A. A. F., Mollinari, M., Marconi, T. G., Serang, O. R., Silva, R. R., Vieira,
M. L. C., et al. (2013). SNP genotyping allows an in-depth characterisation of
the genome of sugarcane and other complex autopolyploids. Sci. Rep. 3, 3399. doi:10.1038/srep03399 Chen, F., and Dixon, R. A. (2007). Lignin modification improves fermentable
sugar yields for biofuel production. Nat. Biotechnol. 25, 759–761. doi:10.1038/
nbt1316 Gnansounou, E., and Dauriat, A. (2010). Techno-economic analysis of lignocel-
lulosic ethanol: a review. Bioresour. Technol. 101, 4980–4991. doi:10.1016/j. REFERENCES Effects of production and market
factors on ethanol profitability for an integrated first and second generation
ethanol plant using the whole sugarcane as feedstock. Biotechnol. Biofuels 7, 26. doi:10.1186/1754-6834-7-26 Ingelbrecht, I. L., Irvine, J. E., and Mirkov, T. E. (1999). Posttranscriptional gene
silencing in transgenic sugarcane. Dissection of homology-dependent virus
resistance in a monocot that has a complex polyploid genome. Plant Physiol. 119,
1187–1198. doi:10.1104/pp.119.4.1187 Macrelli, S., Mogensen, J., and Zacchi, G. (2012). Techno-economic evaluation
of 2nd generation bioethanol production from sugar cane bagasse and leaves
integrated with the sugar-based ethanol process. Biotechnol. Biofuels 5, 22. doi:
10.1186/1754-6834-5-22 Ingvarsson, P. K., and Street, N. R. (2011). Association genetics of complex traits in
plants. New Phytol. 189, 909–922. doi:10.1111/j.1469-8137.2010.03593.x Jakob, K., Zhou, F., and Paterson, A. (2009). Genetic improvement of C4 grasses as
cellulosic biofuel feedstocks. In vitro Cell. Dev. Biol. Plant 45, 291–305. doi:10. 1007/s11627-009-9214-x Manickavasagam, M., Ganapathi, A., Anbazhagan, V. R., Sudhakar, B., Selvaraj,
N., Vasudevan, A., et al. (2004). Agrobacterium-mediated genetic transformation
and development of herbicide-resistant sugarcane (Saccharum species hybrids)
using axillary buds. Plant Cell Rep. 23, 134–143. doi:10.1007/s00299-004-0794-y Jannoo, N., Grivet, L., Chantret, N., Garsmeur, O., Glaszmann, J. C., Arruda, P., et al. (2007). Orthologous comparison in a gene-rich region among grasses reveals
stability in the sugarcane polyploid genome. Plant J. 50, 574–585. doi:10.1111/j. 1365-313X.2007.03082.x Manners, J., McIntyre, L., Casu, R., Cordeiro, G., Jackson, M., Aitken, K., et al. (2004). “Can genomics revolutionize genetics and breeding in sugarcane,” in
Proceedings of the 4th International Crop Science Congress (Brisbane, QLD). Johansson, T. B., and Burnham, L. (1993). Renewable Energy: Sources for Fuels and
Electricity. Washington, DC: Island Press. Matsuoka, S., Ferro, J., and Arruda, P. (2009). The Brazilian experience of sugarcane
ethanol industry. In vitro Cell. Dev. Biol. Plant 45, 372–381. doi:10.1007/s11627-
009-9220-z Joyce, P., McQualter, R., Handley, J., Dale, J., Harding, R., and Smith, G. (1998). “Transgenic sugarcane resistant to sugarcane mosaic virus,” in Proceedings-
Australian Society of Sugar Cane Technologists (Salisbury: Watson Ferguson and
Company), 204–210. McIntyre, C. L., Goode, M. L., Cordeiro, G., Bundock, P., Eliott, F., Henry, R. J.,
et al. (2015). Characterisation of alleles of the sucrose phosphate synthase gene
family in sugarcane and their association with sugar-related traits. Mol. Breed. 35, 1–14. doi:10.1007/s11032-015-0286-5 Jung, H. G. (1989). Forage lignins and their effects on fiber digestibility. Agronomy
Journal 81, 33–38. doi:10.2134/agronj1989.00021962008100010006x McIntyre, C. L., Jackson, M., Cordeiro, G. REFERENCES R., et al. (2012). Application of large-scale sequencing to marker discovery in
plants. J. Biosci. 37, 829–841. doi:10.1007/s12038-012-9253-z T. R., et al. (2012). Application of large-scale sequencing to marker discovery in
plants. J. Biosci. 37, 829–841. doi:10.1007/s12038-012-9253-z Loureiro, M., Barbosa, M. P., Lopes, F. F., and Silvério, F. (2011). “Sugarcane breed-
ing and selection for more efficient biomass conversion in cellulosic ethanol,”
in Routes to Cellulosic Ethanol, eds M. S. Buckeridge and G. H. Goldman (New
York, NY: Springer), 199–239. Higuchi, T. (1981). “Biosynthesis of lignin,” in Plant Carbohydrates II, eds W. Tanner and F. Loewus (Berlin: Springer), 194–224. Himmel, M., Ding, S., Johnson, D., Adney, W., Nimlos, M., Brady, J., et al. (2007). Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315, 804–807. doi:10.1126/science.1137016 Lupoi, J., Singh, S., Simmons, B., and Henry, R. (2013). Assessment of lignocellulosic
biomass using analytical spectroscopy: an evolution to high-throughput tech-
niques. Bioenerg. Res. 7, 1–23. doi:10.1007/s12155-013-9352-1 Hirschhorn, J. N., and Daly, M. J. (2005). Genome-wide association studies for
common diseases and complex traits. Nat. Rev. Genet. 6, 95–108. doi:10.1038/
nrg1521 Lupoi, J. S., Singh, S., Parthasarathi, R., Simmons, B. A., and Henry, R. J. (2015). Recent innovations in analytical methods for the qualitative and quantitative
assessment of lignin. Renew. Sustain. Energ. Rev. 49, 871–906. doi:10.1016/j.rser. 2015.04.091 Hoarau, J.-Y., Grivet, L., Offmann, B., Raboin, L.-M., Diorflar, J.-P., Payet, J., et al. (2002). Genetic dissection of a modern sugarcane cultivar (Saccharum spp.). II. Detection of QTLs for yield components. Theor. Appl. Genet. 105, 1027–1037. doi:10.1007/s00122-002-1047-5 Ma, H., Albert, H. H., Paull, R., and Moore, P. H. (2000). Metabolic engineering
of invertase activities in different subcellular compartments affects sucrose
accumulation in sugarcane cells. Funct. Plant Biol. 27, 1021–1030. doi:10.1071/
PP00029 Hotta, C., Lembke, C., Domingues, D., Ochoa, E., Cruz, G. Q., Melotto-Passarin,
D., et al. (2010). The biotechnology roadmap for sugarcane improvement. Trop. Plant Biol. 3, 75–87. doi:10.1007/s12042-010-9050-5 Ma, H. M., Schulze, S., Lee, S., Yang, M., Mirkov, E., Irvine, J., et al. (2004). An
EST survey of the sugarcane transcriptome. Theor. Appl. Genet. 108, 851–863. doi:10.1007/s00122-003-1510-y Huang, E., Aitken, K., and George, A. (2010). “Association Studies,” in Genetics,
Genomics and Breeding of Sugarcane, eds. R. Henry and C. Kole (Enfield, NH:
Science Publishers), 43–68. Macrelli, S., Galbe, M., and Wallberg, O. (2014). REFERENCES M., Amouyal, O., Hermann, S., Aitken,
K. S., et al. (2006). The identification and characterisation of alleles of sucrose
phosphate synthase gene family III in sugarcane. Mol. Breed. 18, 39–50. doi:10. 1007/s11032-006-9012-7 Jung, J. H., Fouad, W. M., Vermerris, W., Gallo, M., and Altpeter, F. (2012). RNAi
suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel
production from lignocellulosic biomass. Plant Biotechnol. J. 10, 1067–1076. doi:10.1111/j.1467-7652.2012.00734.x McIntyre, C. L., Whan, V. A., Croft, B., Magarey, R. and Smith, G. R. (2005)
Identification and validation of molecular markers associated with pachymetra
root rot and brown rust resistance in sugarcane using Map- and association-
based approaches. Molecular Breeding 16, 151–161. doi:10.1007/s11032-005-
7492-5 Kim, C., Lee, T. H., Compton, R. O., Robertson, J. S., Pierce, G. J., and Paterson,
A. H. (2013). A genome-wide BAC end-sequence survey of sugarcane elucidates
genome composition, and identifies BACs covering much of the euchromatin. Plant Mol. Biol. 81, 139–147. doi:10.1007/s11103-012-9987-x Lam, E., Shine, J., Da Silva, J., Lawton, M., Bonos, S., Calvino, M., et al. (2009). Improving sugarcane for biofuel: engineering for an even better feed-
stock. Glob. Change Biol. Bioenergy 1, 251–255. doi:10.1111/j.1757-1707.2009. 01016.x Mertes, F., Elsharawy, A., Sauer, S., Van Helvoort, J. M. L. M., Van Der Zaag, P. J., Franke, A., et al. (2011). Targeted enrichment of genomic DNA regions for
next-generation sequencing. Brief. Funct. Genomics 10, 374–386. doi:10.1093/
bfgp/elr033 Le Cunff, L., Garsmeur, O., Raboin, L. M., Pauquet, J., Telismart, H., Selvi, A.,
et al. (2008). Diploid/polyploid syntenic shuttle mapping and haplotype-specific
chromosome walking toward a rust resistance gene (Bru1) in highly polyploid
sugarcane (2n ∼12x ∼115). Genetics 180, 649–660. doi:10.1534/genetics.108. 091355 Ming, R., Liu, S.-C., Lin, Y.-R., Da Silva, J., Wilson, W., Braga, D., et al. (1998). Detailed alignment of Saccharum and Sorghum chromosomes: comparative
organization of closely related diploid and polyploid genomes. Genetics 150,
1663–1682. Ming, R., Liu, S. C., Moore, P. H., Irvine, J. E., and Paterson, A. H. (2001). QTL analysis in a complex autopolyploid: genetic control of sugar content in
sugarcane. Genome Res. 11, 2075–2084. doi:10.1101/gr.198801 Li, C., Sun, L., Simmons, B., and Singh, S. (2013). Comparing the recalcitrance of
Eucalyptus, Pine, and Switchgrass using ionic liquid and dilute acid pretreat-
ments. Bioenerg. Res. 6, 14–23. doi:10.1007/s12155-012-9220-4 Li, X., Ximenes, E., Kim, Y., Slininger, M., Meilan, R., Ladisch, M., et al. (2010). Lignin monomer composition affects Arabidopsis cell-wall degradability after
liquid hot water pretreatment. Biotechnol. REFERENCES “Wheat and barley genome sequenc-
ing,” in Genetics and Genomics of the Triticeae, eds G. J. Muehlbauer and C. Feuillet (New York, NY: Springer), 713–742. Harrison, M. D., Geijskes, J., Coleman, H. D., Shand, K., Kinkema, M., Palupe,
A., et al. (2011). Accumulation of recombinant cellobiohydrolase and endoglu-
canase in the leaves of mature transgenic sugar cane. Plant Biotechnol. J. 9,
884–896. doi:10.1111/j.1467-7652.2011.00597.x Falco, M. C., Tulmann Neto, A., and Ulian, E. C. (2000). Transformation and
expression of a gene for herbicide resistance in a Brazilian sugarcane. Plant Cell
Rep. 19, 1188–1194. doi:10.1007/s002990000253 884–896. doi:10.1111/j.1467-7652.2011.00597.x FAOSTAT. (2015). Available at: http://faostat3.fao.org/home/E [accessed May 25,
2015]. Harvey, M., Huckett, B., and Botha, F. (1994). “Use of polymerase chain reac-
tion (PCR) and random amplification of polymorphic DNAs (RAPDs) for
the determination of genetic distances between 21 sugarcane varieties,” in
Proceedings of the South African Sugar Technologists Association (Citeseer),
36–40. Fedenko, J., Erickson, J., Woodard, K., Sollenberger, L., Vendramini, J. B., Gilbert,
R. A., et al. (2013). Biomass production and composition of perennial grasses
grown for bioenergy in a subtropical climate across Florida, USA. BioEnergy Res. 6, 1082–1093. doi:10.1007/s12155-013-9342-3 Hattori, T., and Morita, S. (2010). Energy crops for sustainable bioethanol
production; which, where and how? Plant Prod. Sci. 13, 221–234. doi:10.1626/
pps.13.221 Figueira, T., Okura, V., Rodrigues Da Silva, F., Jose Da Silva, M., Kudrna, D.,
Ammiraju, J. S. et al. (2012). A BAC library of the SP80-3280 sugarcane variety
(Saccharum sp.) and its inferred microsynteny with the Sorghum genome. BMC
Res. Notes 5:185. doi:10.1186/1756-0500-5-185 Henry, R. J. (2010a). Evaluation of plant biomass resources available for replace-
ment of fossil oil. Plant Biotechnol. J. 8, 288–293. doi:10.1111/j.1467-7652.2009. 00482.x Furtado, A., Lupoi, J. S., Hoang, N. V., Healey, A., Singh, S., Simmons, B. A.,
et al. (2014). Modifying plants for biofuel and biomaterial production. Plant
Biotechnol. J. 12, 1246–1258. doi:10.1111/pbi.12300 Henry, R. J. (2010b). Plant Resources for Food, Fuel and Conservation. London:
Earthscan. November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 12 Sugarcane Genetic Improvement for Biofuels Hoang et al. Henry, R. J., Edwards, M., Waters, D. L., Gopala Krishnan, S., Bundock, P., Sexton, parentage among genotypes of sugar cane (Saccharum spp.). Theor. Appl. Genet. 104, 30–38. doi:10.1007/s001220200003 parentage among genotypes of sugar cane (Saccharum spp.). Theor. Appl. Genet. 104, 30–38. doi:10.1007/s001220200003 Henry, R. J., Edwards, M., Waters, D. L., Gopala Krishnan, S., Bundock, P., Sexton,
T. REFERENCES Biofuels: the past, present, and a new vision for the future. Bioscience 59, 926–927. doi:10.1525/bio.2009.59.11.2 Park, J. W., Benatti, T. R., Marconi, T., Yu, Q., Solis-Gracia, N., Mora, V., et al. (2015). Cold responsive gene expression profiling of sugarcane and Saccharum
spontaneum with functional analysis of a cold inducible Saccharum homolog of
NOD26-like intrinsic protein to salt and water stress. PLoS ONE 10:e0125810. doi:10.1371/journal.pone.0125810 Sims, R. E. H., Hastings, A., Schlamadinger, B., Taylor, G., and Smith, P. (2006). Energy crops: current status and future prospects. Glob. Chang. Biol. 12,
2054–2076. doi:10.1111/j.1365-2486.2006.01163.x Somerville, C., Youngs, H., Taylor, C., Davis, S. C., and Long, S. P. (2010). Feedstocks for lignocellulosic biofuels. Science 329, 790–792. doi:10.1126/
science.1189268 Paterson, A., Bowers, J., Bruggmann, R., Dubchak, I., Grimwood, J., Gundlach, H.,
et al. (2009). The Sorghum bicolor genome and the diversification of grasses. Nature 457, 551–556. doi:10.1038/nature07723 Souza, A., Leite, D. C., Pattathil, S., Hahn, M., and Buckeridge, M. (2013). Com-
position and structure of sugarcane cell wall polysaccharides: implications for
second-generation bioethanol production. Bioenerg. Res. 6, 564–579. doi:10. 1007/s12155-012-9268-1 Paterson, A., Souza, G., Sluys, M. V., Ming, R., D’Hont, A., Henry, R., et al. (2010). “Structural genomics and genome sequencing,” in Genetics, Genomics
and Breeding of Sugarcane, eds. R. Henry and C. Kole (Enfield, NH: Science
Publishers), 149–165. Souza, G. M., Berges, H., Bocs, S., Casu, R., D’Hont, A., Ferreira, J. E., et al. (2011). The sugarcane genome challenge: strategies for sequencing a highly complex
genome. Trop. Plant Biol. 4, 145–156. doi:10.1007/s12042-011-9079-0 Pauly, M., Gille, S., Liu, L., Mansoori, N., De Souza, A., Schultink, A., et al. (2013). Hemicellulose biosynthesis. Planta 238, 627–642. doi:10.1007/s00425-
013-1921-1 Sreedhar, A., and Collins, A. K. (2010). “Molecular genetic linkage mapping in
saccharum,” in Genetics, Genomics and Breeding of Sugarcane, eds. R. Henry
and
C. Kole
(Enfield,
NH:
Science
Publishers),
69–96. doi:10.1201/
EBK1578086849-6 Pereira, S. C., Maehara, L., Machado, C. M. M., and Farinas, C. S. (2015). 2G ethanol
from the whole sugarcane lignocellulosic biomass. Biotechnol. Biofuels 8, 44. doi:10.1186/s13068-015-0224-0 Sreenivasan, T. V., Ahloowalia, B. S., and Heinz, D. J. (1987). “Chapter 5 – cytoge-
netics,” in Developments in Crop Science, ed. J. H. Don (Amsterdam: Elsevier),
211–253. Pinto, L. R., Oliveira, K. M., Ulian, E. C., Garcia, A. A., and De Souza, A. P. (2004). Survey in the sugarcane expressed sequence tag database (SUCEST) for simple
sequence repeats. Genome 47, 795–804. REFERENCES doi:10.1139/g04-055 Steuernagel, B., Taudien, S., Gundlach, H., Seidel, M., Ariyadasa, R., Schulte, D.,
et al. (2009). De novo 454 sequencing of barcoded BAC pools for comprehensive
gene survey and genome analysis in the complex genome of barley. BMC
Genomics 10:547. doi:10.1186/1471-2164-10-547 Piperidis, G., D’Hont, A., and Hogarth, D. (2001). “Chromosome composition anal-
ysis of various Saccharum interspecific hybrids by genomic in situ hybridisation
(GISH),” in International Society of Sugar Cane Technologists. Proceedings of the
XXIV Congress, Vol. 2 ed. D. M. Hogarth (Brisbane: Australian Society of Sugar
Cane Technologists), 565–566. Sticklen, M. (2006). Plant genetic engineering to improve biomass characteristics for
biofuels. Curr. Opin. Biotechnol. 17, 315–319. doi:10.1016/j.copbio.2006.05.003 Piperidis, G., Piperidis, N., and D’Hont, A. (2010). Molecular cytogenetic investi-
gation of chromosome composition and transmission in sugarcane. Mol. Genet. Genomics 284, 65–73. doi:10.1007/s00438-010-0546-3 Sugesi. (2015). The Sugarcane Genome Sequencing Initiative. Available at: http://
cnrgv.toulouse.inra.fr/fr/Projets/Canne-a-sucre/The-Sugarcane-Genome-
Sequencing-Initiative-SUGESI-Strategies-for-Sequencing-a-Highly-Complex-
Genome. Accessed on 25 May 2015 Rabemanolontsoa, H., and Saka, S. (2013). Comparative study on chemical com-
position of various biomass species. RSC Adv. 3, 3946–3956. doi:10.1039/
c3ra22958k Sun, N., Liu, H., Sathitsuksanoh, N., Stavila, V., Sawant, M., Bonito, A., et al. (2013).Production andextraction ofsugarsfrom switchgrasshydrolyzed in ionic
liquids. Biotechnol. Biofuels 6, 39. doi:10.1186/1754-6834-6-39 Raboin, L. M., Pauquet, J., Butterfield, M., D’Hont, A., and Glaszmann, J. C. (2008). Analysis of genome-wide linkage disequilibrium in the highly polyploid
sugarcane. Theor. Appl. Genet. 116, 701–714. doi:10.1007/s00122-007-0703-1 Tabasum, S., Khan, F. A., Nawaz, S., Iqbal, M. Z., and Saeed, A. (2010). DNA
profiling of sugarcane genotypes using randomly amplified polymorphic DNA. Genet. Mol. Res. 9, 471–483. doi:10.4238/vol9-1gmr709 Ramos, R. L. B., Tovar, F. J., Junqueira, R. M., Lino, F. B., and Sachetto-Martins, G. (2001). Sugarcane expressed sequences tags (ESTs) encoding enzymes involved
in lignin biosynthesis pathways. Genet. Mol. Biol. 24, 235–241. doi:10.1590/
S1415-47572001000100031 Tai, P., and Miller, J. (2001). A core collection for Saccharum spontaneum L. from the
world collection of sugarcane. Crop Sci. 41, 879–885. doi:10.2135/cropsci2001. 413879x Renewable Fuels Association. (2015). World Fuel Ethanol Production. Available at:
http://ethanolrfa.org/pages/World-Fuel-Ethanol-Production. Accessed 25 May
2015 Taylor, L. E. II, Dai, Z., Decker, S. R., Brunecky, R., Adney, W. S., Ding, S. Y.,
et al. (2008). Heterologous expression of glycosyl hydrolases in planta: a new
departure for biofuels. Trends Biotechnol. 26, 413–424. doi:10.1016/j.tibtech. 2008.05.002 Roach, B. (1989). “Origin and improvement of the genetic base of sugarcane,”
in Proceedings Australian Society of Sugar Cane Technologists, eds. B. T. REFERENCES Biofuels 3, 27. doi:10.1186/1754-6834-
3-27 Ming, R., Moore, P. H., Wu, K.-K., D’Hont, A., Glaszmann, J. C., Tew, T. L., et al. (2010). “Sugarcane improvement through breeding and biotechnology,” in Plant
Breeding Reviews, ed. J. Janick (Oxford, UK: John Wiley & Sons, Inc), 15–118. doi:10.1002/9780470650349.ch2 Lima, M. L., Garcia, A. A., Oliveira, K. M., Matsuoka, S., Arizono, H., De Souza, C. L. Jr., et al. (2002). Analysis of genetic similarity detected by AFLP and coefficient of Moore, P. H. (2009). “Sugarcane biology, yield, and potential for improvement,” in
Workshop BIOEN on Sugarcane Improvement (San Pablo). November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 13 Sugarcane Genetic Improvement for Biofuels Hoang et al. Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y., Holtzapple, M., et al. (2005). Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 96, 673–686. doi:10.1016/j.biortech.2004.06.025 Sato, K., Motoi, Y., Yamaji, N., and Yoshida, H. (2011). 454 sequencing of pooled
BAC clones on chromosome 3H of barley. BMC Genomics 12:246. doi:10.1186/
1471-2164-12-246 Mutwil, M., Debolt, S., and Persson, S. (2008). Cellulose synthesis: a complex
complex. Curr. Opin. Plant Biol. 11, 252–257. doi:10.1016/j.pbi.2008.03.007 Saxena, I. M., and Brown, R. M. Jr. (2000). Cellulose synthases and related enzymes. Curr. Opin. Plant Biol. 3, 523–531. doi:10.1016/S1369-5266(00)00125-4 Nagaraj, S. H., Gasser, R. B., and Ranganathan, S. (2007). A hitchhiker’s guide to
expressed sequence tag (EST) analysis. Brief. Bioinformatics 8, 6–21. doi:10.1093/
bib/bbl015 Schubert, C. (2006). Can biofuels finally take center stage? Nat. Biotechnol. 24,
777–784. doi:10.1038/nbt0706-777 Seabra, J. E. A., Tao, L., Chum, H. L., and Macedo, I. C. (2010). A techno-economic
evaluation of the effects of centralized cellulosic ethanol and co-products refin-
ery options with sugarcane mill clustering. Biomass Bioenergy 34, 1065–1078. doi:10.1016/j.biombioe.2010.01.042 Neale, D. B., and Savolainen, O. (2004). Association genetics of complex traits in
conifers. Trends Plant Sci. 9, 325–330. doi:10.1016/j.tplants.2004.05.006 Pandey, A., Soccol, C. R., Nigam, P., and Soccol, V. T. (2000). Biotechnological
potential of agro-industrial residues. I: sugarcane bagasse. Bioresour. Technol. 74,
69–80. doi:10.1016/s0960-8524(99)00142-x Shi, J., Gladden, J. M., Sathitsuksanoh, N., Kambam, P., Sandoval, L., Mitra, D., et al. (2013). One-pot ionic liquid pretreatment and saccharification of switchgrass. Green Chem. 15, 2579–2589. doi:10.1039/C3GC40545A Panje, R. R. and Babu, C. N. (1960). Studies in saccharum spontaneum distribution
and geographical association of chromosome numbers. Cytologia 25, 152–72,
doi:10.1508/cytologia.25.152 Simpson, T. (2009). REFERENCES Egan
(Salisbury: Ferguson and Company), 34–47. Tew, T., and Cobill, R. (2008). “Genetic improvement of sugarcane (Saccharum spp.)
as an energy crop,” in Genetic Improvement of Bioenergy Crops, ed. W. Vermerris
(New York, NY: Springer), 273–294. Saathoff, A., Sarath, G., Chow, E., Dien, B., and Tobias, C. (2011). Downregulation
of cinnamyl-alcohol dehydrogenase in switchgrass by RNA silencing results in
enhanced glucose release after cellulase treatment. PLoS ONE 6:e16416. doi:10. 1371/journal.pone.0016416 Todd, J., Wang, J., Glaz, B., Sood, S., Ayala-Silva, T., Nayak, S., et al. (2014). Pheno-
typic characterization of the Miami world collection of sugarcane (Saccharum
spp.) and related grasses for selecting a representative core. Genet. Resour. Crop
Evol. 61, 1581–1596. doi:10.1007/s10722-014-0132-3 Santos Brito, M., Nobile, P., Bottcher, A., Dos Santos, A., Creste, S., De Landell,
M., et al. (2015). Expression profile of sugarcane transcription factor genes
involved in lignin biosynthesis. Trop. Plant Biol. 8, 19–30. doi:10.1007/s12042-
015-9147-y Tomkins, J. P., Yu, Y., Miller-Smith, H., Frisch, D. A., Woo, S. S., and Wing, R. A. (1999). A bacterial artificial chromosome library for sugarcane. Theor. Appl. Genet. 99, 419–424. doi:10.1007/s001220051252 November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 14 Sugarcane Genetic Improvement for Biofuels Hoang et al. effects of population substructure. Theor. Appl. Genet. 114, 155–164. doi:10. 1007/s00122-006-0418-8 effects of population substructure. Theor. Appl. Genet. 114, 155–164. doi:10. 1007/s00122-006-0418-8 Van Der Weijde, T., Alvim Kamei, C. L., Torres, A. F., Vermerris, W., Dolstra, O.,
Visser, R. G., et al. (2013). The potential of C4 grasses for cellulosic biofuel
production. Front. Plant Sci. 4:107. doi:10.3389/fpls.2013.00107 Whetten, R., and Ron, S. (1995). Lignin biosynthesis. Plant Cell 7, 1001–1013. doi:10.2307/3870053 Van Dijk, E. L., Auger, H., Jaszczyszyn, Y., and Thermes, C. (2014). Ten years of next-
generation sequencing technology. Trends Genet. 30, 418–426. doi:10.1016/j.tig. 2014.07.001 Whittaker, A., and Botha, F. C. (1997). Carbon partitioning during sucrose accu-
mulation in sugarcane internodal tissue. Plant Physiol. 115, 1651–1659. Xu, Y., Lu, Y., Xie, C., Gao, S., Wan, J., and Prasanna, B. (2012). Whole-genome
strategies for marker-assisted plant breeding. Mol. Breed. 29, 833–854. doi:10. 1007/s11032-012-9699-6 Vargas, L., Santa Brígida, A. B., Mota Filho, J. P., De Carvalho, T. G., Rojas,
C. A., Vaneechoutte, D., et al. (2014). Drought tolerance conferred to sug-
arcane by association with Gluconacetobacter diazotrophicus: a transcriptomic
view of hormone pathways. PLoS ONE 9:e114744. doi:10.1371/journal.pone. REFERENCES 0114744 Yan, L., Xu, C., Kang, Y., Gu, T., Wang, D., Zhao, S., et al. (2013). The heterologous
expression in Arabidopsis thaliana of Sorghum transcription factor SbbHLH1
downregulates lignin synthesis. J. Exp. Bot. 64, 3021–3032. doi:10.1093/jxb/
ert150 Verheye, W. (2010). “Growth and production of sugarcane,” in Soils, Plant Growth
and Production Volume II in Encyclopedia of Life Support Systems (EOLSS),
Developed under the Auspices of the UNESCO, Vol. 2. Paris: Eolss Publishers. Available at: www.eolss.net/sample-chapters/c10/e1-05a-22-00.pdf [Accessed 15
July 15]. Yoshida, K., Sakamoto, S., Kawai, T., Kobayashi, Y., Sato, K., Ichinose, Y., et al. (2013). Engineering the Oryza sativa cell wall with rice NAC transcription
factors regulating secondary wall formation. Front. Plant Sci. 4:383. doi:10.3389/
fpls.2013.00383 Vettore, A. L., Da Silva, F. R., Kemper, E. L., Souza, G. M., Da Silva, A. M.,
Ferro, M. I., et al. (2003). Analysis and functional annotation of an expressed
sequence tag collection for tropical crop sugarcane. Genome Res. 13, 2725–2735. doi:10.1101/gr.1532103 Yuan, J. S., Tiller, K. H., Al-Ahmad, H., Stewart, N. R., and Stewart, C. N. Jr. (2008). Plants to power: bioenergy to fuel the future. Trends Plant Sci. 13, 421–429. doi:10.1016/j.tplants.2008.06.001 g
Vettore, A. L., Silva, F. R. D., Kemper, E. L., and Arruda, P. (2001). The
libraries that made SUCEST. Genet. Mol. Biol. 24, 1–7. doi:10.1590/S1415-
47572001000100002 Zhang, J. S., Nagai, C., Yu, Q. Y., Pan, Y. B., Ayala-Silva, T., Schnell, R. J., et al. (2012). Genome size variation in three Saccharum species. Euphytica 185, 511–519. doi:10.1007/s10681-012-0664-6 Viikari, L., Vehmaanpera, J., and Koivula, A. (2012). Lignocellulosic ethanol:
from science to industry. Biomass Bioeng. 46, 13–24. doi:10.1016/j.biombioe. 2012.05.008 Zhang, L., Xu, J., and Birch, R. G. (1999). Engineered detoxification confers resis-
tance against a pathogenic bacterium. Nat. Biotechnol. 17, 1021–1024. doi:10. 1038/13721 Vogel, J. (2008). Unique aspects of the grass cell wall. Curr. Opin. Plant Biol. 11,
301–307. doi:10.1016/j.pbi.2008.03.002 Zhang, S.-Z., Yang, B.-P., Feng, C.-L., Chen, R.-K., Luo, J.-P., Cai, W.-W., et al. (2006). Expression of the Grifola frondosa trehalose synthase gene and improvement of
drought-tolerance in sugarcane (Saccharum officinarum L.). J. Integr. Plant Biol. 48, 453–459. doi:10.1111/j.1744-7909.2006.00246.x Waclawovsky, A. J., Sato, P. M., Lembke, C. G., Moore, P. H., and Souza, G. M. (2010). Sugarcane for bioenergy production: an assessment of yield and
regulation of sucrose content. Plant Biotechnol. J. 8, 263–276. doi:10.1111/j. 1467-7652.2009.00491.x Zhu, C., Gore, M., Buckler, E. S., and Yu, J. (2008). November 2015 | Volume 3 | Article 182 REFERENCES Status and prospects of associ-
ation mapping in plants. Plant Genome 1, 5–20. doi:10.3835/plantgenome2008. 02.0089 Wang, J., Roe, B., Macmil, S., Yu, Q., Murray, J. E., Tang, H., et al. (2010). Micro-
collinearity between autopolyploid sugarcane and diploid Sorghum genomes. BMC Genomics 11:261. doi:10.1186/1471-2164-11-261 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. Wang, X., and Paterson, A. (2013). “Comparative genomic analysis of C4 photosyn-
thesis pathway evolution in grasses,” in Genomics of the Saccharinae, ed. A. H. Paterson (New York, NY: Springer), 447–477. Copyright © 2015 Hoang, Furtado, Botha, Simmons and Henry. This is an open-access
article distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) or licensor are credited and that the original publication in this
journal is cited, in accordance with accepted academic practice. No use, distribution
or reproduction is permitted which does not comply with these terms. Copyright © 2015 Hoang, Furtado, Botha, Simmons and Henry. This is an open-access
article distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) or licensor are credited and that the original publication in this
journal is cited, in accordance with accepted academic practice. No use, distribution
or reproduction is permitted which does not comply with these terms. Wei, X., Jackson, P. A., Hermann, S., Kilian, A., Heller-Uszynska, K., and Deomano,
E. (2010). Simultaneously accounting for population structure, genotype by
environment interaction, and spatial variation in marker-trait associations in
sugarcane. Genome 53, 973–981. doi:10.1139/g10-050 Wei, X., Jackson, P. A., McIntyre, C. L., Aitken, K. S., and Croft, B. (2006). Associations between DNA markers and resistance to diseases in sugarcane and November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 15
|
https://openalex.org/W4281940932
|
https://www.preprints.org/manuscript/202205.0285/v1/download
|
English
| null |
Cervical Cancer Prevention in El Salvador: Gains to Date and Challenges for the Future
|
Cancers
| 2,022
|
cc-by
| 5,411
|
Article
Cervical Cancer Prevention in El Salvador: Gains to Date and
Challenges for the Future Article Karla Alfaro 1, Montserrat Soler 1,2,*, Mauricio Maza 3, Mauricio Flores 4, Leticia López 1, Juan C. Rauda 1, Andrea
Chacón 5, Patricia Erazo 5, Nora Villatoro 5, Eveline Mumenthaler 1, Rachel Masch 1, Gabriel Conzuelo 1, Juan C. Felix 6 and Miriam Cremer 1,2 1 Basic Health International, San Salvador, C.P. 01101El Salvador and Pittsburgh, 15205, USA; kalfaro@basi-
chealth.org (K.A.); llopez@basichealth.org (L.L.); jcrauda@basichealth.org (J.C.R.); emumenthaler@basi-
chealth.org (E.M.); rmasch@basichealth.org (R.M.); gconzuelo@basichealth.org (G.C.); mcremer@basi-
chealth.org (M.C.) 1 Basic Health International, San Salvador, C.P. 01101El Salvador and Pittsburgh, 15205, USA; kalfaro@basi-
chealth.org (K.A.); llopez@basichealth.org (L.L.); jcrauda@basichealth.org (J.C.R.); emumenthaler@basi-
chealth.org (E.M.); rmasch@basichealth.org (R.M.); gconzuelo@basichealth.org (G.C.); mcremer@basi-
chealth.org (M.C.) g
2 Ob/Gyn and Women’s Health Institute, Cleveland Clinic, Cleveland, OH 44195, USA 3 Cancer Program, Pan American Health Organization, Washington, DC, 20037 USA; mazamau@paho.org g
g
g
p
g
4 Comité Asesor de Prácticas en Inmunizaciones de El Salvador, San Salvador, C.P. 01101, El Salvador;
mernesto.flores@salud.gob.sv g
5 Unidad de Cáncer, Ministerio de Salud de El Salvador, San Salvador, C.P. 01101, El Salvador;
marina.chacon@salud.gob.sv (A.C.); mayra.erazo@salud.gob.sv (P.E.); nora.villatoro@salud.gob.sv (N.V. g
(
)
y
g
(
)
g
(
6 Department of Pathology, Medical College of Wisconsin, Milwaukee, WI 53226, USA; jcfelix@mcw.edu
* Correspondence: msoler@basichealth.org. * Correspondence: msoler@basichealth.org. Simple Summary: Cervical cancer is a leading cause of death for women in low-resource areas
around the world. However, the disease can be prevented through vaccination, screening, and early
treatment. Recently, the World Health Organization released a strategy with the goal of eliminating
cervical cancer through a combination of these three preventive strategies. In El Salvador, a screen-
ing program has been in place for some years, and vaccination is in its early stages. Here we describe
the Salvadoran experience and discuss successes to date and challenges for the future. We also pro-
vide recommendations that may be helpful for other countries working to meet the WHO elimina-
tion goals. Abstract: Cervical cancer is preventable through vaccination, early detection, and treatment of pre-
cancerous lesions. However, global inequalities mean that the disease remains a leading cause of
cancer death around the world, with over 80% of new cases and 90% of deaths occurring in low and
middle-income countries (LMICs). In El Salvador, joint efforts between the Ministry of Health
(MoH) and the non-profit organization Basic Health International (BHI) have been in place since
2008 with the goal of reducing the country’s disease burden. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 Article
Cervical Cancer Prevention in El Salvador: Gains to Date and
Challenges for the Future While the World Health Organization’s
(WHO) call to action to eliminate cervical cancer provided worldwide momentum to implement
new public health initiatives, the COVID-19 pandemic disrupted ongoing programs and jeopard-
ized plans for the future. The purpose of this manuscript is to describe the progress that El Salvador
has achieved in improving cervical cancer prevention, the impact of the pandemic on current strat-
egies, and potential solutions that can help the country meet the WHO’s strategic targets by 2030 to
accelerate the elimination of cervical cancer. Keywords: El Salvador; cervical cancer; cervical precancer; human papillomavirus (HPV); screen-
and-treat Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 2 of 8 LMICs, and these numbers are expected to grow in the next decade [2]. In light of these
considerations, the World Health Organization (WHO) released a call to action in 2018 to
eliminate cervical cancer as a public health problem [3]. The WHO strategy sets three
global targets to be reached by 2030 in order to accelerate elimination: (1) vaccinating 90%
of girls by age 15 with the human papillomavirus (HPV) vaccine, the causal agent of the
disease, (2) screen 70% of women at age 35 and again at age 45 with a high-performance
test, and (3) treat 90% of women with underlying cervical disease [4]. Meeting these goals
would save 300,000 lives by 2030, 13.4 million lives by 2070, and 62 million lives by 2120
[5]. While the WHO’s strategy presents challenges to resource-poor countries with com-
peting health priorities, the call to action has provided momentum to increase access to
cervical cancer preventive services. El Salvador is well-positioned to demonstrate an ef-
fective implementation of cervical cancer control programs. The country has historically
high rates of incidence and mortality (13.1 and 7.43 in 100,000, age-adjusted, respectively)
[1]. The sociocultural context complicates access to cancer prevention services. Almost
40% of the population lives in rural areas, and gang-related violence, poverty and out-
migration remain pervasive problems. The Ministry of Health operates a network of mu-
nicipal clinics that offer free-of-cost primary care services, although medications must of-
ten be purchased out of pocket. More specialized care can only be found at regional hos-
pitals in urban areas or in expensive private facilities. For many women, transportation to
clinics and hospitals is a significant hurdle. Since 2008, a collaboration between the Ministry of Health (MoH) and Basic Health
International (BHI), a non-profit organization, has resulted in a range of initiatives to
strengthen cervical cancer prevention in the country [6]. Previously, the national screening
program relied on colposcopy and/or cytology (Pap test), which involved multiple visits
for each patient and resulted in very low national coverage (estimates range from 19% [7]
to 44% [8]). In 2012, a partnership between the MoH and BHI resulted in a donation of
low-cost HPV tests (careHPV, Qiagen, Gaithersburg, MD, USA) to the country. The tests
were utilized to conduct a demonstration project that eventually reached 28,000 women. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 In the first stage, 2000 women were assigned to either screen-and-treat or colposcopy man-
agement [9]. Screen-and-treat approaches, where all screen-positive women are offered
treatment without biopsy-confirmed diagnosis, are recommended by the WHO in low-
resource settings [10]. In El Salvador, the initial pilot was gradually expanded to an addi-
tional 8000 women. Outcomes revealed significant differences in loss to follow-up, with
88.3% of HPV positive women in the screen-and-treat group completing treatment in 6
months, compared to 44.2% of HPV positive women who were referred to colposcopy
management within the same time period [11]. Following the success of this project, the screen-and-treat program was scaled-up in
the Paracentral region [12]. Cost-effectiveness analyses demonstrated the long-term ad-
vantages of this approach in the Salvadoran context [13,14]. Findings were presented to
public health officials and resulted in the incorporation of HPV testing into the national
cervical cancer control guidelines [15], making El Salvador one of the first countries to do
so in the region. In 2020, the COVID 19 pandemic disrupted health systems worldwide,
but secondary prevention services continued as local regulations allowed. The purpose of
this manuscript is to describe the progress of cervical cancer prevention programs in El
Salvador, review the challenges presented by the COVID-19 pandemic, and outline po-
tential future directions for the country. 1. Background Cervical cancer is a disease of inequality. Wealthy countries have access to preventive
strategies that have significantly reduce incidence and mortality rates in the last 50 years,
but low-and-middle-income countries (LMICs) continue to bear a large burden of disease
[1]. It is estimated that 80% of new cases and 90% of deaths from cervical cancer occur in © 2. The HPV Screening Program in El Salvador In 2017, after the initial success of a primary HPV-based screen-and-treat demonstra-
tion project, El Salvador was able to obtain grant funding for further HPV tests in addition
to HPV vaccines. The MoH included the procurement of HPV tests in its 2022 annual
budget, cementing the screen-and-treat program as part of the national cervical cancer
control effort. Today, HPV screening is available in a network of 750 primary care units Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 3 of 8 distributed in all five regions of the country (Figure 1). Provider trainings for implemen-
tation in the fifth and final region, which includes the metropolitan area of San Salvador,
began in March 2022. Since its inception, the program was designed to rely on the existing
public health infrastructure. As technical advisors, BHI has followed a “train the trainer”
approach with MoH personnel to ensure long-term sustainability. distributed in all five regions of the country (Figure 1). Provider trainings for implemen-
tation in the fifth and final region, which includes the metropolitan area of San Salvador,
began in March 2022. Since its inception, the program was designed to rely on the existing
public health infrastructure. As technical advisors, BHI has followed a “train the trainer”
approach with MoH personnel to ensure long-term sustainability. Figure 1. Gradual scale-up of the primary HPV screen-and-treat program in El Salvador. Figure 1. Gradual scale-up of the primary HPV screen-and-treat program in El Salvador. Community health teams, or ECOs in Spanish (Equipos Comunitarios de Salud),
form the backbone of the HPV screening program. ECOs consist of healthcare providers
at three levels: general practitioners that head municipal health clinics, nurses at the clin-
ics, and health promoters who are usually community members with no formal clinical
training. Health promoters are the first line of advocacy and education. They are respon-
sible for contacting women due for screening to set up appointments at local clinics. Cur-
rently, El Salvador guidelines recommend HPV testing for women aged 30–59 and cytol-
ogy for those between 20 to 29 and over 59 years of age. Participants of the HPV program
receive an educational session taught by nurses on arrival at the local clinic. Women then
undergo a provider sample collection using careHPV, which detects 14 types of high-risk
HPV (pooled, not genotyped). Screened women are given an appointment to return after
30 days for their test results. 2. The HPV Screening Program in El Salvador Since not all clinics offer cryotherapy, some women must
schedule the second appointment at the nearest treatment center. There are 74 such cen-
ters strategically distributed throughout the country. On the second visit, those who test
positive are offered an immediate pelvic exam to visually assess eligibility for ablation
treatment and, if eligible, gas-based cryotherapy. HPV testing, visual assessment, and
treatment are performed by trained general practitioners or gynecologists. Women who
are deemed ineligible for treatment during the visual assessment are referred to col-
poscopy and biopsy by colposcopists at regional hospitals. Criteria for ablation treatment
ineligibility are drawn from WHO guidelines and include a lesion that is larger than 75%
of the cervix, enters the endocervical canal, or is suspicious for cancer, or cases where the
squamocolumnar junction is not fully visible. To date, over 4500 healthcare providers have been trained as part of the screen-and-
treat program (Table 1) and 145,000 women have been tested. The HPV positivity rate is
approximately 14%. Women who test negative return for re-testing after five years. Those
who test positive return at one year after ablation treatment, and if they test positive again,
they are referred to colposcopy and biopsy. Although we know that approximately 88% Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 4 of 8 of HPV-screened women have received cryotherapy treatment to date, data on treatment
refusals, adherence to colposcopy and biopsy, or treatment follow-up are not currently
available. While El Salvador began to add outcomes of its cervical cancer prevention pro-
grams to its epidemiological surveillance system since 2012, data entry across health units
and hospitals is not consistent. Efforts are underway to improve surveillance, including
training of data entry specialists. Figure 2.. ECO team members train in El Salvador as part of the national HPV screening program. Figure 2.. ECO team members train in El Salvador as part of the national HPV screening program. After the WHO call for elimination was released, the MoH engaged in further efforts
to maximize the impact of this opportunity in El Salvador with two important outcomes. First, the MoH released a new screening target to increase the number of eligible women
screened to 15% per year for the next 5 years, which would achieve the WHO target of
70% of eligible women screened by 2030. 2. The HPV Screening Program in El Salvador Second, after a series of stakeholder meetings
that included MoH and BHI personnel, changes are planned to the national cervical cancer
guidelines. BHI had successfully completed several projects on the feasibility of HPV self-
collection in underserved areas [16–18]. Self-collection allows women to collect their own
sample using a small brush, circumventing the need for a speculum exam. BHI had also
participated in the review of WHO guidelines for thermal ablation [19], an alternative
treatment to cryotherapy that does not require a consumable (i.e., compressed gas) and
can be applied with a portable, battery-operated device. The BHI team introduced thermal
ablation to El Salvador through two ongoing research protocols (NCT NCT03084081 and
NCT03429582). Both HPV self-sampling and thermal ablation are expected to be included
in guidelines to be released in 2022. These innovations have the potential to increase access
to screening and treatment, particularly among rural and hard-to-reach populations. After the WHO call for elimination was released, the MoH engaged in further efforts
to maximize the impact of this opportunity in El Salvador with two important outcomes. First, the MoH released a new screening target to increase the number of eligible women
screened to 15% per year for the next 5 years, which would achieve the WHO target of
70% of eligible women screened by 2030. Second, after a series of stakeholder meetings
that included MoH and BHI personnel, changes are planned to the national cervical cancer
guidelines. BHI had successfully completed several projects on the feasibility of HPV self-
collection in underserved areas [16–18]. Self-collection allows women to collect their own
sample using a small brush, circumventing the need for a speculum exam. BHI had also
participated in the review of WHO guidelines for thermal ablation [19], an alternative
treatment to cryotherapy that does not require a consumable (i.e., compressed gas) and
can be applied with a portable, battery-operated device. The BHI team introduced thermal
ablation to El Salvador through two ongoing research protocols (NCT NCT03084081 and
NCT03429582). Both HPV self-sampling and thermal ablation are expected to be included
in guidelines to be released in 2022. These innovations have the potential to increase access
to screening and treatment, particularly among rural and hard-to-reach populations. 4. The Impact of COVID-19 In March 2020, as COVID-19 cases began to rise nationally, non-emergency health
services were suspended throughout the El Salvador public health system. Most MoH
personnel, including the ECOs that implemented the HPV screening program, were re-
directed to pandemic containment efforts. Thousands of women who had been screened
as part of the HPV screen-and-treat program had follow-up care delayed for several
months. In addition to this growing backlog of patients, a large number of HPV tests were
left in storage and expired during the lockdown period, rendering them unusable. The
planned April start date for the new HPV vaccination campaign was delayed to Novem-
ber 2020 when it was hoped in-person schooling would return. However, as non-essential
medical services slowly resumed in late 2020 and early 2021, an urgent COVID-19 vac-
cination campaign was prioritized. Thus, screening and vaccination initiatives for cervical
cancer were further interrupted. The true impact of the pandemic on routine medical services has only recently begun
to surface. In the Occidental and Oriental regions of El Salvador, the MoH estimated that
approximately 5000 HPV-positive women were awaiting follow-up care since the begin-
ning of lockdowns in 2020. Thus, in collaboration with BHI, a funding opportunity was
identified and a project initiated with the goal of locating these women and providing
follow-up care. Since MoH providers and laboratories remained at capacity with COVID-
related care, private colposcopists and pathology laboratories were engaged to conduct
this intervention. From March 2021 to March 2022, 3017/5000 (60%) screen-positive
women have been identified and efforts are ongoing to locate the remaining 1983/5000
(40%). Of those accounted for, 702/3017 (23%) had already received follow-up care at pri-
vate clinics, 210/3017 (7%) refused further care, and 96/3017 (3%) were lost to follow-up
(had moved, migrated out of the country, died, etc.). The remaining 2009/3017 (66.5%)
have undergone colposcopy and biopsy as part of this project. Laboratory analysis have
been completed for 1990/2009 (99%). Results show alarmingly high rates of high-grade
precancer at 23% (452/1990) and invasive cancer at 1.3% (26/1990). Although there are no
comparable pre-pandemic figures available, findings from the original HPV screen-and-
treat demonstration project provide a frame of reference. Among HPV positive women in
the colposcopy management group who completed biopsy within 6 months, 56/385 (15%)
were diagnosed with high-grade precancer and 1 (<1%) with invasive cancer [11].These
women were similarly recruited from a semi-rural screening population. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 5 of 8 and the MoH settled on a school-based strategy targeting 9-year old girls for a first dose,
with a second dose after six months. Quadrivalent Gardasil (Merck &Co., Kenilworth, NJ,
USA) was the vaccine of choice. Usually, childhood vaccines are given at local health
units, but given the need for two doses and the target ages, it was determined that a
school-based approach would result in better follow-up. HPV vaccination was projected
to start in April 2020 during the Americas Vaccination Week with the goal of vaccinating
approximately 55,000 girls the first year. The HPV vaccine was also included in El Salva-
dor’s vaccination schedule, ensuring HPV vaccine doses would be included in future na-
tional budgets. Finally, the vaccine data collection system was prepared to receive general
outcomes of the HPV vaccination effort. As the program was about to begin, the COVID-
19 was declared a pandemic by the WHO. HPV screening and vaccination programs were
immediately halted. 3. HPV Vaccination in El Salvador The MoH planned to introduce HPV vaccination into the country since 2015. The
WHO call to action coincided with a donation from the World Bank, providing the neces-
sary momentum to begin implementation efforts. An advisory committee was convened 5. Challenges for the Future El Salvador has achieved significant gains over the last decade to reduce its cervical
cancer burden. There is now a scalable screening program in all regions of the country
and, for the first time in 2022, HPV tests were purchased solely with government funds. The WHO call for elimination has provided additional motivation to set defined screening
targets and begin the implementation of a new HPV vaccination program despite the chal-
lenges of the pandemic. However, significant barriers remain, many of which represent
broader issues that can impede elimination goals globally. Although HPV screening is
highly acceptable to patients and providers, there are limited low-cost options. In El Sal-
vador, budgetary constraints currently allow for the procurement of only 100,000 test kits
per year, which is projected to cover 15% of the eligible population. In addition, many
existing HPV tests require complex equipment or long processing times. Inexpensive,
rapid HPV tests are in development, but none are yet available that allow for the mainte-
nance of a nation-wide, single-day screen-and-treat program. There is also an urgent need
for affordable and accessible triage options that can minimize overtreatment and more
efficiently channel resources. Better follow-up options for women ineligible for ablation,
who may be at greater risk for invasive cancer, are also lacking. Innovative and affordable
point-of-care screening and treatment alternatives will be necessary to meet elimination
goals. g
In terms of primary prevention, the HPV vaccination program in El Salvador is still
in early adoption. It is a positive sign that the vaccine has been included in the national
vaccination schedule, which will greatly assist in ensuring its sustainability. However, a
recent survey in two regions of the country revealed that only 60% of adult respondents
would vaccinate a daughter or close relative between the ages of 9 and 12, even if such a
program was free of cost [20]. Thus, even if vaccines become widely available effective
interventions to overcome vaccine hesitancy will be essential. On the other hand, if a sin-
gle-dose schedule is eventually introduced, this would facilitate implementation by re-
ducing costs and avoiding loss to follow-up. The COVID-19 pandemic has also highlighted the need to strengthen health systems
globally, and cervical cancer programs in particular. In El Salvador, the return of screening
has been gradual and irregular due to recurring infection waves, and there is a significant
backlog of women waiting for results or treatment. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 6 of 8 HPV vaccine in the national budget means that procurement of HPV doses in the next few
years is assured. This, in turn, will facilitate meeting elimination goals. HPV vaccine in the national budget means that procurement of HPV doses in the next few
years is assured. This, in turn, will facilitate meeting elimination goals. 5. Challenges for the Future While other health emergencies will
always exist (for example, at the time of writing El Salvador was undergoing a dengue
breakout that suspended training for cervical cancer screening providers), there are strat-
egies that can help mitigate these challenges. For example, HPV self-collection can allow
for at-home testing and including HPV vaccination in national schedules can help priori-
tize cervical cancer prevention. 4. The Impact of COVID-19 Thus, unless
rapid action is taken, the suspension of routine screening services is expected to negatively
impact the achievement of elimination goals. The HPV vaccination effort has also faced challenges. Since public schools were shut
down for most of 2020 and part of 2021, the MoH decided to shift its strategy to an at-
home approach. The age range was also expanded to encompass girls aged 9 to 11 years. Despite these adjustments, the total number of first doses provided was 15,286, 28% of the
original target. Although these figures fall short of initial projections, the inclusion of the Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Conflicts of Interest: M.C. is a speaker and on the advisory board of Merck & Co. and Organon
International and the President and Founder of Basic Health International. M.S. is a paid consultant
for Basic Health International. 5.
Brisson, M.; Kim, J.J.; Canfell, K.; Drolet, M.; Gingras, G.; A Burger, E.; Martin, D.; Simms, K.T.; Bénard, E.; Boily, M.-C.; et al.
Impact of HPV vaccination and cervical screening on cervical cancer elimination: A comparative modelling analysis in 78 low-
income and lower-middle-income countries. Lancet 2020, 395, 575–590. https://doi.org/10.1016/S0140-6736(20)30068-4. 6.
Maza, M.; Alfaro, K.; Garai, J.; Velado, M.M.; Gage, J.C.; Castle, P.E.; Felix, J.; Luciani, S.; Campos, N.; Kim, J.; et al. Cervical
cancer prevention in El Salvador (CAPE)—An HPV testing-based demonstration project: Changing the secondary prevention
paradigm in a lower middle-income country. Gynecol. Oncol. Rep. 2017, 20, 58–61. https://doi.org/10.1016/j.gore.2017.02.011. p
g
y
y
p
p
g
j g
7.
Murillo, R.; Almonte, M.; Pereira, A.; Ferrer, E.; Gamboa, O.A.; Jerónimo, J.; Lazcano-Ponce, E. Cervical cancer screening pro-
grams in Latin America and the Caribbean. Vaccine 2008, 26 (Suppl. 11), L37–L48. https://doi.org/10.1016/j.vaccine.2008.06.013. 4.
World Health Organization. Global Strategy to Accelerate the Elimination of Cervical Cancer as a Public Health Problem; World Health
Organization: Geneva, Switzerland, 2020. 6. Conclusions In order for LMICs to meet the WHO’s elimination goals by 2030, more affordable
and accessible vaccination and screening modalities are necessary but not sufficient. It is
also essential to follow evidence-based strategies and recommendations to implement and
sustain population-level programs. Our experiences in El Salvador have taught us valua-
ble lessons in this regard. First and foremost, it is crucial to engage stakeholders at the
early planning stages of any project. At the local level, a joint BHI and MoH team orga-
nized meetings and workshops with other MoH staff, professional organizations, and
other actors working in the cervical cancer space in order to build interest in a pilot
demonstration. We also activated an international network of academic connections,
which proved valuable in bringing expertise to the design and implementation of the pro-
ject. The MoH has been extremely receptive of these contributions and this has helped to Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 7 of 8 build in-country capacity across multiple sectors. We would encourage any government
team involved in screening or vaccination initiatives to seek out partnerships with re-
search or non-profit groups that can bring flexible skills and resources to the development
of a new initiative. Once a program is ready for implementation, it is crucial to design an education and
awareness plan, not only at the community level, but also targeting the staff that will run
it on a day-to-day basis. In El Salvador, there are separate training and refresher for clini-
cians and health promoters, which ensures that providers at all levels are knowledgeable
and motivated to recruit participants. On the more practical side, negotiating with local
distributors to supply tests and necessary materials can be helpful in avoiding customs
delays and cutting down costs. Finally, surveillance and data collection, at any scale that
is feasible, are absolutely necessary to gauge program success and detect areas where
more work is needed. In our case, data have also been essential to effect guideline change
and ensure meaningful and sustainable impact. The WHO call for elimination has placed
cervical cancer prevention at the front and center of global health. It is crucial that gov-
ernments, funding agencies, researchers, and other stakeholders take advantage of the
current moment to meet elimination targets and ensure that future generations are free of
this preventable disease. Author Contributions: Conceptualization, K.A., M.S., M.M., M.F., A.C., P.E., N.V., R.M., J.C.F. and
M.C.; Data curation, M.S., L.L., J.C.R., A.C. and G.C.; Formal analysis, M.S. and G.C.; Funding ac-
quisition, K.A., M.M., E.M., R.M. and M.C.; Investigation, K.A., M.S., M.M., L.L., E.M., R.M., G.C.,
J.C.F. and M.C.; Project administration, K.A., L.L., J.C.R., A.C., P.E. and N.V.; Supervision, K.A.,
M.M., M.F., L.L., J.C.R., A.C., P.E., N.V., R.M., J.C.F. and M.C.; Writing—original draft, M.S.; Writ-
ing—review & editing, K.A., M.M., M.F., J.C.R., A.C., P.E., N.V., E.M., R.M., G.C., J.C.F. and M.C. All authors have read and agreed to the published version of the manuscript. Funding: The Freedom Philanthropic Fund, Einhorn Collaborative. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. References 1. Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global cancer statistics 2020: GLOBOCAN
estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA A Cancer J. Clin. 2021, 71, 209–249. https://doi.org/10.3322/caac.21660. p
g
2. Hull, R.; Mbele, M.; Makhafola, T.; Hicks, C.; Wang, S.; Reis, R.M.; Mehrotra, R.; Mkhize‑Kwitshana, Z.; Kibiki, G.; Bates, D.O.;
et
al. Cervical
cancer
in
low
and
middle.income
countries
(Review). Oncol. Lett. 2020,
20,
2058–2074. https://doi.org/10.3892/ol.2020.11754. p
g
3. World Health Organization. WHO Director-General Calls for All Countries to Take Action to Help End the Suffering Caused
by Cervical Cancer. Published 2018. Available online: https://www.who.int/reproductivehealth/call-to-action-elimination-cer-
vical-cancer/en/ (accessed on 29 August 2021). p
g
3. World Health Organization. WHO Director-General Calls for All Countries to Take Action to Help End the Suffering Caused
by Cervical Cancer. Published 2018. Available online: https://www.who.int/reproductivehealth/call-to-action-elimination-cer-
vical-cancer/en/ (accessed on 29 August 2021). g
4. World Health Organization. Global Strategy to Accelerate the Elimination of Cervical Cancer as a Public Health Problem; World Health
Organization: Geneva, Switzerland, 2020. 5. Brisson, M.; Kim, J.J.; Canfell, K.; Drolet, M.; Gingras, G.; A Burger, E.; Martin, D.; Simms, K.T.; Bénard, E.; Boily, M.-C.; et al. Impact of HPV vaccination and cervical screening on cervical cancer elimination: A comparative modelling analysis in 78 low-
income and lower-middle-income countries. Lancet 2020, 395, 575–590. https://doi.org/10.1016/S0140-6736(20)30068-4. 6. Maza, M.; Alfaro, K.; Garai, J.; Velado, M.M.; Gage, J.C.; Castle, P.E.; Felix, J.; Luciani, S.; Campos, N.; Kim, J.; et al. Cervical
cancer prevention in El Salvador (CAPE)—An HPV testing-based demonstration project: Changing the secondary prevention
paradigm in a lower middle-income country. Gynecol. Oncol. Rep. 2017, 20, 58–61. https://doi.org/10.1016/j.gore.2017.02.011. p
g
y
y
p
p
g
j g
7. Murillo, R.; Almonte, M.; Pereira, A.; Ferrer, E.; Gamboa, O.A.; Jerónimo, J.; Lazcano-Ponce, E. Cervical cancer screening pro-
grams in Latin America and the Caribbean. Vaccine 2008, 26 (Suppl. 11), L37–L48. https://doi.org/10.1016/j.vaccine.2008.06.013. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 8 of 8 8. Maza, M.; Matesanz, S.; Alfaro, K.; Alonzo, T.; Masch, R.; Calderon, S.; Velis, J.; Jamshidi, R.; Cremer, M. Adherence to recom-
mended
follow-up
care
after
high-grade
cytology
in
El
Salvador. Int. J. Healthc. 2016,
2,
31–36. https://doi.org/10.5430/ijh.v2n2p31. p //
g/
/ j
p
9. Cremer, M.L.; Maza, M.; Alfaro, K.M.; Kim, J.J.; Ditzian, L.R.; Villalta, S.; Alonzo, T.A.; Felix, J.C.; Castle, P.E.; Gage, J.C. References Intro-
ducing a high-risk HPV DNA test into a public sector screening program in El Salvador. J. Low. Genit. Tract Dis. 2016, 20, 145–
150. https://doi.org/10.1097/LGT.0000000000000188. p
g
10. World Health Organization. WHO Guidelines for Screening and Treatment of Precancerous Lesions for Cervical Cancer Pre-
vention. Published
Online
2013. Available
online:
https://apps.who.int/iris/bitstream/han-
dle/10665/94830/9789241548694_eng.pdf?sequence=1 (accessed on 29 August 2021). 11. Cremer, M.; Maza, M.; Alfaro, K.; Velado, M.M.; Felix, J.; Castle, P.E.; Kim, J.; Gage, J.C. Scale-up of an human papillomavirus
testing
implementation
program
in
El
Salvador. J. Low. Genit. Tract
Dis. 2017,
21,
26–32. https://doi.org/10.1097/LGT.0000000000000280. p
g
12. Alfaro, K.; Maza, M.; Felix, J.C.; Castle, P.E.; Alonzo, T.A.; Chacón, A.; González, E.; Soler, M.; Conzuelo-Rodriguez, G.; et al. Outcomes for step-wise implementation of a human papillomavirus testing-based cervical screen-and-treat program in El Sal-
vador. JCO Glob. Oncol. 2020, 6, 1519–1530. https://doi.org/10.1200/GO.20.00206. p
g
13. Campos, N.G.; Maza, M.; Alfaro, K.; Gage, J.C.; Castle, P.E.; Felix, J.C.; Masch, R.; Cremer, M.; Kim, J.J. The cost-effectiveness of
implementing HPV testing for cervical cancer screening in El Salvador. Int. J. Gynecol. Obstet. 2019, 145, 40–46. https://doi.org/10.1002/ijgo.12773. p
g
jg
14. Campos, N.G.; Maza, M.; Alfaro, K.; Gage, J.C.; Castle, P.E.; Felix, J.C.; Cremer, M.L.; Kim, J.J. The comparative and cost-effec-
tiveness of HPV-based cervical cancer screening algorithms in El Salvador. Int. J. Cancer 2015, 137, 893–902. https://doi.org/10.1002/ijc.29438. p
g
j
15. Ministerio de Salud Gobierno de El Salvador. Lineamientos Técnicos Para la Prevención y Control Del Cáncer Cérvico Uterino
y de Mama. Published Online 2015. Available online: http://asp.salud.gob.sv/regulacion/pdf/lineamientos/lineamientos_pre-
vencion_cancer_cervico_uterino_y_de_mama_v3.pdf (accessed on 29 August 2021). 16. Rosenbaum, A.J.; Gage, J.C.; Alfaro, K.M.; Ditzian, L.R.; Maza, M.; Scarinci, I.C.; Felix, J.C.; Castle, P.E.; Villalta, S.; Miranda, E.;
et al. Acceptability of self-collected versus provider-collected sampling for HPV DNA testing among women in rural El Salva-
dor. Int. J. Gynecol. Obstet. 2014, 126, 156–160. https://doi.org/10.1016/j.ijgo.2014.02.026. y
p
g
j jg
17. Maza, M.; Melendez, M.; Masch, R.; Alfaro, K.; Chacon, A.; Gonzalez, E.; Soler, M.; Conzuelo-Rodriguez, G.; Gage, J.; Alonzo,
T.; et al. Acceptability of self-sampling and human papillomavirus testing among non-attenders of cervical cancer screening
programs in El Salvador. Prev. Med. 2018, 114, 149–155. https://doi.org/10.1016/j.ypmed.2018.06.017. p
g
p
g
j yp
18. Laskow, B.; Figueroa, R.; Alfaro, K.M.; Scarinci, I.C.; Conlisk, E.; Maza, M.; Chang, J.C.; Cremer, M. References A pilot study of community-
based self-sampling for HPV testing among non-attenders of cervical cancer screening programs in El Salvador. Int. J. Gynecol. Obstet. 2017, 138, 194–200. https://doi.org/10.1002/ijgo.12204. p
g
jg
19. Guidelines Review Committee. WHO Guidelines for the Use of Thermal Ablation for Cervical Pre-Cancer Lesions. Who.int. Published Online September 2019. Available online: https://www.who.int/publications/i/item/9789241550598 (accessed on 29
August 2021). p
g
jg
19. Guidelines Review Committee. WHO Guidelines for the Use of Thermal Ablation for Cervical Pre-Cancer Lesions. Who.int. Published Online September 2019. Available online: https://www.who.int/publications/i/item/9789241550598 (accessed on 29
August 2021). 20. Conocimientos, Actitudes y Practicas Sobre El Cáncer Cervicouterino, El Virus Del Papiloma Humano y Sus Formas de Preven-
ción, En Los Departamentos de Santa Ana y Sonsonate, El Salvador, 2014. Published 2014. Available online: https://www.iccp-
portal.org/system/files/resources/Estudio%20CAPs%20sobre%20el%20CCU%20%20-%20El%20Salvador%202014%202.pdf (ac-
cessed on 4 April 2022). 20. Conocimientos, Actitudes y Practicas Sobre El Cáncer Cervicouterino, El Virus Del Papiloma Humano y Sus Formas de Preven-
ción, En Los Departamentos de Santa Ana y Sonsonate, El Salvador, 2014. Published 2014. Available online: https://www.iccp-
portal.org/system/files/resources/Estudio%20CAPs%20sobre%20el%20CCU%20%20-%20El%20Salvador%202014%202.pdf (ac-
cessed on 4 April 2022).
|
https://openalex.org/W2018711649
|
https://www.scielo.br/j/paz/a/q5KtqfwhN3wRGLTjbYq4SnB/?lang=en&format=pdf
|
English
| null |
Historical Biogeography of a Clade of Liolaemus (Iguania: Liolaemidae) based on ancestral areas and dispersal-vicariance analysis (DIVA)
|
Papéis Avulsos de Zoologia
| 2,006
|
cc-by
| 7,652
|
Cátedra de Anatomía Comparada. Facultad de Ciencias Naturales, Universidad Nacional de Salta. Avda. Bolivia 5150, 4400, Salta,
Argentina. E-mail: jmdiaz@unsa.edu.ar
CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas). IBIGEO (Instituto de Bio y Geociencias), IBIGEO. HISTORICAL BIOGEOGRAPHY OF A CLADE OF LIOLAEMUS
(IGUANIA: LIOLAEMIDAE) BASED ON ANCESTRAL AREAS
AND DISPERSAL-VICARIANCE ANALYSIS (DIVA) JUAN MANUEL DÍAZ GÓMEZ
FERNANDO LOBO ABSTRACT Although many phylogenetic studies have been made on the genus Liolaemus, they are inappropriate for
a biogeographic study because of incomplete taxon sampling. Here we develop a biogeographic analysis
using a new phylogenetic study of the chiliensis group, which differs from the other studies in having his
members fully represented. The biogeographic analysis presented here consists in reconstructing the
ancestral area of this clade, under three different methods of analysis: Fitch Optimisation, Dispersal
Vicariance Analysis and Weighted Ancestral Area Analysis. The analyses were made on phylogenies
from parsimony under equally weighted characters, as well as under implied weighting. The equally
weighted tree recovers most of the groupings proposed in previous studies. The strict consensus of the
implied weighting trees recovers fifteen groups, but there is no agreement of the relationships between
those groups. The ancestral area analyses agree in an Andean-Patagonian origin for the chiliensis clade,
and are congruent with previous hypotheses and paleontological data. KEYWORDS: Liolaemus, chiliensis-group, biogeography, ancestral area, DIVA, Fitch, Patagonia,
Andes. Volume 46(24):261-274, 2006 Volume 46(24):261-274, 2006 INTRODUCTION orous climates, in very high latitudes or at high altitudes
(over 4000 m above sea level). This diversity and wide
distribution has allowed the development of very di-
verse biological characteristics, like oviparous and vivipa-
rous species (perhaps related to cold conditions, Schulte
et al. 2000) and a diverse diet, existing omnivorous, her-
bivorous and insectivorous species. The genus Liolaemus includes about one hundred
and sixty species (Espinoza et al., 2004), and is the sec-
ond most specious genus of Lizards. Its distribution
extends from Tierra del Fuego (Liolaemus magellanicus)
in the southernmost extreme of South America, to the
Peruvian Andes (L. walkeri). Liolaemus species occupy
diverse habitats, including terrestrial and arboreal, some
are saxicolous, some arenicolous, others live in very rig- Some authors included several statements regard-
ing the distributional patterns of this genus in their
taxonomic studies. One of them is Hellmich (1951) DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS 262 of dispersal events could be just a problem of taxon
sampling. who described the distribution of six species groups
from Chile, which occupy very different areas. Hellmich
explained these current distributions as a consequence
of expansions and contractions of the original distri-
butions. According to him, this happened during the
interglacial and glacial periods of the Tertiary, and this
would have given rise to the ‘races’ recognized. For
Hellmich, the final explanation was dispersal, a state-
ment which was also used by Lamborot et al. (1981) to
explain the chromosomic variation in the monticola spe-
cies complex. Although in the last years many works have been
focused on the phylogeny of Liolaemus (Ortiz, 1981;
Etheridge, 1995, Halloy et al. 1998; Young-Downey,
1998; Etheridge, 2000; Schulte et al. 2000), they are not
useful for a biogegraphic analysis because they are fo-
cused on subgroups or the genus is not fully repre-
sented. The cladistic analysis on the chiliensis group re-
cently performed by Lobo (2001, 2005) differs from the
other studies in having the chiliensis group, which is half
the genus Liolaemus, fully represented. Laurent (1983, 1985, 1992) also included biogeo-
graphic comments when dividing the genus into two
major subgroups (subgenus Eulaemus or Argentine
group and subgenus Liolaemus or Chilean group), divi-
sion attributed to the uplift of the Andes. INTRODUCTION In this paper we present a new cladistic analysis
of the chiliensis group, based on already published data
as well as evidence not previously considered. We in-
clude species recently described and ethological charac-
ters, and use parsimony to obtain a new hypothesis for
the clade, and study its historical biogeography based
on the topologies obtained. We apply three different
methods of ancestral area analysis: Fitch optimisation
(Ronquist, 1994), Weighted Ancestral Area Analysis
(WAAA, Hausdorf, 1998) and Dispersal Vicariance
Analysis (DIVA, Ronquist, 1997). The results are com-
pared with previous hypothesis, and the methods are
discussed. Cei (1979) described the composition of the
Patagonian and Andean herpetofauna, characterising
these regions by their high endemicity, and described
centres of speciation and dispersal by using paleonto-
logical and paleoclimatic data. Lobo (2001) described the areas of distribution
on the phylogeny of the chiliensis group, following the
biogeographic characterization of Roig-Juñent (1994)
and adding areas not considered previously (i.e: Puna). There are only two works where the historical bio-
geography of Liolaemus is discussed using some cladis-
tic methodology. Young-Downey (1998) performed a
Brooks Parsimony Analysis by using areas defined by
Cabrera & Willink (1980) and Brown & Gibson (1983),
and compared her results with the hypothesis of Solbrig
(1976) on the origin and evolution of those areas. Schulte
et al. (2000) proposed a hypothesis in which the species
inhabiting three general areas, Andes, West of the Andes
and East of the Andes were monophyletic. Using a
molecular-based phylogeny, they optimized the distri-
butions of the species, and ultimately rejected that hy-
pothesis as the shortest phylogenetic estimate required
multiple invasions of each of the areas. The pattern of
distribution of Liolaemus is explained by recurring
vicariance with subsequent dispersal between the areas
that allowed further vicariance. Both studies had the
problem that the areas of distribution were represented
by biogeographic units (provinces) or arbitrarily defined,
and in many cases, those units were not fully occupied
by the species. Using areas much larger than the ranges
of the species used in the analysis, as is the case with
biogeographic provinces, will cause a loss of resolution
in the analysis, because sometimes species separated by
hundreds of kilometres will be included in the same
province. Even worse, the sampling of species in the
cited studies was not complete, then, the identification MATERIALS AND METHODS Taxa and Characters Biogeographic Analysis Ancestral Area Analysis was proposed by
Bremer (1992) as a way for identifying the area of
distribution of the ancestor of a monophyletic taxon,
which he named the ancestral area. The analytical meth-
ods for ancestral areas are based on the assumption
that there is information about the ancestral area of a
taxon in the topology of its area cladogram, that is,
the probability for an area of being part of the ances-
tral area increases with its presence in plesiomorphic
branches and its general presence in the cladogram
(Hausdorf, 1998). We applied three methods pro-
posed in the last years: Fitch optimisation (Ronquist,
1994), Weighted Ancestral Area Analysis (WAAA)
(Hausdorf, 1998) and Dispersal Vicariance Analysis
(DIVA) (Ronquist, 1997). All these methods use
optimisations with reversible parsimony for estimat-
ing ancestral areas. Fitch Optimisation was proposed
by Ronquist (1994) to avoid the problems of Camin-
Sokal (irreversible) parsimony originally proposed by
Bremer (1992). WAAA uses Fitch parsimony with a
weighting scheme that weights favourably areas lo-
cated in plesiomorphic branches, and areas more com-
mon as terminals. In this method, a Probability In-
dex (PI) is calculated to give a measure of the likeli-
hood of a particular area of being part of the ances-
tral area. The index is calculated for each area from the
ratio of gains (assuming that the area was not part of
the ancestral area) over losses (assuming that the area Cladistic Analysis was part of the ancestral area). DIVA searches ances-
tral areas using a three dimensional cost matrix that
gives different costs to events, minimizing the dis-
persal events needed for explaining the distributions. In this approach, the vicariance events have no cost,
while the dispersals and extinctions cost one per area
unit added to the distribution. Parsimony analysis was made under implied
weighting (Goloboff, 1993). This weighting method
weights each character according to the number of extra
or homoplasious steps in a tree, using a concave func-
tion that gives lower weights to the characters with high
homoplasy (Kitching et al. 1998). For the implied
weighting analysis 6 concavity values were used (K, 1 to
6). Also we made a parsimony analysis under equal
weights. For both analyses TNT version 1.0b4
(Goloboff et al. 2003) was used. We searched for opti-
mal trees using TBR branch swapping, making 500
replications saving up to 10 trees for both the equally
weighted and implied weights analysis. A strict consen-
sus tree was generated using the topologies obtained
from the implied weights. Continuous characters were
coded using the gap weighting method (Thiele, 1993). Characters 0-20, 22-34, 37-82, 115 and 116 were coded
as additive. Ancestral area analysis has been considered by
some authors as a reimplementation of the centre of
origin concept, and recently criticized on the basis that it
utilizes paralogy (widespread taxa and redundant dis-
tributions) instead of reducing it, as is the usual proce-
dure for methods of cladistic biogeography (Ebach,
1999). However, ancestral area methods rely on the fact
that all taxa have an area where their ancestor was dis-
tributed, and the search for that area is a valid proce-
dure. The ancestral area of a taxon needs not to be
smaller than the actual distribution of its descendants,
it can be larger or equal (Bremer, 1992), and dispersal
needs not to be postulated as the only possible expla-
nation. Ancestral area analysis tries to identify areas that
may have been part of the ancestral area, and to distin-
guish vicariance events from possible dispersals
(Hausdorf, 1998). As Hovenkamp (1997) pointed out,
methods that try to elucidate Earth history search for
common patterns in cladograms, discard the incongru-
ences, opposed to methods that try to search for taxon
histories (as ancestral area methods), which have differ-
ent methodologies and does not need to discard
paralogies. Taxa and Characters The biogeographic analyses are based on a new
hypothesis for the chiliensis group, and include all the
species currently known for this group (83 species). We
constructed a matrix using the morphological data from
Lobo (2005), which was updated by adding three new
species and 35 characters. The new species are Liolaemus
yanalcu, (Martinez Oliver & Lobo, 2002), L. puna and
L. chaltin (Lobo & Espinoza, 2004). Three new charac-
ters are original to this study: gravid coloration, plant-
microhabitat preference and lateral black spots; character
states are included in appendix 1. We also included in
the morphological matrix the allozyme data set of
Young-Downey (1998) (32 characters for 26 taxa of the
chiliensis group); the species L. alticolor of Young-
Downey (op.cit.) analysis is actually L. puna (Lobo &
Espinoza, 2004). Sp 1 is a new Liolaemus under descrip-
tion (Vega et al. pers. comm), Sp 2 is a new species (Lobo
et al. submitted). Being Phymaturus the closest living rela-
tive to Liolaemus (Espinoza et al. 2004), we used
Phymaturus indisctintus as outgroup. Data matrix is avail-
able at www.unsa.edu.ar/acunsa/index02.html. PAP. AVULS ZOOL. 46(24), 2006 263 Selection of Areas The areas used were taken from Roig-Juñent
(1994). To better represent the distribution of several
species, we decided to include some new areas not con-
sidered in that study like Puna, Atacama, Cordillera Ori-
ental and Andes. This last area was divided into Andes,
Andes Centrales and Andes Meridionales. We also di-
vided the Monte area from Roig-Juñent (1994) into the
new areas Sierras Pampeanas and Sierras Subandinas. The areas used are (Fig. 1): Andes, Andes Centrales,
Andes Meridionales, Atacama, Coquimbo, Cordillera
Oriental, Maulina, Monte, Patagonia, Puna, Selva
Valdiviana, Sierras Pampeanas, Sierras Subandinas, and
Valle Central. To make comparisons with a previous biogeo-
graphic hypothesis, we also replicated the topology from
Schulte et al. (2000) for the chiliensis group, and applied
the three methods replacing the areas used by Schulte
et al. with the areas used in this study. DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS 264 FIGURE 1: Areas used in the Ancestral Area Analysis. AND:
Andes; ANDC: Andes Centrales; ANDM: Andes Meridionales;
ATA: Atacama; COQ: Coquimbo; COR: Cordillera Oriental;
MAU: Maulina; MON: Monte; PAT: Patagonia; PUN: Puna; SEV:
Selva Valdiviana; SPA: Sierras Pampeanas; SSA: Sierras Subandinas;
VAC: Valle Central TABLE 1: List of groups and species belonging to each group
according to the parsimony analysis under equal weights and
implied weighting. The species not included were found not
belonging to any group. g g
y g
p
Group
Equally
Implied
weighted
weighting
analysis
analysis
alticolor
L. chaltin
L. alticolor
L. alticolor
L. paulinae
L. puna
L. puna
L. walkeri
L. variegatus
L. tacnae
L. walkeri
bibronii
L. exploratorum
L. hernani
L. bibronii
L. yanalcu
L. ramirezae
L. bitaeniatus
L. chaltin
capillitas
L. capillitas
L. buergueri
L. heliodermis
L. heliodermis
L. dicktracyi
L. capillitas
L. umbrifer
L. dicktracyi
L. umbrifer
ceii-kriegi
L. ceii
L. ceii
L. kriegii
L. kriegii
L. buergueri
elongatus
L. petrophilus
L. austromendocinus
L. elongatusRN
gravenhorsti
L. gravenhorsti
L. brattstroemi
L. cyanogaster
L. cyanogaster
L. schroederi
L. gravenhorsti
L. schroederi
leopardinus
L. leopardinus
L. leopardinus
L. ramonensis
L. ramonensis
L. valdesianus
nigroviridis
L. maldonadae
L. campanae
L. nigroviridis
nigromaculatus
L. donosoi
L. platei
L. monticola
L. atacamensis
L. tacnae
L. silvai
L. platei
L. sieversi
L. velosoi
L. nigromaculatus
L. atacamensis
L. kuhlmanni
L. ater
L. copiapensis
L. bisignatus
L. bisignatus
L. copiapensis
L. ater
L. nigromaculatus
L. sieversi
L. zapallarensis
L. kuhlmanni
L. hellmichi
L. silvai
pictus
L. pictus
L. Liolaemus systematics Four different topologies were obtained using the
six concavity values (K). The strict consensus indicates
that these four topologies have in common thirteen
groups (Fig. 2). The analysis under equal weights yielded
one tree (Fig. 3). Eight groups of species are recovered. The list of groups and the species included are in Table 1. Selection of Areas talcanensis
L. argentinus
L. chiloensis
robertmertensi
L. robertmertensi
L. curicensis
L. chiliensis
L. chiliensis
L. nitidus
L. nitidus
L. robertmertensi
L. saxatilis
L. gracilis
L. sp 2
tenuis
L. tenuis
L. coerulus
L. punctatissimus
L. neuquensis
L. argentinus
L. punctatissimus
L. pictus
L. tenuis
L. talcanensis
L. brattstroemi
L. chiloensis
L. coerulus
L. neuquensis FIGURE 1: Areas used in the Ancestral Area Analysis. AND:
Andes; ANDC: Andes Centrales; ANDM: Andes Meridionales;
ATA: Atacama; COQ: Coquimbo; COR: Cordillera Oriental;
MAU: Maulina; MON: Monte; PAT: Patagonia; PUN: Puna; SEV:
Selva Valdiviana; SPA: Sierras Pampeanas; SSA: Sierras Subandinas;
VAC: Valle Central Liolaemus biogeography The results for the three methods utilized are re-
sumed on Table 2. These assignations refer to the most
basal node of the tree. Fitch Optimisation. The Fitch optimization on the con-
sensus tree (Fig. 2) assigns the most basal node Andes
as the ancestral area. The Fitch optimisation on the
equally weighted tree (Fig. 3) assigns for the most basal
node the areas Patagonia, Andes or Monte as the ances-
tral area. PAP. AVULS ZOOL. 46(24), 2006 265 2: Strict consensus from the four topologies obtained with the implied weights analysis. The area assignations are as
rmal, Fitch optimization; Italics, Weighted Ancestral Area Analysis. FIGURE 2: Strict consensus from the four topologies obtained with the implied weights analysis. The area assignations are as
follow: Normal, Fitch optimization; Italics, Weighted Ancestral Area Analysis. DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS 266 FIGURE 3: Tree from the equally weighted analysis. The area assignations are as follow: Normal, Fitch optimization; Italics,
Weighted Ancestral Area Analysis; Bold, DIVA (constrained to 2 maximum areas) FIGURE 3: Tree from the equally weighted analysis. The area assignations are as follow: Normal, Fitch optimization; Italics
Weighted Ancestral Area Analysis; Bold, DIVA (constrained to 2 maximum areas) Dispersal Vicariance analysis (DIVA). The DIVA on the
equally weighted tree achieved an exact solution, with 6
optimal reconstructions, requiring 45 dispersals each. (Table 2). For the most basal node, the areas Puna,
Atacama, Monte, Andes, Valle Central, Coquimbo, Sier-
ras Pampeanas, Andes Meridionales y Andes Centrales
are repeated in all reconstructions. Maulina appears in
four of six reconstructions, and Selva Valdiviana in three. The only areas that don’t appear in any reconstruction
are Sierras Subandinas and Patagonia. The DIVA could not be done on the consensus
tree, as the software cannot handle trees with polytomies. We selected for DIVA the trees corresponding to K = 1
and 2, and K = 5 and 6, because those hypothesis repre-
sented two different weighting schemes: a stronger for
K = 1,2 and weaker for K = 5, 6. Because of time con- PAP. AVULS ZOOL. 46(24), 2006 267 TABLE 2: Results of the ancestral area analysis. The abbreviations for the areas are from Fig. 1. Liolaemus biogeography Equally Weighted Analysis
Consensus
K = 1,2
K = 5,6
Fitch
PAT, AND, MON
AND
AND
AND
WAAA
AND, MON
AND
AND, PAT
AND, SPA
Constrained
Max areas = 2
Max areas = 3
—
Max areas = 2,3,4
Max areas = 2,3,4,5,6
DIVA
PAT, MON
PAT, AND, MON
PAT, AND
PAT, AND
PAT, AND, MON
Unconstrained DIVA
PUN ATA MON AND ANDM ANDC
PUN ATA MON AND ANDM ANDC
ATA MON AND ANDM VAC
VAC COQ SPA
VAC COQ MAU SEV SPA COR
COQ MAU SEV SPA COR
PUN ATA MON AND ANDM ANDC
PAT PUN ATA MON AND ANDM ANDC
PAT PUN ATA MON AND ANDM VAC
VAC COQ MAU SPA
VAC COQ MAU SEV SPA COR
COQ MAU SEV SPA COR
PUN ATA MON AND ANDM ANDC
PUN ATA MON AND ANDM ANDC
PUN ATA MON AND ANDM VAC
VAC COQ SEV SPA
VAC COQ MAU SEV SPA SSA COR
COQ MAU SEV SPA SSA COR
PUN ATA MON AND ANDM ANDC
PAT PUN ATA MON AND ANDM ANDC
PAT PUN ATA MON AND ANDM VAC
VAC COQ MAU SEV SPA
VAC COQ MAU SEV SPA SSA COR
COQ MAU SEV SPA SSA COR
PUN ATA MON AND ANDM ANDC
PUN ATA MON AND ANDM ANDC VAC
VAC COQ MAU SPA COR
COQ MAU SEV SPA COR
PUN ATA MON AND ANDM ANDC
PAT PUN ATA MON AND ANDM ANDC VAC
VAC COQ MAU SEV SPA COR
COQ MAU SEV SPA COR
PUN ATA MON AND ANDM ANDC VAC
COQ MAU SEV SPA SSA COR
PAT PUN ATA MON AND ANDM ANDC VAC
COQ MAU SEV SPA SSA COR DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS 268 Implied weighting analysis. The WAAA analysis was made
on the strict consensus tree as well as the topologies
corresponding to K = 1, 2 and K = 5, 6 (Table 2). The
areas with higher PI in the consensus were Andes (0.48)
and Coquimbo (0.23). Monte has a low PI of 0.10. The
areas with higher PI for the tree with K 1 and 2 are
Andes (0.69) and Patagonia (0.30). Again Monte has a
very low PI (0.07). Sierras Pampeanas (0.19) and
Coquimbo (0.17) are areas that present higher values of
PI than the rest. Liolaemus biogeography The tree resulting from K 5 and 6 has
only Andes with a high PI of 0.59, the values for other
areas are: 0.16 (Atacama), 0.16 (Sierras Pampeanas), 0.16
(Selva Valdiviana) and 0.12 (Coquimbo). straints the solution was heuristic, yielding 4 optimal
reconstructions that required 41 dispersals each. These
reconstructions are both essentially the same, except that
Andes and Sierras Subandinas are not present in three
of them: in one reconstruction both areas are missing,
and one of them is absent from each of the other two. The last reconstruction includes all the areas. The analy-
sis on the tree resulting form K = 5 and 6, also heuris-
tic, produced 8 optimal reconstructions, requiring 42
dispersals. One of the optimal reconstructions involves
all the areas, the other seven differ in the presence or
absence of Patagonia, Sierras Subandinas, and Andes
Centrales. These results are summarized in Table 2. Given
the number of possible reconstructions, it’s hard to
identify unambiguous vicariance events, making diffi-
cult to hypothesize a sequence of events for the distri-
bution of Liolaemus. Ancestral Area analysis on the topology of
Schulte et al. (2000) When constraints were used on the maximum
number of areas allowed for the ancestral area, on the
equally weighted analysis, for a maximum of 2 areas the
reconstruction is Patagonia-Monte or Patagonia-Andes;
for a maximum of 3 to 6 areas the reconstruction is
Patagonia, Andes, Monte (Table 2). For the trees with
implied weights, when making DIVA on the topolo-
gies corresponding to K = 1 and 2, the reconstruction
for a maximum of 2 to 4 areas is Patagonia and Andes;
for K = 5 and 6 with a maximum of 2 to 6 areas, the
reconstruction is Patagonia and Andes (Table 2). In all
cases, the optimizations for a maximum number of
areas superior to 6 showed several ambiguous recon-
structions (data not shown). The topology of Schulte et al. (2000) (Fig. 4) has a
polytomy with the taxons Liolaemus bellii, L. chiliensis
and L. cyanogaster; the polytomy was resolved, and the
two resolutions used for the DIVA analysis. For the
Fitch optimization (11 steps) on the topology of Schulte
et al. (2000) assigns Andes as the ancestral area. Uncon-
strained DIVA (8 equally optimal reconstructions) as-
signs to the basal node all the areas, requiring 15 dis-
persal events. When using constraints to a maximum
of 2 areas, it requires 18 dispersal events, with two equally
optimal reconstructions between Andes and Sierras
Pampeanas. Weighted Ancestral Area Analysis found
Andes with the higher PI (0.44) followed by Sierras
Pampeanas and Maulina (0.39), and Valle Central in Chile
(0.38). Weighted Ancestral Area Analysis (WAAA). The PI val-
ues for the WAAA analysis were calculated for the most
basal node of the tree, and for the nodes that give rise to
groups of species. Liolaemus systematics Equally weighted analysis. The areas with higher PI are
Andes and Monte (0.67 and 0.44 respectively), and have
more probability of being part of the ancestral area
(Table 2). This result is congruent with Fitch
optimisation, which has Patagonia, Andes or Monte as
equally probable reconstructions for the basal node, and
Andes as the ancestral reconstruction for the nodes as
we go up in the tree. Nevertheless, the area Patagonia
has a very low PI (0.04), discarding it as part of the
ancestral area. There are other areas that have low values
of PI, but slightly higher than the remaining, these ar-
eas are Sierras Pampeanas and Selva Valdiviana (0.13 and
0.12 respectively). This may be reflecting the fact that
these areas appear in more plesiomorfic branches, and
are given more weight accordingly. Lobo (2005) found six different resolutions
among subclades of the chiliensis group, some of those
resolutions are recovered here, i.e. the basal position of
leopardinus and kriegi groups is recovered in every
analysis,and the clade formed by the robertmertensi,
gravenhorsti and alticolor groups appears only on the
equally weighted tree. The strict consensus of the im-
plied weighting analysis recovered 15 groups, most of
them found in previous studies, but the relationships
among these groups remain unresolved. Cei (1986, 1993) and Avila et al. (1992) defined the
bibronii group based on a character combination includ-
ing as members of this group the following species: 269 PAP. AVULS ZOOL. 46(24), 2006 FIGURE 4: Tree from Schulte et al. (2000), showing the possible resolutions of the polytomy. The area assignations are as follow:
Normal, Fitch optimization; Italics, Weighted Ancestral Area Analysis; Bold, DIVA (constrained to 2 maximum areas). Liolaemus
alticolor is actually Liolaemus puna (Lobo & Espinoza, 2004). FIGURE 4: Tree from Schulte et al. (2000), showing the possible resolutions of the polytomy. The area assignations are as follow:
Normal, Fitch optimization; Italics, Weighted Ancestral Area Analysis; Bold, DIVA (constrained to 2 maximum areas). Liolaemus
alticolor is actually Liolaemus puna (Lobo & Espinoza, 2004). Liolaemus bibronii, L. exploratorum, L. sanjuanensis,
L. saxatilis, L. gracilis, L. robertmertensi and L. fuscus. In
this study this group is paraphyletic. In the equally
weighted analysis L. bibronii is sister taxon of
L. sanjuanensis included in a subclade related to the most
terminal grouping formed by robertmertensi, gravenhorsti
and alticolor groups; L. saxatilis, L. bitaeniatus, L. fuscus
and L. Liolaemus systematics gracilis are nested within this clade but not form-
ing a monophyletic group. In the Implied weighting
analysis species belonging to the traditional bibronii group
are separated in two groups which here we redefine as
bibronii (including only L. exploratorum, L. bibronii,
L. hernani and northern Argentina species: L. bitaeniatus,
L. chaltin, L. yanalcu and L. ramirezae) and robertmertensi
group including now L. curicensis, L. chiliensis, L. nitidus,
L. robertmertensi, L. saxatilis, L. gracilis and L. tandilensis. Liolaemus robertmertensi, L. saxatilis and L. tandilensis are
distributed following the mountain arc formed by Sier-
ras Pampeanas, and Sierras de Tandil in eastern Argen-
tina, L. robertmertensi in the slopes of Sierras of Catamarca
and La Rioja, L. saxatilis in Sierras de San Luis and Córdoba, L. tandilensis in Sierra de Tandil. Liolaemus
sanjuanensis lives in Sierra Pie de Palo (San Juan province,
Argentina) a chain of mountains related to the same
system of Sierras Pampeanas but in this analysis is not
found related to this group of species, perhaps this is
due to problems of taxon sampling, because only two
paratypes of this species were available for our study. The alticolor-bibronii group of Espinoza et al. (2004)
includes 10 species in two clades one formed by Liolaemus
bibronii, L. gracilis, L. robertmertensi, L. yanalcu,
L. ramirezae, and other clade with L. bitaeniatus,
L. pagaburoi, L. chaltin, L. puna, and L. cf. walkeri, which
is actually L. puna according to Lobo & Espinoza (2004). Lobo (2005) also found the alticolor-bibronii group, with
between 14 and 17 species in different analysis. In this study the alticolor-bibronii is not recovered. In the equally weighted analysis is only formed by 4
species (Fig. 3), and in the consensus the alticolor and
bibronii groups appear separated, with species inhabiting
Northern Argentina (L. puna), northeastern Chile
(L. paulinae), Puna and high mountains in Bolivia DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS 270 following climate effects (desertification of the
Patagonian steppe). Unfortunately, the areas indicated
as occupied by the ancestor of Liolaemus are not clearly
described by Young-Downey and is difficult to estab-
lish direct correspondences with the areas used here. Young-Downey (1998) found Atacama region as more
related to Coquimbo and Chilean (Valle Central) in con-
gruence with Solbrig (1976). Liolaemus systematics The current distributions
of the species and the phylogenetic analysis used here as
well as the cladogram of Lobo (2005) suggest different
area relationships. The nigromaculatus group from the
Northern deserts in Chile (Coquimbo and Atamaca ar-
eas) in that analysis is sister taxon of the alticolor group
(Puna); interestingly Morrone (1996) found Puna and
the Northern deserts forming a monophyletic unit. (L. variegatus and L. alticolor) and Perú (L. tacnae and
L. walkeri) forming the alticolor group, and the remain-
ing species of alticolor-bibronii which are oviparous
(L. ramirezae, L. yanalcu, L. bitaeniatus and L. chaltin) are
included in the bibronii group. Liolaemus fuscus and
L. lemniscatus form a separated monophyletic group,
lemniscatus group (congruent with the fuscus-lemniscatus
group of Ortiz (1981), and lemniscatus group of Cei,
1986). The petrophilus group recovered in the analysis with
implied weights includes the capillitas group; this is con-
gruent with the results of Morando et al. (2003). The
morphological information does not support the in-
clusion of the ceii-kriegi within the petrophilus group as
suggested by the mitochondrial DNA analysis. Comparisons with the distribution of the groups
more related with Liolaemus may prove useful. The sis-
ter clade of Liolaemus, Phymaturus (Schulte et al. 2003;
Espinoza et al. 2004), is distributed in Patagonia and in
both sides of the Andes, reaching north to the Puna of
Catamarca Province in Argentina, distribution that is
shared with many species of Liolaemus and does not
contradict the hypothesis of ancestral areas for the chiliensis
group. Nevertheless, Ctenoblepharis, the basal taxa for
Liolaemidae (Schulte et al. 2003; Espinoza et al. 2004), is
found only in coastal deserts of Peru, thus making nec-
essary further studies including these two genera. Liolaemus Biogeography The results are summarized in Table 2. There is
congruence between the three methods because all sus-
tain the hypothesis of an Andean-Patagonian origin for
the chiliensis group. The common area to all the analyses
is Andes, followed by Patagonia, and Monte. This results show some degree of congruence with
previous studies. Cei (1979) sustained that Patagonia
was a center of origin for at least four groups of
Liolaemus, describing two major faunal regions in
Patagonia (see Cei 1979, Fig. 13:3): the northern or an-
cient Patagonian region and the southern or Santa Cruz
region, the former includes part of the Patagonia and
Andes areas used in this study. The first fossil attrib-
uted to Liolaemus (Albino, 1998) comes from the Mi-
ocene of Patagonia, found in sediments of the Miembro
Trelew, Formación Sarmiento, near Gaiman in Chubut
Province, this locality is included in the area Patagonia. These studies, although not focused on the chiliensis
group but on the entire genus, are congruent with the
ancestral areas found here. Recently, Albino (2005) found
new fossils attributed to Liolaemus from the late Quater-
nary in the Southwest of Buenos Aires Province, in-
cluded in the Monte Area of this study. Fitch optimisation. The Fitch optimisation on the equally
weighted analysis yielded Andes, Patagonia and Monte. This shows the perhaps excessive effect that Fitch opti-
mization places in the more plesiomorfic areas, because,
Patagonia only appears again in more terminal branches
of the tree. Fitch optimisation. The Fitch optimisation on the equally
weighted analysis yielded Andes, Patagonia and Monte. This shows the perhaps excessive effect that Fitch opti-
mization places in the more plesiomorfic areas, because,
Patagonia only appears again in more terminal branches
of the tree. Effect of constraining the number of areas Ronquist (1997) noted that when using DIVA
for reconstruct the ancestral area of a taxon, the root
node will tend to include all the areas occupied by the
terminals, making it very uninformative. The solutions
to this suggested by Ronquist are two: the inclusion of
more outgroups, thus making the root node no longer
the root node, and constraining the maximum number
of areas that may form part of the ancestral area. The
first of this possible solutions is, at least in this case,
not useful: for example, the inclusion of other
outgroups related to Liolaemus, i.e. Phymaturus and
Ctenoblepharis, will not solve the problem, given that
Phymaturus is distributed in the same areas used for
Liolaemus, and Ctenoblepharis, distributed in coastal Peru, Because the BPA results of Young-Downey (1998)
had incongruences with the hypothesis of Solbrig (1976),
she evaluated the area cladogram of Liolaemus regarding
ancestral distributions and evaluating possible dispers-
als, extinctions, and lack of response to vicariance. Thus,
Young-Downey (1998) postulates that the ancestor of
Liolaemus inhabited areas comprising the Southern
Nothofagus forest (Subantartic Province), Central Argen-
tina and Central-Northern Chile, and from there, it
vicariated by the appearance of the Andes, and by the PAP. AVULS ZOOL. 46(24), 2006 271 areas, making the results uninformative, given the size
of the areas. probably will only add this new area to the ancestral area
reconstruction in the root, leaving the ancestral area for
Liolaemus without major changes. The second alterna-
tive presents the problem that there is no criteria for
choosing the number of areas for constraining. The
sympatry of sister groups is evidence for dispersal
(Hausdorf, 1998), so it can be argued that the ancestral
distribution may have been larger o smaller than the
present area occupied by the taxon. Examples of such
sympatry in sister species are L. chiliensis and L. nitidus,
both living in Valle Central (Chile); L. cyanogaster and
L. schroederi, both distributed in Maulina (Chile). On
this basis, one may assume that the ancestral area may
have been smaller in the past, and this provides a ratio-
nale for constraining the maximum number of areas
allowed in the ancestral distribution, taking into account
that this practice remains ad hoc. A last remark is needed about the areas used for
this study. Commentaries about Schulte et al. (2000) Schulte et al. (2000) postulated that the ancestral
area for the subgenus Liolaemus (equivalent to the chiliensis
group) in their reconstruction is equivocal between
Andes, western lowlands and eastern lowlands, the three
areas used for the analysis. With the exception of Andes,
the other areas found in the analysis (Sierras Pampeanas,
Maulina and Chile Central) are not hypothesized as an-
cestral in the biogeographic analysis of the more com-
plete phylogeny herein. This shows the effects of a lim-
ited taxon sampling for making biogeographic infer-
ences about ancestral areas. There are areas not repre-
sented in the analysis, and the species located
plesiomorphically on the cladogram have a strong influ-
ence in the result of the optimization. There are only
three species inhabiting Sierras Pampeanas (L. bitaeniatus,
L. robertmertensi and L. capillitas) but ten in Andes, but
both areas appear as equally probable ancestral areas. Also, there is an oversimplification of the distribution
of the species used by Schulte et al. (2000); any ambigu-
ity at any node will involve at least two of the three Effect of constraining the number of areas These areas from Roig-Juñent (1994) are based
on distributions of arthropods, and despite they are
accurate respect to the distribution of Liolaemus, some
regions are missing (i.e. Puna), and others do not repre-
sent very well the distributions of Liolaemus, like Monte,
and would need a reformulation. The ancestral area of a taxon, as defined by Bremer
(1992), needs not to be limited to a single place, it may
be equal or larger than the current area occupied by the
taxa. However, as implemented by the methods, the
results of the ancestral area analysis will be an ancestral
area for a taxon at least equal or smaller than the com-
bined distributions of his descendants. The only pos-
sible way to find an ancestral area larger than the sum of
the distributions used would be with the inclusion of
fossil groups distributed in areas not currently occupied
by the taxa in study. For many groups, this is very diffi-
cult. For example, the fossil record may be absent, or the
fossils may be found only in the areas already present in
the analysis, as is the case with the fossils attributed to
Liolaemus (Albino, 1998, 2005). Even in the case that a
fossil distribution could be used, the area occupied by
the fossil taxa would have to be present in deep branches
of the cladogram or in numerous terminals or it would
have no influence in the analysis and could not be hy-
pothesized as being part of the ancestral area. Thus,
although the methods contemplate the possibility of
identifying an ancestral area larger than the current distri-
bution using data from fossils, is very difficult to this
situation to effectively appear in an analysis. When using constrained DIVA (Table 2), con-
straining the maximum number of areas that may form
the ancestral distribution from 3 to 6 areas, yield as op-
timal reconstruction Andes, Patagonia and Monte, the
same areas that yield the Fitch optimization on the equally
weighted tree (when constraining to 2 areas, the recon-
struction is Andes and Monte). Andes also have the
highest PI in the WAAA analysis, being Monte the sec-
ond. Sadly, there are no criteria for selecting a number of
area units by constraining the ancestral distributions,
making any further statements speculative. CONCLUSIONS In recent years several studies have dealed with the
phylogenetic relationships of the chiliensis group (Schulte
et al. 2000; Lobo, 2001; Espinoza et al. 2004; Lobo, 2005;
this study). Some groups are recovered in this analysis,
but others do not, as the alticolor-bibronii, which appears
splitted. The consensus of the implied weighting analy-
sis shows that the relationships between the groups are
not yet resolved, and would require further studies to
clarify the intern relationships inside the chiliensis group. As with any cladistic biogeographic analysis, the results
of the ancestral area methods may vary according to the
different hypothesis. However, if the degree of varia-
tion is only on the terminal groups, the ancestral area
assignations will likely not be affected. Nevertheless, some
area relationships found in other studies (Morrone, 1996; DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS 272 Lobo, 2005) are recovered here, like the sister relation-
ship between Puna and the Northern deserts of Chile
(Coquimbo and Atacama). ancestral, resultado este, congruente com hipóteses prévias e com
informação paleontológica. (
q
)
Of the studies that include biogeographic consid-
erations about Liolaemus, only Young-Downey (1998)
applied an explicit methodology. However, these stud-
ies have the problem of limited taxon sampling and
area definitions, making difficult to make comparisons
between the results. However, the results of the ances-
tral area analysis of this work are congruent with previ-
ous studies and paleontological data, hypothesizing an
Andean-Patagonian ancestral area for the chiliensis group. A large part of the genus is not included in the chiliensis
group (the Eulaemus subgenus or Argentine group),
and its inclusion is necessary for the completion of the
ancestral area analysis of the genus. This work is under-
way and will be published elsewhere. PALABRAS-CHAVE: Liolaemus, chilinesis, Dispersão-
Vicariância, Fitch, Áreas Ancestrais Pesadas, Andes,
Patagonia. PALABRAS-CHAVE: Liolaemus, chilinesis, Dispersão-
Vicariância, Fitch, Áreas Ancestrais Pesadas, Andes,
Patagonia. ACKNOWLEDGMENTS We thank Claudia Szumik and Sebastián
Quinteros for making suggestions to an earlier draft of
the manuscript. We also thank Sonia Kretzschmar for
help with the translation of the abstract to Portuguese. Cristian Abdala, Sebastián Quinteros, Federico Arias and
Soledad Vadecantos helped with disscussions and with
friendship. The senior author would like to thank Elisa
Núñez for her invaluable help and patience. This study
was supported by Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET – PIP Nº 5982). A last consideration is needed regarding the ances-
tral area methodologies. Although they erroneously are
criticized as an absolute dispersalist approach, they are a
valid procedure for studying the historical biogeogra-
phy of a taxon. These methods, however, present some
problems, as the sometimes uninformative multiple
reconstructions of DIVA, or the excessive importance
of the basal areas for the Fitch optimization. REFERENCES Avila, L.J.; Cei, J.M.; Martori, R.A. & Acosta, J.C. 1992. A new
species of Liolaemus of the bibronii group from granitic
ravines of Achiras, Sierra de Comechingones, Córdoba,
Argentina (Reptilia: Tropiduridae). Bolletino del Museo Regionale
di Scienze Naturali, Torino, 10(1):101-111. This work represents the first study of the his-
torical biogeography of the chiliensis group, a major part
of the genus Liolaemus using explicit methodology and
with a complete taxon sampling. Further studies need
to be carried out, especially concerning the areas used,
particularly its unambiguous definition, and using geo-
morphologic data to contrast the hypothesis produced. z
( )
Albino, A.M. 1998. Primer registro fósil de Liolaemus (Squamata:
Iguania: Tropiduridae). In: VII Congreso Argentino de
Paleontología y Bioestratigrafía, Bahía Blanca. Actas, p. 57. Albino, A.M. 2005. A late Quartenary lizard assemblage from
the Southern Pampean region of Argentina. Journal of
Vertebrate Paleontology, 25(1):185-191. Bremer, K. 1992. Ancestral Areas: a cladistic reinterpretation of
the center of origin concept. Systematic Biology, 41:436-445. RESUMO puna salteña, Argentina. Cuadernos de Herpetología, 16(1):47-64. Goloboff, P. 1993. Estimating character weights during tree
search. Cladistics, 9:83-91. Morando M.; Avila, L.J. & Sites Jr., J.W. 2003. Sampling strategies
for delimiting species: genes, individulas, and populations
in the Liolaemus elongatus-kriegi complex (Squamata:
Liolaemidae) in andean-patagonian South America. Systematic
Biology, 52(2):159-185. Goloboff, P.; Farris, S. & Nixon, K. 2003. T.N.T.: Tree analysi Goloboff, P.; Farris, S. & Nixon, K. 2003. T.N.T.: Tree analysis
using New Technology. Available at: <www.zmuc.dk./
public/phylogeny>. y
using New Technology. Available at: <www.zmuc.dk./
public/phylogeny>. Morrone, J.J. 1996. The biogeographical Andean subregion: a
proposal exemplified by Arthropod taxa (Arachnida,
Crustacea and Hexapoda). Neotropica, 42:103-114. Halloy, M.; Etheridge, R. & Burghardt, G.M. 1998. To bury in
sand: Phylogenetic relationships among lizard species of
the boulengeri group, Liolaemus (Reptilia: Squamata:
Tropiduridae), based on behavioral characters. Herpetological
Monographs, 12:1-37. Ortiz, J.C. 1981. Révision taxonomique et biologique des Liolaemus du
groupe nigromaculatus (Squamata, Iguanidae). Université de Paris
VII. (Thèse Doctorat d’Etat ès Sciences Naturelles). Hausdorf, B. 1998. Weighted ancestral area analysis and the
solution of the redundant distribution problem. Systematic
Biology, 47(3):445-456. Roig-Juñent, S. 1994. Historia Biogeográfica de América del Sur
Austral. Multequina. 3:167-203. Hellmich, W. 1951. On ecotypic and autotypic characters, a
con-tribution to the knowledge of the evolution of the
genus Liolaemus (Iguanidae). Evolution, 5:359-369. Ronquist, F. 1994. Ancestral areas and parsimony. Systematic Biology,
43:267-274. g
genus Liolaemus (Iguanidae). Evolution, 5:359-369 Ronquist, F. 1997. Dispersal-vicariance analysis: a new approach
to the quantification of historical biogeography. Systematic
Biology, 46:195-203. Hovenkamp, P. 1997. Vicariance events, not Areas, should be
used in Biogeographical analysis. Cladistics, 13:67-79. Kitching, I.J.; Forey, P.L.; Humphries, C.J. & Williams, D.M. 1998. Cladistics: The Theory and Practice of Parsimony Analysis. Oxford University Press, Oxford. Schulte, J.A.; Macey, J.R.; Espinoza, R.E. & Larson, A. 2000. Phylogenetic relationships in the iguanid lizard genus
Liolaemus: multiple origins of viviparous reproduction and
evidence for recurring andean vicariance and dispersal. Biological Journal of the Linnean Society, 69:75-102. Lamborot, M.; Alvarez, E.; Campos, I. & Espinoza, A. 1981. Karyotypic characterization of three chilean subspecies
of Liolaemus monticola. The Journal of Heredity, 72:328-334. Schulte II, J.A.; Valladares, J.P. & Larson, A. 2003. Phylogenetic
relationships within Iguanidae using molecular and
morphological data and a phylogenetic taxonomy of
iguanian lizards. Herpetologica, 59:399-419. Laurent, R.F. 1983. Contribución al conocimiento de la estructura
taxonómica del género Liolaemus Wiegmann (Iguanidae). Boletin de la Asociación Herpetologica Argentina, 1:16-18. Solbrig, O.T. 1976. RESUMO The origin and floristic affinities of the
South American temperate desert and semidesert regions. In: Goodall, D.W. (Ed.), Evolution of desert biota. University
Texas Press, Austin, p. 7-49. Laurent, R.F. 1985. Segunda contribución al conocimiento de la
estructura taxonómica del género Liolaemus Wiegmann
(Iguanidae). Cuadernos de Herpetología, 1:1-37. Laurent, R.F. 1992. On some overlooked species of the genus
Liolaemus Wiegmann (Reptilia Tropidurinae) from Peru. Breviora, 494:1-33. Thiele, K. 1993. The holy grail of the perfect character: the
cladistic treatment of morphometric data. Cladistics,
9:275-304. Lobo, F. 2001. A phylogenetic analysis of lizards of the Liolaemus
chiliensis group (Iguania: Tropiduridae). Herpetological Journal,
11(4):137-150 Young-Downey, A.R. 1998. Phylogenetic studies on Liolaemus (Sauria:
Tropiduridae): an interpretation based on molecular data and
a biochemical test of a biogeographic hypothesis. Univestity
of Miami, Coral Gables, Florida. (Ph.D. Dissertation). Lobo, F. 2005. Las relaciones filogenéticas dentro del grupo
chiliensis (Iguania: Liolaemidae) sumando nuevos caracteres
y taxones. Acta Zoológica Lilloana, 49(1-2):67-89. of Miami, Coral Gables, Florida. (Ph.D. Dissertation). Lobo, F. & Espinoza, R.E. 2004. Further Resolution of Purported
Reproductive Bimodality in Liolaemus alticolor (Iguania:
Tropiduridae) with descriptions of Two New Species from
the Puna Region of Argentina and Chile. Copeia, (4):850-867. Recebido em: 21.08.2006
Aceito em: 19.12.2006 Recebido em: 21.08.2006
Aceito em: 19.12.2006 Recebido em: 21.08.2006
Aceito em: 19.12.2006 RESUMO the center of origin concept. Systematic Biology, 41:436-445. Brown, J.H. & Gibson, A.C. 1983. Biogeography. C.V. Mosby,
Toronto. Ainda que tenham sido feito muitos estudos filogenéticos
no gênero Liolaemus, os mesmos não são apropriados para uma
análise biogeográfica, porque não possuem uma amostragem
taxonômica adequada. Desenvolve-se neste trabalho, uma análi-
se biogeográfica do grupo de L. chiliensis, que se diferencia das
contribuições prévias por representar a todos os membros do gru-
po. A análise biogeográfica consiste em reconstruir a área ances-
tral deste clado, utilizando três métodos diferentes: Otimização
de Fitch, Análise de Dispersão-Vicariância e Áreas Ancestrais
Pesadas. As análises foram realizadas em filogenias feitas com o
critério de parsimônia sob pesos iguais e pesos implicados. A
árvore obtida sob pesos iguais, recupera a maioria dos grupos
encontrados em análises prévias. O consenso estrito das árvores
de pesos implicados, recupera quinze grupos, mas não pode-se
estabelecer as relações entre eles. A análise de áreas ancestrais
permite postular a região Andino-Patagônica como provável área Cabrera, A.L. & Willink, A. 1980. Biogeografía de América Latina. Secretaría General de la OEA. Washington. Cei, J.M. 1979. The Patagonian Herpetofauna. In: Duellman, W.E. (Ed.), The South American Herpetofauna: Its Origin, Evolution,
and Dispersal. Museum of Natural History University of
Kansas, Monograph 7, p. 309-339. Cei, J.M. 1986. Reptiles del centro, centro-oeste y sur de la Argentina. Museo Regionale di Scienze Naturali, Torino, Monografie
4. Cei, J.M. 1993. Reptiles del noroeste, nordeste y este de la Argentina. Museo
Regionale di Scienze Naturali, Torino, Monografie 14. Ebach, M.C. 1999. Paralogy and the Centre of Origin Concept. Cladistics, 15(4):387-391. Espinoza, R.E.; Wiens, J.J. & Tracy, C.R. 2004. Recurrent evolution
of herbivory in small, cold-climate lizards: breaking the
ecophysiological rules of reptilian herbivory. Proceedings of
the National Academy of Sciences, DOI: 101:16819-16824. Etheridge, R. 1995. Redescription of Ctenoblepharys adspersa
Tschudi, 1845, and the taxonomy of Liolaeminae (Reptilia: PAP. AVULS ZOOL. 46(24), 2006 273 Squamata: Tropiduridae). American Museum Novitates,
3142:1-34. Lobo, F; Quinteros, S. & Díaz Gómez, J.M. In press. Description
of a new species of the liolaemus alticolor group (Iguania:
Liolaemidae) from Cuzco, Peru. Etheridge, R. 2000. A review of lizards of the Liolaemus
wiegmanniii group (Squamata, Iguania, Tropiduridae), and a
history of morphological change in the sand-dwelling
species. Herpertological Monographs, 14:293-352. Martinez Oliver, I. & Lobo, F. 2002. Una nueva especie de
Liolaemus del grupo alticolor (Iguania: Liolaemidae) de la
puna salteña, Argentina. Cuadernos de Herpetología, 16(1):47-64. 274 DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS APPENDIX 1 New characters used:
1. Gravid Coloration: (0) Absent; (1) Present
2. Plant-Microhabitat: (0) Bunch grass (Festuca sp); (1) Spiny shrub (Parastrephia); (2) Both
3. Lateral Black Spots: (0) Absent; (1) Present New characters used: 1. Gravid Coloration: (0) Absent; (1) Present 1. Gravid Coloration: (0) Absent; (1) Present 2. Plant-Microhabitat: (0) Bunch grass (Festuca sp); (1) Spiny shrub (Parastrephia); (2) Both
3 Lateral Black Spots (0) Absent (1) Present 2. Plant-Microhabitat: (0) Bunch grass (Festuca sp); (1) Spiny shrub (Parastrephia); (2) Both 3. Lateral Black Spots: (0) Absent; (1) Present
|
https://openalex.org/W3201585393
|
https://www.epj-conferences.org/articles/epjconf/pdf/2021/06/epjconf_hinpw62021_04001.pdf
|
English
| null |
Recent experimental activity on heavy-ion induced reactions within the NUMEN project
|
EPJ web of conferences
| 2,021
|
cc-by
| 4,286
|
for the NUMEN Collaboration 1Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali del Sud, Catania, Italy
2Dipartimento di Fisica e Astronomia “Ettore Majorana”, Università di Catania, Italy
3Instituto de Fısica, Universidad Nacional Autonoma de Mexico - Mexico City, Mexico
4Instituto Nacional de Investigaciones Nucleares - Ocoyoacac, Mexico
5Department of Physics, Akdeniz University - Antalya, Turkey
6Istituto Nazionale di Fisica Nucleare, Sezione di Torino, Italy
7DISAT, Politecnico di Torino, Italy
8LPC Caen, Normandie Universite, ENSICAEN, UNICAEN, CNRS/IN2P3 Caen, France
9Ankara University, Institute of Accelerator Technologies, Turkey
10Centro Universitario FEI - Sao Bernardo do Campo, Brazil
11Institute of Natural Sciences, Karadeniz Teknik University - Trabzon, Turkey
12Università degli Studi di Enna “Kore” - Enna, Italy
13Instituto de Fisica, Universidade Federal Fluminense - Niteroi, Brazil
14Instituto de Fisica, Universidade de Sao Paulo - Sao Paulo, Brazil
15Instituto de Pesquisas Energeticas e Nucleares IPEN/CNEN - Sao Paulo, Brazil
16Department of Physics and HINP, University of Ioannina - Ioannina, Greece
17IFIN-HH - Magurele, Romania
18Department of Chemistry, National and Kapodistrian University of Athens, Athens, Greece 1Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali del Sud, Catania, Italy
2Dipartimento di Fisica e Astronomia “Ettore Majorana”, Università di Catania, Italy
3Instituto de Fısica, Universidad Nacional Autonoma de Mexico - Mexico City, Mexico
4Instituto Nacional de Investigaciones Nucleares - Ocoyoacac, Mexico
5Department of Physics, Akdeniz University - Antalya, Turkey
6Istituto Nazionale di Fisica Nucleare, Sezione di Torino, Italy
7DISAT, Politecnico di Torino, Italy
8LPC Caen, Normandie Universite, ENSICAEN, UNICAEN, CNRS/IN2P3 Caen, France
9Ankara University, Institute of Accelerator Technologies, Turkey
10Centro Universitario FEI - Sao Bernardo do Campo, Brazil
11Institute of Natural Sciences, Karadeniz Teknik University - Trabzon, Turkey
12Università degli Studi di Enna “Kore” - Enna, Italy
13Instituto de Fisica, Universidade Federal Fluminense - Niteroi, Brazil
14Instituto de Fisica, Universidade de Sao Paulo - Sao Paulo, Brazil
15Instituto de Pesquisas Energeticas e Nucleares IPEN/CNEN - Sao Paulo, Brazil
16Department of Physics and HINP, University of Ioannina - Ioannina, Greece
17IFIN-HH - Magurele, Romania
18Department of Chemistry, National and Kapodistrian University of Athens, Athens, Greece Abstract. The possibility to use a special class of heavy-ion induced direct reactions, such as double charge
exchange reactions, is discussed in view of their application to extract information that may be helpful to
determinate the nuclear matrix elements entering in the expression of neutrinoless double beta decay half-life. for the NUMEN Collaboration The strategies adopted in the experimental campaigns performed at INFN - Laboratori Nazionali del Sud are
briefly described, emphasizing the advantages of the multi-channel approach to nuclear reaction data analysis. 1 Introduction aspect is the possibility to use CE to investigate the spin-
isospin-dependent response of nuclei. Charge exchange (CE) reactions are nuclear processes in
which the individual balance of neutrons and protons
inside the projectile and the target is modified, while
keeping the overall number of nucleons unchanged. In the
isospin formalism the CE promote isovector excitations in
both the projectile and target. CE are thus selective tools
to emphasize the role of isospin in both nuclear structure
and reaction studies. Relevant aspects of single charge
exchange (SCE) induced by light projectile have been
discussed in several articles, among which excellent
reviews are in ref. [1] [2] [3]. For heavy-ion induced CE
we refer to the recent review [4]. An especially appealing Recently, particular interest has been given to double
charge exchange (DCE) processes [5] [6] [7] as they can
give access to the nuclear response to second order isospin
operators, which also promote double beta decays. SCE and DCE studies have a genuine nuclear physics
interest, since they reflect a key aspect of nuclear
dynamics. However, beyond that, there is an even broader
impact of such investigations because of their relevance
for nuclear beta decay, thus connecting strong and weak
interactions. In this context, the NUMEN and NURE projects at INFN-
LNS laboratory in Catania [8] [9] aim at exploring heavy-
ion induced single and double charge exchange (DCE) Recent experimental activity on heavy-ion induced reactions
within the NUMEN project Francesco Cappuzzello1,2,*, Luis Acosta3, Clementina Agodi1, Carmen Altana1, Paulina Amador-Valenzuela4, Ismail
Boztosun5, Giuseppe A. Brischetto1,2, Salvatore Calabrese1,2, Daniela Calvo6, Vittoria Capirossi6,7, Diana Carbone1, Manuela
Cavallaro1, Efrain R. Chávez Lomeli3, Irene Ciraldo1,2, Mauro Cutuli1,2, Franck Delaunay1,2,8, Haris Djapo9, Canel Eke5,
Paolo Finocchiaro1, Suna Firat5, Maria Fisichella1, Marcilei A. Guazzelli da Silveira10, Aylin Hacisalihoglu11, Felice Iazzi6,7,
Gaetano Lanzalone1,12, Laura La Fauci1,2, Roberto Linares13, Nilberto H. Medina14, Mauricio Moralles15, José R. B. Oliveira14, Athena Pakou16, Luciano Pandola1, Horia Petrascu17, Federico Pinna6,7, Giuseppe Russo1, Roberto B. B. Santos10,
Onoufrios Sgouros1, Selçuk O. Solakci5, George Souliotis18, Vasileios Soukeras1,2, Alessandro Spatafora1,2, Domenico
Torresi1, Salvatore Tudisco1, Aydin Yildirin5, Vinicius A. B. Zagatto13 https://doi.org/10.1051/epjconf/202125204001 https://doi.org/10.1051/epjconf/202125204001 EPJ Web of Conferences 252, 04001 (2021)
HINPw6 © 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/). * Corresponding author: cappuzzello@lns.infn.it DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
ses/by/4 0/) 3 The experiments A crucial feature is the implementation of a technique of
trajectory reconstruction, based on differential algebraic
techniques and the accurate mapping of the spectrometer
magnetic fields [29] [30] [31] [32], which solves the
equation of motion of each detected particle to 10th order
[33]. This is a unique characteristic of MAGNEX, which
guarantees the above mentioned performances and its
relevance in the worldwide scenario of heavy-ion physics. The possible connections of DCE measured cross-
sections with double beta decay NMEs and, consequently,
with the still unsolved neutrino puzzles are among the
goals of the present studies. 3 The experiments The NUMEN experiments are performed at INFN-LNS
using the K800 Superconducting Cyclotron to accelerate
beams and the MAGNEX large acceptance magnetic
spectrometer for the detection of the ejectiles. The NUMEN experiments are performed at INFN-LNS
using the K800 Superconducting Cyclotron to accelerate
beams and the MAGNEX large acceptance magnetic
spectrometer for the detection of the ejectiles. MAGNEX is a large acceptance magnetic device
consisting of a large aperture vertically focusing
quadrupole and a horizontally bending dipole magnet. A
hybrid Focal Plane detector (FPD) featuring a gaseous
tracker and a wall of silicon pad sensors is mounted at the
focal plane to detect the reaction ejectiles [25] [26]. MAGNEX was designed to investigate processes
characterized by very low yields, guaranteeing the
identification of heavy ions with quite high mass
(ΔA/A∼1/160), angle (Δθ∼0.2°) and energy resolutions
(ΔE/E∼1/1000), within a large solid angle (Ω∼50 msr)
and momentum range (−14% < Δp/p < +10%). High-
resolution measurements for quasi-elastic processes,
characterized by differential cross-sections as low as tens
of nb/sr, were already performed with this setup [27] [28]. A crucial feature is the implementation of a technique of
trajectory reconstruction, based on differential algebraic
techniques and the accurate mapping of the spectrometer
magnetic fields [29] [30] [31] [32], which solves the
equation of motion of each detected particle to 10th order
[33]. This is a unique characteristic of MAGNEX, which
guarantees the above mentioned performances and its
relevance in the worldwide scenario of heavy-ion physics. MAGNEX is a large acceptance magnetic device
consisting of a large aperture vertically focusing
quadrupole and a horizontally bending dipole magnet. A
hybrid Focal Plane detector (FPD) featuring a gaseous
tracker and a wall of silicon pad sensors is mounted at the
focal plane to detect the reaction ejectiles [25] [26]. MAGNEX was designed to investigate processes
characterized by very low yields, guaranteeing the
identification of heavy ions with quite high mass
(ΔA/A∼1/160), angle (Δθ∼0.2°) and energy resolutions
(ΔE/E∼1/1000), within a large solid angle (Ω∼50 msr)
and momentum range (−14% < Δp/p < +10%). High-
resolution measurements for quasi-elastic processes,
characterized by differential cross-sections as low as tens
of nb/sr, were already performed with this setup [27] [28]. 2 The motivation Recently, accurate cross sections for heavy-ion DCE
reactions have been measured at the INFN-LNS
laboratory, thanks to the use of the MAGNEX
spectrometer. Neutrinoless double beta decay (0νββ) is an hypothetical
process in which a “parent” nucleus is spontaneously
transformed into a “daughter” isobar by the conversion of
two neutron/protons into two protons/neutrons. The
process is accompanied by the emission of two electrons
(or positrons), which guarantee also electric charge
conservation. If detected, 0νββ would provide evidence
for the Majorana nature of the neutrino (i.e. the
equivalence of neutrino and antineutrino) and give access
to the neutrino mass [13] [14] [15] [16]. Under the
hypothesis that the neutrino, electrons and nuclear degrees
of freedom can be separated in 0νββ, the decay rate
explicitly depends on the nuclear transition probability,
accounted for in the so-called Nuclear Matrix Element
(NME). The 0νββ NMEs are very sensitive to nuclear
physics involved in decay, since they probe the nuclear
wave functions of the initial and final states. In particular,
the nuclear response to high-momentum second-order
isospin operators is probed in 0νββ, similarly to what
happen in DCE reactions [17]. However, there are no
experimental methods to directly measure 0νββ NMEs
and accurate theoretical calculations are challenging,
leading to discrepancy factors larger than two, as
presently reported in literature [13]. Thus experimental
inputs from SCE and DCE studies are useful to help
evaluate the 0νββ NMEs and to constrain the calculations. In this context, an experimental campaign has started at
the INFN-Laboratori Nazionali del Sud in Catania using
the MAGNEX large acceptance magnetic spectrometer
[18]. It focuses on the study of heavy-ion induced DCE
reactions and competing reaction channels involving the
nuclei of interest for 0νββ decay. The accurate description of heavy-ion induced DCE is a
demanding task also for nuclear theory. From the reaction
theory side initial and final state interactions of projectile
and target systems need to be under control and the
reaction amplitude must be described properly. In this
context direct reaction theory can provide the suitable
toolbox. Nuclear structure theory is asked to provide
accurate input for modelling both projectile and target
[20]. Recently, new theoretical developments for the reaction
mechanism involved in heavy ion induced reactions have
been achieved alongside. Presently this activity is in
progress within the NUMEN project [21] [22] [23] [24]. * Corresponding author: cappuzzello@lns.infn.it © 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/). https://doi.org/10.1051/epjconf/202125204001 https://doi.org/10.1051/epjconf/202125204001 EPJ Web of Conferences 252, 04001 (2021)
HINPw6 EPJ Web of Conferences 252, 04001 (2021) transfer range. In addition, the rather tiny DCE cross
sections (down to few nb) demand for high experimental
sensitivity, which strongly depends on the rejection
capability against unwanted spurious events. These latter
may be generated by competing reaction processes, some
of which are more likely to occur than DCE. For this
reason highly selective particle identification is a
necessary prerequisite for these experiments. In addition,
spurious data associated to a wrong determination of
detection parameters could casually be misinterpreted as
valid DCE data. Characterizing the relevant background
sources can be subtle and require stringent conditions for
the experimental set-up as well as a careful study of the
detection response [19]. reactions in a systematic approach. The main goal is to
extract information on nuclear matrix elements for SCE
and DCE processes and to identify the possible
connections with double beta decay. Detailed information about these projects are found in
recent articles, which we refer to [10] [11] [12] for both
scientific and technological aspects. In the present paper, the
global strategy and
methodologies of the above mentioned programmes, the
challenges of the experimental activities together with the
status of the already performed experimental runs are
briefly outlined. reactions in a systematic approach. The main goal is to
extract information on nuclear matrix elements for SCE
and DCE processes and to identify the possible
connections with double beta decay. Detailed information about these projects are found in
recent articles, which we refer to [10] [11] [12] for both
scientific and technological aspects. In the present paper, the
global strategy and
methodologies of the above mentioned programmes, the
challenges of the experimental activities together with the
status of the already performed experimental runs are
briefly outlined. 2
Heavy-ion induced double charge
exchange reactions All
of these information are provided in a consistent
experimental framework and analysed in a multi-channel
approach, based on quantum scattering theory of coupled
channels. A unique opportunity is thus searched by
NUMEN to build an over-constrained set of nuclear
structure information, promoting the development of a
new approach to 0νββ NMEs calculations. • (20Ne,18O) two-proton stripping reaction (
)
p
pp g
• (20Ne,19F) one-proton stripping reaction
• (20Ne 22Ne) two-neutron pickup reaction • (20Ne,19F) one-proton stripping reaction • (20Ne,22Ne) two-neutron pickup reaction • (20Ne,21Ne) one-neutron pickup reaction. For these experiments, the incident beam (20Ne10+) has a
lower magnetic rigidity compared to to the ejectiles of
interest. Thus, for a fixed magnetic field setting, the beam
is more bent than the ejectiles of interest. The
spectrometer optical axis is typically placed at −3°, thus
the covered angular range is −8° < θlab < +3°. The
quadrupole and dipole magnetic fields are set in order that
the 20Ne10+ beam reaches the low-Bρ region besides the
FPD. The 20Ne9+ and 20Ne8+ components of the beam
emerging for the target could be a source of background
in these experiments. They are significantly reduced by
the use of a supplementary stripper foil mounted after the
target itself [38]. Investigations of the two classes of experiments have been
started in the recent years with the present facility. These
studies highlighted the strengths and the limiting aspects
of the adopted technique, providing first valuable
information on relevant nuclear structure aspects for a
number of double beta decay emitter candidates [8]. This
activity has also given precious information to guide the
major upgrade of the facility toward much higher beam
intensity, which is presently going on. The systems already experimentally explored using the
(20Ne,20O) reaction at 15 AMeV are the 116Cd target (to
study the 116Cd → 116Sn transition), the 130Te (for the 130Te
→ 130Xe) and the 76Ge (for the 76Ge → 76Se). The data
reduction and analysis is in progress and the first results
from some reaction channels have been recently
published [39] [40] [41] [42]. 2
Heavy-ion induced double charge
exchange reactions In this way, we can get
supplementary information from the detailed study of the
reaction network, profiting of the strong selectivity of
quasi-elastic processes. The nucleus-nucleus interaction
can be extracted from elastic scattering data and applied
to the evaluation of the distorted waves in each direct
reaction channel. Inelastic scattering data give selective
access to the role of the deformation in the coupled
channel reaction network equations. Single nucleon
transfer reactions shed light on the role of single particle
configurations in the involved nuclear many body states. Two nucleon transfer reactions probe pairing correlations,
while SCE give access to 1particle-1hole excitations. All
of these information are provided in a consistent
experimental framework and analysed in a multi-channel
approach, based on quantum scattering theory of coupled
channels. A unique opportunity is thus searched by
NUMEN to build an over-constrained set of nuclear
structure information, promoting the development of a
new approach to 0νββ NMEs calculations. In each case, the experiments are organized in order to
explore together with the DCE reactions of interest, all the
relevant quasi-elastic processes occurring in the
projectile-target collision. In this way, we can get
supplementary information from the detailed study of the
reaction network, profiting of the strong selectivity of
quasi-elastic processes. The nucleus-nucleus interaction
can be extracted from elastic scattering data and applied
to the evaluation of the distorted waves in each direct
reaction channel. Inelastic scattering data give selective
access to the role of the deformation in the coupled
channel reaction network equations. Single nucleon
transfer reactions shed light on the role of single particle
configurations in the involved nuclear many body states. 3.2. Experiments with 20Ne beam (β−β−direction)
In the class of experiments triggered by 20Ne10+ beams, the
reaction channels we are interested are the following: • (20Ne,20Ne) elastic and inelastic scattering • (20Ne,20Ne) elastic and inelastic scattering • (20Ne,20O) DCE reaction • (20Ne,20F) SCE reaction Two nucleon transfer reactions probe pairing correlations,
while SCE give access to 1particle-1hole excitations. All
of these information are provided in a consistent
experimental framework and analysed in a multi-channel
approach, based on quantum scattering theory of coupled
channels. A unique opportunity is thus searched by
NUMEN to build an over-constrained set of nuclear
structure information, promoting the development of a
new approach to 0νββ NMEs calculations. Two nucleon transfer reactions probe pairing correlations,
while SCE give access to 1particle-1hole excitations. 2
Heavy-ion induced double charge
exchange reactions Challenging experimental issues must be addressed to
measure heavy-ion induced DCE cross sections. Indeed
one needs to detect heavy ions with good isotopic
separation and energy resolution in a wide angular range,
including zero-degree, in order to distinguish transitions
to individual states and explore a wide momentum 2 https://doi.org/10.1051/epjconf/202125204001 EPJ Web of Conferences 252, 04001 (2021)
HINPw6 EPJ Web of Conferences 252, 04001 (2021)
6 θlab < 9° is explored. The MAGNEX quadrupole and
dipole magnetic fields are set in order that the incident
beam, after passing through the magnets, reaches a region
besides the FPD. For this class of experiments, in fact, the
incident ion beam (18O8+) has higher magnetic rigidity
(Bρ) than the ejectiles of interest (namely fully stripped
18Ne, 18F, 20Ne, 19F, 16O, 17O ions). The beam stops in a
Faraday cup, placed in the high-Bρ region besides the
FPD, which measures the collected charge for each run. The experimental activity with accelerated beams
proposed and presently in progress consists of two main
classes of experiments, aiming at the exploration of the
two directions of isospin lowering τ− τ− and rising τ+ τ+,
characteristic of β−β− and β+β+ decays, respectively. In particular, the β+β+ direction in the target excitation
modes is investigated using a 18O8+ beam and measuring
the (18O,18Ne) DCE transitions, together with other
reaction channels involving same beam and target. Similarly, the β−β− direction is explored via the (20Ne,20O)
reaction, using a 20Ne10+ beam and detecting the reaction
products from the DCE channel and from the other open
reaction channels. 116Sn, 76Se and 48Ti are the targets of interest for 0νββ,
which have already been explored by NUMEN, via
(18O,18Ne) reaction at 15 and 22 AMeV. The purpose was
to study the 116Sn → 116Cd, 76Se → 76Ge and 48Ti → 48Ca
transitions and the competing channels as shown in Fig. 2. A dedicated run was also performed on 12C target, in
order to compare reaction models with an easier light
system. The reduction and analysis of the collected data
from these campaigns is presently in progress. First
results have been already published in ref. [34] [35] [36]
[37]. reaction channels. In each case, the experiments are organized in order to
explore together with the DCE reactions of interest, all the
relevant quasi-elastic processes occurring in the
projectile-target collision. Acknowledgments This project has received funding from the European
Research Council (ERC) under the European Union’s
Horizon 2020 research and innovation programme (grant
agreement No 714625). This project has received funding from the European
Research Council (ERC) under the European Union’s
Horizon 2020 research and innovation programme (grant
agreement No 714625). References [1] Alford, W.P.; Spicer, B.M. Nucleon charge-exchange
reactions at intermediate energy. Adv. Nucl. Phys. 1998,
1, 24. Fig. 2 Scheme of the reaction channels already studied
within the NURE and NUMEN experiments. [2] Osterfeld, F. Nuclear spin and isospin excitations. Rev. Mod. Phys. 1992, 64, 491.. [3] Taddeucci, T.N.; et al. The (p, n) reaction as a probe of
beta decay strength. Nucl. Phys. A 1987, 469, 125.. [4] Lenske, H.; Cappuzzello, F.; Cavallaro, M.; Colonna, M. Heavy ion charge exchange reactions as probes for
nuclear β-decay. Prog. Part. Nucl. Phys. 2019, 109,
103716.. [5] F. Cappuzzello et al., Eur. Phys. J. A 51, 145 (2015). [6] Cappuzzello, F.; Cavallaro, M.; Agodi, C.; Bondì, M.;
Carbone, D.; Cunsolo, A.;, Foti, A. Heavy-ion double
charge exchange reactions: A tool toward 0νββ nuclear
matrix elements. Eur. Phys. J. A 2015, 51, 145. [7] Cappuzzello, F., and Agodi, C. (2021). The NUMEN
Project: Shedding Light on Neutrinoless Double Beta
Decay by Heavy-Ion Nuclear Reactions. Nucl, Phys. News, in press, DOI 10.1080/10619127.2021.1881368. [8] Cappuzzello, F., Agodi, C., Cavallaro, M., Carbone, D.,
Tudisco, S., Lo Presti, D., et al.. (2018). The NUMEN
project: NUclear Matrix Elements. Eur. Phys. J. A. 54,
72. [9] Cavallaro, M., Aciksoz, E., Acosta, L., Agodi, C.,
Auerbach, N., Bellone, J. I., et al., (2017). “NURE: an
ERC project to study nuclear reactions,” in Proceedings,
55th International Winter Meeting on Nuclear Physics
Bormio, (Bormio). doi: 10.22323/1.302.. [10] P. Finocchiaro et al., Universe 2020, 6, 129;
doi:10.3390/universe6090129. [11] C. Agodi et al., Universe 2021, 7, 72. https://doi.org/10.3390/universe7030072. [12] F. Cappuzzello et al., The NUMEN Project: An Update
of the Experimental Campaigns. Front. Astron. Space
Sci. 8:668587. doi: 10.3389/fspas.2021.668587. [13] H. Ejiri, J. Suhonen, K. Zuber, Physics Reports 797
(2019) 1. [14] J. Engel, J. Menéndez, Rep. Progr. Phys. 60 (2017)
046301. [15] S. Dell’Oro, S. Marcocci, and F. Vissani, Phys. Rev. D
90, 033005 (2014). [16] J. Vergados, H. Ejiri, F. Šimkovic, Rep. Progr. Phys. 75
(2012) 106301. Fig. 2 Scheme of the reaction channels already studied
within the NURE and NUMEN experiments. [17] Cappuzzello, F., and Cavallaro, M. (2020). Nuclear
Response to Second-Order Isospin Probes in Connection
to Double Beta Decay. Universe. 6, 217. 3.1. Experiments with 18O beam study the
Cd →
Sn transition), the
Te (for the
Te
→ 130Xe) and the 76Ge (for the 76Ge → 76Se). The data
reduction and analysis is in progress and the first results
from some reaction channels have been recently
published [39] [40] [41] [42]. For the experiments of this class, the reaction channels of
our interest are listed below: For the experiments of this class, the reaction channels of
our interest are listed below: • (18O,18O) elastic and inelastic scattering
18
18 • (18O,18Ne) DCE reaction A scheme of the transitions already studied in the
experimental runs is shown in Fig. 2. • (18O,18F) SCE reaction • (18O,20Ne) two-proton pickup reaction
18
19 • (18O,19F) one-proton pickup reaction • (18O,16O) two-neutron stripping reaction • (18O,17O) one-neutron stripping reaction One of the main challenges of such experiments is the
measurement at very forward angles, including zero-
degree. This is performed by placing the spectrometer
with its optical axis at +3° with respect to the beam axis. Thanks to its large angular acceptance, the range −2° < One of the main challenges of such experiments is the
measurement at very forward angles, including zero-
degree. This is performed by placing the spectrometer
with its optical axis at +3° with respect to the beam axis. Thanks to its large angular acceptance, the range −2° < 3 https://doi.org/10.1051/epjconf/202125204001 EPJ Web of Conferences 252, 04001 (2021)
HINPw6 EPJ Web of Conferences 252, 04001 (2021) ig. 2 Scheme of the reaction channels already
within the NURE and NUMEN experiments. Conclusions [18] F. Cappuzzello et al. The MAGNEX spectrometer:
Results and perspectives. Eur. Phys. J. A (2015) 51:145. A systematic study of heavy-ion induced DCE reactions
and of the other reaction channels characterized by the
same projectile and target is in progress at INFN-LNS. The main goal is to investigate the nuclear response to
DCE reactions for all the isotopes candidate for 0νββ
decay towards the determination of 0νββ NMEs. [19] S. Calabrese et al. Analysis of the background on cross
section measurements with the MAGNEX spectrometer:
The (20Ne,20O) Double Charge Exchange case. Nuclear
Inst. and Methods in Physics Research, A 980 (2020)
164500. 4 4 EPJ Web of Conferences 252, 04001 (2021)
HINPw6 https://doi.org/10.1051/epjconf/202125204001 EPJ Web of Conferences 252, 04001 (2021)
HINP 6 [20] N. Shimizu, J. Menendez, and K. Yako, Phys. Rev. Lett. 120, 142502 (2018).. [21] H. Lenske, J.I. Bellone, M. Colonna, J. A. Lay, Phys. Rev. C 98 (4), 044620 (2018).. [22] E. Santopinto, H. García-Tecocoatzi, RIM
Vsevolodovna, J. Ferretti, Phys. Rev. C 98 (6), 061601
(2018). [22] E. Santopinto, H. García-Tecocoatzi, RIM [23] J.I. Bellone, S. Burrello, M. Colonna, J. A. Lay, H. Lenske, Phys. Lett. B 807, 135528 (2020). [24] Lenske, H.; Bellone, J.; Colonna, M.; Gambacurta, D. Nuclear Matrix Elements for Heavy Ion Sequential
Double Charge Exchange Reactions. Universe 2021, 7,
98. https://doi.org/10.3390/universe7040098. [25] Torresi, D., Sgouros, O., Soukeras, V., Cavallaro, M.,
Cappuzzello, F., Carbone, D., et al. (2021). An upgraded
focal plane detector for the MAGNEX spectrometer. Nucl. Instr. and Meth. A. 989, 164918. [26] M. Cavallaro et al. The low-pressure focal plane detector
of the MAGNEX. Eur. Phys. J. A (2012) 48: 59. DOI
10.1140/epja/i2012-12059-8. [27] J.R.B. Oliveira et al., Journal of Physics G: Nuclear and
Particle Physics, 40 (2013) 105101. [28] D. Pereira et al. Phys. Lett. B 710 (2012) 426. [29] A. Lazzaro et al., Nucl. Instr. and Methods A 591, 394
(2008).. [30] A. Lazzaro et al., Nucl. Instr. and Methods A 585, 136
(2008). [31] A. Lazzaro et al., Nucl. Instr. and Methods A 570, 192
(2007). [32] A. Lazzaro et al., Nucl. Instr. and Methods A 602, 494
(2009). [33] F. Cappuzzello et al., Eur. Phys. J. A 52 (2016) 169. [34] O. Sgouros et al., Phys. Rev. C, accepted. [35] D. Carbone et al., Universe 7 (2021) 58.. [36] J. L. Ferreira et al., Phys. Rev. C 103 (2021) 054604. [37] M. Conclusions Cavallaro et al., Frontiers in Astron. and Space
Science 8 (2021) 659815. [38] M. Cavallaro et al., Results in Phys. 13 (2019) 102191. [39] A. Spatafora et al., Phys. Rev. C 100 (2019) 034620. [40] V. Soukeras et al., Results in Physics, (2021) submitted. [41] S. Calabrese et al., Nucl. Instr. and Meth. A, 980 (2020)
164500. [42] S. Calabrese et al., Acta Phys. Pol. B 49 (2018) 275. 5 5
|
https://openalex.org/W2561924782
|
https://scindeks-clanci.ceon.rs/data/pdf/0354-3471/2016/0354-34711602129V.pdf
|
Croatian
| null |
Evaluation of brand from the perspective of consumers
|
Marketing
| 2,016
|
cc-by-sa
| 5,114
|
UVOD Globalizacija i kapitalizam kao osnovne odlike savremenog poslovnog
okruženja, podigle su pojam konkurencije na jedan potpuno novi nivo. Nadmetanje na globalnom tržištu postaje sve intenzivnije i složenije. Kako bi opstale i napredovale u ovakvim uslovima, kompanije teže ka
maksimalnom iskorišćavanju sopstvenih potencijala, razvijanju kom-
parativnih prednosti i diferenciranju u odnosu na konkurente. Upravo
brend, postaje sve značajniji instrument diferenciranja i sticanja konku-
rentske prednosti kako u proizvodnom, tako i u uslužnom sektoru. Prvi
korak koji kompanija mora načiniti na putu ka izgradnji snažnog brenda
je identifikovanje jedinstvenih prednosti proizvoda/usluga na kojima će
bazirati brend. Međutim, brendiranje kao proces, ne treba precenjivati. Napor ulo-
žen u brendiranje, sam po sebi, ne može prikriti nedostatke proizvoda. S
tim u vezi, uspešan proces brendiranja treba da teče u smeru: proizvod
– brend. Drugim rečima, brendiranje treba posmatrati kao završni mo-
menat. U domaćoj literaturi, autori su se bavili merenjem vrednosti brenda,
ali isključivo sa teorijskog stanovišta u smislu objašnjavanja modela za
merenje performansi. Cilj ovog rada je identifikovanje i analiza fakto-
ra koji imaju uticaj na preferenciju brenda. Na ovaj način, sprovedeno
empirijsko istraživanje pruža doprinos postojećoj literaturi. Pored toga,
nalazi ovog istraživanja mogu poslužiti kompanijama kao koristan alat za
upravljanje brendom, te alat za unapređenje efikasnosti marketing strate-
gije i konkurentske pozicije preduzeća. Ključne reči: dimenzije vrednosti
brenda, merenje vrednosti brenda, prefe-
rencija brenda Originalni naučni rad, UDK 003.65:339.138 Originalni naučni rad, UDK 003.65:339.138 Vrednovanje brenda iz
perspektive potrošača
Marija Vranješ, Dragoljub Jovičić,
Dragana Drinić Marija Vranješ, Dragoljub Jovičić,
Dragana Drinić JEL klasifikacija: M31, D12 JEL klasifikacija: M31, D12 JEL klasifikacija: M31, D12 Apstrakt: Brend je izuzetno važna
intelektualna svojina kompanije. Kako bi
privukle nove potrošače i zadržale što više
postojećih, kompanije moraju neprestano
da rade na unapređenju vrednosti brenda,
što u krajnjoj liniji dovodi do rasta tržišnog
učešća, prihoda i profita. Cilj rada je da
istraži na koji način određene dimenzije
vrednosti brenda utiču na preferenciju
brenda. U skladu sa navedenim ciljem,
korišćene su mere deskriptivne statistike,
analiza pouzdanosti, korelaciona i regresi-
ona analiza. Rezultati istraživanja ukazuju
da dve dimenzije vrednosti brenda: me-
đuljudski odnosi i komparativna prednost
brenda, imaju statistički značajan uticaj
na povećanje nivoa preferencije. S druge
strane, razumevanje i istorija, kao dimen-
zije vrednosti brenda, nemaju statistički
značajan uticaj. Nalazi sprovedene studije
od koristi su menadžmentu kompanija
prilikom formiranja odgovarajućih okvira
za upravljanje brendom u cilju povećanja
preferencije brenda, što bi u krajnjoj liniji
trebalo da dovede do unapređenja konku-
rentske pozicije kompanije. Ključne reči: dimenzije vrednosti
brenda, merenje vrednosti brenda, prefe-
rencija brenda 1.1 Brend Savremeno poslovno okruženje karakteriše sve jači intenzitet konkuren-
cije. U takvim uslovima, kompanije nastoje da se što više diferenciraju
u odnosu na konkurente. S tim u vezi, kompanije mogu diferencirati
svoj proizvod, fokusirajući se na bilo koje materijalno svojstvo proizvo- 129 da (npr. ukus, dizajn) ili nematerijalno svojstvo (npr. cena, brend, zemlja porekla) (Zeugner-Roth, Dia-
mantopoulos & Montesinos, 2008). Upravo brend po-
staje sve značajniji instrument diferenciranja i sticanja
konkurentske prednosti. Postojeća literatura obiluje
definicijama ovog pojma. Prema definiciji američ-
kog udruženja za marketing, brend predstavlja „ime,
pojam, znak, simbol, dizajn, ili pak, kombinacija na-
vedenog, čime se identifikuje roba ili usluge jednog
prodavca/grupe prodavaca i diferencira u odnosu na
konkurentske“ (Kotler & Keller, 2006, p. 274). Vrednost brenda, moguće je posmatrati iz dve per-
spektive: potrošača i kompanije. Ove dve perspektive
su povezane na način da finansijska vrednost brenda
počiva na vrednosti brenda za potrošače. Upravo la-
nac vrednosti brenda, na odgovarajući način pred-
stavlja navedeni odnos. Naime, proces kreiranja bren-
da započinje investiranjem kompanije u marketing
program čiji je osnovni cilj da utiče na stav kupaca
o brendu. Stav velikog broja kupaca dalje utiče na tr-
žišni učinak, koji definiše vrednost brenda na tržištu
(Kotler & Keller, 2006). U daljem radu biće razmatra-
na vrednost brenda iz perspektive potrošača. Jedna od osnovnih uloga brenda je da potrošačima
stvori sliku o vrednosti proizvoda, pa je brend moguće
posmatrati i kao izvestan „sertifikat o kvalitetu proi-
zvoda“ (Keegan & Green, 2008, p. 328). S tim u vezi,
„brendovi omogućavaju potrošačima da skrate vreme
potrebno za izbor pravih alternativa dok istovreme-
no predstavljaju garanciju da će proizvodi i usluge
zadovoljiti njihova očekivanja“ (Veljković i Đorđević,
2010, p. 10). p
p
p
„Brend treba da omogući funkcionalne, simbo-
ličke i iskustvene koristi potrošaču“ (Helgeson &
Supphellen, 2004, p. 205). To praktično znači da brend,
s jedne strane, treba da omogući rešavanje problema
potrošača, dok s druge strane, treba da pruži i određe-
ne simboličke koristi, odnosno odgovarajuće značenje
za potrošača. Kada se govori o simboličkim koristima,
veoma je važno da postoji podudarnost ličnosti brenda
i ličnosti potrošača. Ova podudarnost definiše se kao
veza ovih koncepata i pokazuje koliko se potrošačeva
predstava o sebi poklapa sa imidžom tipičnog korisni-
ka brenda (Escalas & Bettman, 2005). Na kraju, brend
treba da omogući i iskustvene koristi, koje se vezuju za
čulna zadovoljstvo ili kognitivna uzbuđenja. 1.1 Brend S obzirom na navedene koristi, kompanije sve više
pažnje posvećuju dizajniranju brenda. „Brend dizaj-
niran prema zahtevima ciljnog tržišta mora imati sle-
deće atribute: jedinstvenost, posebnost, originalnost i
kompatibilnost sa atributima proizvoda“ (Kocić, Šapić
i Kijevčanin, 2013, p. 220). Zavisno od toga kako je
brend dizajniran, reakcije potrošača mogu da se kre-
ću od veoma niskog nivoa na kom postoji samo svest
o brendu ili upoznatost, pa sve do visokog nivoa koji
podrazumeva lojalnost brendu. Međutim, snagu bren-
da ne treba precenjivati. „Ključ uspeha kompanije je,
pre svega, u izvrsnom proizvodu, a zatim u njegovom
brendu“ (Keller, 2011, p. 38). To praktično znači da
„potrošač neće biti zainteresovan za kupovinu proi-
zvoda ili usluga u slučaju kada su fizičke manjkavosti
prikrivene snažnom promotivnom kampanjom“ (Ko-
cić i Senić, 2010, p. 90). Pored percepcija, koje direktno utiču na vrednost
brenda, veoma je važno razmotriti i ponašanja potro-
šača koja počivaju na percepcijama i tako indirektno
definišu vrednost brenda. Upravo šestodimenzionalni
model (Lehmann, Keller & Farley, 2008) ukazuje na
povezanost dimenzija vrednosti brenda, te predstavlja
jedan od indirektnih metoda za određivanje vrednosti
brenda za potrošače. Model je ustanovljen na osnovu
3 izvora (Lehmann et al., 2008): 1. istraživanja koja su sprovodili pomenuti autori i
Fournier (1998) Jednom ustanovljen brend potrebno je kontinui-
rano pratiti i usavršavati. „Uspešnost brenda upravo
zavisi od sposobnosti kompanije da održava njegovu
vrednost, istovremeno uzimajući u obzir i aktivnosti
konkurenata“ (Kocić et al., 2013, p. 220). 2. istraživanja koje je sproveo Ambler (2003), i 3. metodologije BAV, BrandZ i Research
International’s Equity Engine Na osnovu navedenih izvora autori su došli do 27
stavki vezanih za vrednost brenda, koje su, nakon
analize međuzavisnosti faktora, svrstali u šest grupa:
razumevanje, komparativna prednost marke, među-
ljudski odnosi, istorija, preferencija i vezanost potro-
šača za marku. 1.3. Preferencija brenda Pored podataka o ispitanicima, upitnik je obuhva-
tio 5 grupa pitanja, sa ukupno 27 konstatacija koje
odražavaju različite dimenzije vrednosti brenda. Kon-
statacije su odabrane na osnovu pregleda relevantne
literature, pri čemu je svaka varijabla modela merena
putem minimum 4 konstatacije. Teorijski osnov za iz-
bor konstatacija temelji se na modelu za određivanje
vrednosti brenda za potrošače, koji su kreirali Leh-
mann et al., (2008). Sve konstatacije uvršćene u upit-
nik izabrane su iz pomenutog modela i modifikovane
od strane autora za potrebe sprovođenja ovog istaživa-
nja. Ispitanici su stepen slaganja sa navedenim konsta-
tacijama iskazivali na petostepenoj Likertovoj skali (1
– potpuno nesaglasan/a, 2 – delimično nesaglasan/a, 3
– neutralan/a, 4 – delimično saglasan/a i 5 – potpuno
saglasan/a). Preferenciju brenda moguće je definisati na više nači-
na. S tim u vezi, ovaj pojam može se posmatrati kao
mera u kojoj potrošači favorizuju određeni brend u
odnosu na konkurente, koji se takođe razmatraju
(Hellier, Geursen, Carr & Rickard, 2003). Drugi auto-
ri, preferenciju brenda vezuju za pristrasnost potroša-
ča i njegovu nameru da kupi određeni brend (Cobb-
Walgren, Ruble & Donthu, 1995). Osnovni preduslov
stvaranja preferencija i naklonosti potrošača ka bren-
du, upravo je izgradnja imovine brenda (Myers, 2003). Pojedini autori prave jasnu razliku između stavova i
preferencija (Franzen & Bouwman, 2001), dok neki
drugi stavljaju znak jednakosti između ova dva poj-
ma, smatrajući da pozitivni stavovi doprinose jačanju
preferencija (Jun, Cho & Kwon, 2008). Analiza prikupljenih podataka, izvršena je pomo-
ću statističkog paketa SPSS (The Statistical Package for
the Social Sciences). Od statističkih metoda analize
podataka, primenjene su: deskriptivna statistička ana-
liza, analiza vrednosti koeficijenta Cronbach’s alpha,
korelacija, te prosta i višestruka regresiona analiza. U
prvom koraku je primenjena deskriptivna statistička
analiza na celom uzorku, i izračunata je aritmetička
sredina i standardna devijacija za svaku konstataci-
ju. Na taj način, mereni su stavovi potrošača vezani
za pojedine dimenzije vrednosti brenda i utvrđena je Rasprostranjenost, poverenje i diferenciranost
su osnovne snage brenda (Predović, 2007). „Snažan
brend može da poboljša preferencije potrošača i sklo-
nost ka određenom proizvodu, pa čak i hedonistič-
ko iskustvo sa proizvodom i to van njegovih fizičkih
svojstava“ (Ariely & Norton, 2009; Ariely & Berns,
2010). Upravo iz tih razloga, izgradnja jakog brenda
višestruko je korisna za kompaniju. 1.3. Preferencija brenda 1.2 Vrednost brenda Jedna od najpoznatijih definicija vrednosti brenda je-
ste Akerova definicija. Prema ovoj definiciji, vrednost
brenda predstavlja „set sredstava i obaveza vezanih za
marku, njeno ime i simbol, koji povećavaju ili uma-
njuju vrednost omogućenu proizvodom ili uslugom“
(Christodoulides & Chernatony, 2010, p. 45, 46). Poznavanje osnovnih dimenzija koje čine vrednost
brenda, kao i načina na koji one utiču na formiranje
preferencija potrošača te donošenje odluke o kupovi-
ni, predstavalja prvi korak ka uspešnom upravljanju
ovom vrednom imovinom. 130 Marija Vranješ, Dragoljub Jovičić, Dragana Drinić Vrednovanje brenda iz perspektive potrošača 131 2. METODOLOGIJA ISTRAŽIVANJA Empirijsko istraživanje uticaja dimenzija vrednosti
brenda na preferenciju brenda od strane potrošača,
sprovedeno je metodom ankete. U pitanju je jedan
od najčešće korišćenih metoda u terenskim marke-
tinškim istraživanjima. U istraživanje je uključeno
140 ispitanika. Podaci su prikupljeni u periodu 02.10. - 20.10.2015. godine. Upitnik je koncipiran u on-li-
ne formi i distribuiran ispitanicima putem društvene
mreže Facebook. Tabela 1. Struktura uzorka (n=140)
Demografski profil
Broj
ispitanika
(n)
Procenat
(%)
Pol
Muškarci
61
43,6
Žene
79
56,4
Starost
≤ 25 godina
49
35,0
26 – 35 godina
74
52,9
36 – 45 godina
11
7,9
46 – 65 godina
6
4,3
Stepen stručne spreme
Niža stručna sprema
2
1,4
srednja stručna sprema
50
35,7
viša stručna sprema
9
6,4
visoka stručna sprema
79
56,4
Region
Vojvodina
110
78,6
Beograd
12
8,6
Šumadija i Zapadna Srbija
6
4,3
Južna i Istočna Srbija
12
8,6 Tabela 1. Struktura uzorka (n=140) Kako bi se ispitanicima olakšalo odgovaranje na
postavljena pitanja i dobili što precizniji odgovori,
istraživanje je vršeno na određenoj kategoriji proi-
zvoda – zubne paste. Od ispitanika je traženo da se
opredele za brend zubne paste koji najviše kupuju
tj. preferiraju. Ponuđeni su sledeći odgovori: Colga-
te (Colgate Palmolive), Kolynos (Colgate Palmolive),
Blenda-med (Procter&Gamble), Signal (Unilever),
Aqua Fresh (GlaxoSmithKline), Vademecum (Henkel
– Merima), Denta fresh (Henkel – Merima), ostalo. Na ovaj način, izvršeno je istraživanje bazirano na
percepcijama potrošača vezanim za tačno određen
brend zubne paste koju preferiraju. Napred navedene konstatacije, mogu se grupisati u
5 faktora odnosno varijabli. Prvi faktor „Razumeva-
nje“ obuhvata sledeće konstatacije: homogenost stavova potrošača. U narednom koraku,
pouzdanost dimenzija vrednosti brenda i unutrašnja
konzistentnost konstatacija, mereni su na osnovu
vrednosti koeficijenta alfa (Cronbach’s alpha). Na-
kon toga, sprovedena je korelaciona analiza kojom je
utvrđen stepen zavisnosti između varijabli u modelu. Na kraju, ispitivanje pojedinačnog uticaja dimenzija
vrednosti brenda tj. nezavisnih promenljivih, na pre-
ferenciju potrošača, kao zavisnu promenljivu, utvrđe-
no je prostom regresionom analizom. − Brend „X“ je veoma prisutan u kanalima marke-
tinga i lako ga je pronaći. − O brendu „X“, prisutne su i dostupne brojne infor-
macije iz promocije i drugih izvora. − Brend „X“ je jedna od prvih asocijacija na paste za
zube. − Do detalja sam upoznat sa karakteristikama i
načinom upotrebe brenda X“ Istraživanjem je obuhvaćena i višestruka regresi-
ona analiza putem koje je utvrđena statistička zna-
čajnost zajedničkog uticaja sve četiri identifikovane
komponente vrednosti brenda, na nivo preferencije
brenda. 2. METODOLOGIJA ISTRAŽIVANJA Na osnovu vrednosti VIF-a (Variance inflati-
on factor) ispitan je problem multikolinearnosti. načinom upotrebe brenda „X“. − Koristim brend „X“. Drugi faktor „Komparativna prednost brenda“, uk-
ljučuje sledeće konstatacije: Drugi faktor „Komparativna prednost brenda“, uk-
ljučuje sledeće konstatacije: − Brend „X“ se izdvaja od konkurencije. j
Na osnovu tabele 1 može se zaključiti da su u
uzorku žene (56,4%) zastupljene u većem procentu
od anketiranih muškaraca (43,6%). Ako se posmatra
struktura ispitanika prema starosti, primećuje se da
najmanji broj ispitanika starosti od 46 do 65 godina
(4,3%), dok je najviše ispitanika pripadalo grupi oso-
ba starih između 26 i 35 godina (52,9%). Analizom
uzorka prema stručnoj spremi, dolazi se do zaključka
da najveći broj ispitanika ima visoku stručnu spremu
(56,4%), dok je najmanje onih sa nižom stručnom
spremom (1,4%). Najzad, shodno regionu u kom žive,
najveći broj ispitanika bilo je iz Vojvodine (78,6 %),
dok je najmanje njih bilo iz Šumadije i Zapadne Srbije
(4,3%). Na osnovu tabele 1 može se zaključiti da su u
uzorku žene (56,4%) zastupljene u većem procentu
od anketiranih muškaraca (43,6%). Ako se posmatra
struktura ispitanika prema starosti, primećuje se da
najmanji broj ispitanika starosti od 46 do 65 godina
(4,3%), dok je najviše ispitanika pripadalo grupi oso-
ba starih između 26 i 35 godina (52,9%). Analizom
uzorka prema stručnoj spremi, dolazi se do zaključka
da najveći broj ispitanika ima visoku stručnu spremu
(56,4%), dok je najmanje onih sa nižom stručnom
spremom (1,4%). Najzad, shodno regionu u kom žive,
najveći broj ispitanika bilo je iz Vojvodine (78,6 %),
dok je najmanje njih bilo iz Šumadije i Zapadne Srbije
(4,3%). − Imam visoko mišljenje o brendu „X“. − Brend „X“ ima snažnu reputaciju. − Brend „X“ ispunjava moja očekivanja. − Smatram da ne mogu pogrešiti ako izaberem
brend „X“. − Brend „X“ je prihvaćen i od strane mojih prijate-
lja, rođaka, kolega. Treći faktor „Međuljudski odnosi“, obuhvata slede-
će konstatacije: Treći faktor „Međuljudski odnosi“, obuhvata slede-
će konstatacije: − Smatram da je brend „X“ posvećen interesu po-
trošača. − Smatram da je brend „X“ prestižan. − Smatram da je brend „X“ prestižan. − Ukoliko se javi problem vezan za brend „X“, veru-
jem da će ga kompanija brzo rešiti. − Brend „X“ je inovativan i stalno se unapređuje. 132 Marija Vranješ, Dragoljub Jovičić, Dragana Drinić 3. REZULTATI ISTRAŽIVANJA Četvrti faktor „Istorija“ odnosi se na sledeće kon-
statacije: Primenom deskriptivne statistike izračunate su vred-
nosti aritmetičke sredine i standardne devijacije za
27 definisanih konstatacija. Koristeći ceo uzorak, re-
zultati deskriptivne statističke analize ukazuju da is-
pitanici generalno visoko vrednuju preferirani brend. Najpovoljniji stav ispitanika je iskazan kod tvrdnje
da ispitanik koristi odnosno upotrebljava preferira-
ni brend (aritmetička sredina = 4,46), dok je najne-
povoljniji stav dobijen u slučaju konstatacije da za
preferirani brend ispitanik, odnosno potrošač vezuje
radosna sećanja (aritmetička sredina = 2,63). Najveći
stepen neslaganja među ispitanicima zabeležen je kod
konstatacije vezane za upotrebu preferiranog brenda
od strane roditelja ispitanika (standardna devijacija =
1,395), dok se najmanji stepen neslaganja među ispi-
tanicima vidi kod konstatacije koja se odnosi na ge-
neralno mišljenje o preferiranom brendu (standardna
devijacija = 0,771) (Tabela 2). − Brend „X“ ima snažnu istoriju i dugo je prisutan
na tržištu. − Brend „X“ su koristili i moji roditelji. − Sećam se brenda „X“ još iz svoje mladosti/detinj-
stva. − Za brend „X“ vezujem radosna sećanja. Peti faktor „Preferencija“ sadrži sledeće konstata-
cije: Peti faktor „Preferencija“ sadrži sledeće konstata-
cije: − Brend „X“ je zaslužio moje poverenje. − Lojalan sam brendu „X“. − Spreman sam da platim višu cenu za brend „X“. − Brend „X“ je nezamenljiv i u slučaju da ga nema
na određenom prodajnom mestu, potražiću ga na
drugom. 132 Marija Vranješ, Dragoljub Jovičić, Dragana Drinić Tabela 2. Rezultati deskriptivne statističke analize Tabela 2. Rezultati deskriptivne statističke analize
Konstatacije
M
SD
Brend „X“ je veoma prisutan u kanalima marketinga i lako ga je pronaći. 4,29
1,027
O brendu „X“, prisutne su i dostupne brojne informacije iz promocije i drugih izvora. 3,75
1,080
Brend „X“ je jedna od prvih asocijacija na paste za zube. 4,09
1,035
Do detalja sam upoznat sa karakteristikama i načinom upotrebe brenda „X“. 3,66
1,044
Koristim brend „X“. 4,46
0,876
Brend „X“ se izdvaja od konkurencije. 3,61
0,941
Imam visoko mišljenje o brendu „X“. 3,81
0,905
Brend „X“ ima snažnu reputaciju. 3,92
0,930
Brend „X“ ispunjava moja očekivanja. 4,01
0,831
Smatram da ne mogu pogrešiti ako izaberem brend „X“. 3,84
0,954
Brend „X“ je prihvaćen i od strane mojih prijatelja, rođaka, kolega. 3,52
0,925
Smatram da je brend „X“ posvećen interesu potrošača. 3,64
0,858
Smatram da je brend „X“ prestižan. 3,34
0,950
Ukoliko se javi problem vezan za brend „X“, verujem da će ga kompanija brzo rešiti. 3. REZULTATI ISTRAŽIVANJA 3,71
0,884
Brend „X“ je inovativan i stalno se unapređuje. 3,88
0,971
Brend „X“ ima snažnu istoriju i dugo je prisutan na tržištu. 3,84
0,954
Brend „X“ su koristili i moji roditelji. 2,94
1,395
Sećam se brenda „X“ još iz svoje mladosti/detinjstva. 2,85
1,330
Za brend „X“ vezujem radosna sećanja. 2,63
1,183
Brend „X“ je zaslužio moje poverenje. 3,76
0,944
Lojalan sam brendu „X“. 3,57
1,074
Spreman sam da platim višu cenu za brend „X“. 3,03
1,269
Brend „X“ je nezamenljiv i u slučaju da ga nema na određenom prodajnom mestu,
potražiću ga na drugom. 2,81
1,324
Kada kupujem pastu za zube, brend „X“ je moj prvi izbor. 4,06
0,976
Smatram da kupovinom brenda „X“ dobijam odličnu vrednost za utrošen novac. 3,88
0,869
Spreman sam da kupim i probam i druge proizvode brenda „X“. 3,89
0,968
Generalno, imam pozitivno mišljenje o brendu „X“. 4,16
0,771
M
aritmetička sredina; SD
standardna devijacija SD M – aritmetička sredina; SD – standardna devijacija Rezultati korelacione analize prikazani su u tabeli
4. Vrednosti Pirsonovog koeficijenta pokazuju stepen
zavisnosti između varijabli u modelu.i − Kada kupujem pastu za zube, brend „X“ je moj
prvi izbor. p
− Smatram da kupovinom brenda „X“ dobijam
odličnu vrednost za utrošen novac. Interkorelaciona matrica ukazuje na signifikantne
vrednosti Pirsonovog koeficijenta. Između varijabli
modela, javlja se statistički značajan stepen korelaci-
je. Međutim, ta korelacija nije svuda jaka. Vrednost − Spreman sam da kupim i probam i druge proizvo-
de brenda „X“. − Generalno, imam pozitivno mišljenje o brendu „X“. Na osnovu koeficijenta Cronbach’s alpha, utvrđe-
na je pouzdanost i interna konzistentnost konstatacija
u upitniku. Rezultati su prikazani u tabeli 3. Vred-
nosti koeficijenta Cronbach’s alpha kreću se od 0 do
1, pri čemu se smatra da vrednosti veće od 0,7 uka-
zuju na adekvatnu pouzdanost i internu konzisten-
tnost tvrdnji (Nunnally, 1978). Vrednosti koeficijenta
Cronbach’s alpha u istraživanju kreće se u rasponu od
0,774 (razumevanje) do 0,884 (preferencija). Dobije-
ne vrednosti ukazuju na adekvatnu pouzdanost i in-
ternu konzistentnost varijabli. Vrednovanje brenda iz perspektive potrošača 133
Tabela 3. Pouzdanost varijabli - vrednosti koeficijenta
Cronbach’s alpha
Varijable
Cronbach’s alpha
Razumevanje
0,774
Komparativna prednost brenda
0,857
Međuljudski odnosi
0,783
Istorija
0,799
Preferencija
0,884 Tabela 3. Pouzdanost varijabli - vrednosti koeficijenta
Cronbach’s alpha
Varijable
Cronbach’s alpha
Razumevanje
0,774
Komparativna prednost brenda
0,857
Međuljudski odnosi
0,783
Istorija
0,799
Preferencija
0,884 Tabela 3. 3. REZULTATI ISTRAŽIVANJA Pouzdanost varijabli - vrednosti koeficijenta
Cronbach’s alpha Vrednovanje brenda iz perspektive potrošača 133 Tabela 4. Korelaciona analiza
Varijable
1
2
3
4
5
Razumevanje
1
0,655**
0,616**
0,335**
0,583**
Komparativna prednost brenda
0,655**
1
0,724**
0,257**
0,805**
Međuljudski odnosi
0,616**
0,724**
1
0,277**
0,690**
Istorija
0,335**
0,257**
0,277**
1
0,303**
Preferencija brenda
0,583**
0,805**
0,690**
0,303**
1
Napomena: ** Korelacija je značajna na nivou 0,01 Tabela 4. Korelaciona analiza Tabela 5. Prosta regresiona analiza (Zavisna varijabla: Preferencija brenda)
Varijable
β
t
Sig. R²
F
Razumevanje
0,583
8,423
0,000**
0,340
70,954
Komparativna prednost brenda
0,805
15,931
0,000**
0,648
253,802
Međuljudski odnosi
0,690
11,196
0,000**
0,476
125,342
Istorija
0,303
3,735
0,000**
0,092
13,947
Napomena: p <0.01 (**) Tabela 5. Prosta regresiona analiza (Zavisna varijabla: Preferencija brenda) Pirsonovog koeficijenta preko 0,6 ukazuje na visok
stepen korelacije između varijabli, odnosno na jaku
koreleciju; vrednost 0,4 – 0,6 na umerenu korelaciju,
a ispod 0,4 na slabu korelaciju. Rezultati korelacione
analize pokazuju visok stepen korelacije između ra-
zumevanja i komparativne prednosti brenda (0,655),
razumevanja i međuljudskih odnosa (0,616), kom-
parativne prednosti brenda i međuljudskih odnosa
(0,724), komparativne prednosti brenda i preferen-
cije (0,805) kao i međuljudskih odnosa i preferencije
(0,690). Umeren stepen korelacije, zabeležen je izme-
đu razumevanja i preferencije (0,583), dok je nizak
stepen korelacije prisutan između istorije i razume-
vanja (0,335), istorije i komparativne prednosti bren-
da (0,257), istorije i međuljudskih odnosa (0,277), te
istorije i preferencije (0,303). imaju signifikantan uticaj na preferenciju brenda. Pri
tom, najveći uticaj na preferenciju ima komparativna
prednost brenda: ß = 0,805, t = 15,931, p <0,01; dok
je najmanji uticaj zabeležen kod istorije kao dimenzija
vrednosti brenda: ß = 0,303, t = 3,735, p <0,01. Na kraju, sprovedena je i višestruka regresija ko-
jom je analiziran zajednički uticaj nezavisnih varijabli
na nivo preferencije brenda koja je zavisna varijabla. Rezultati analize prikazani su u tabeli 6. Koeficijent
determinacije (R2) iznosi 0,678 što znači da je 67,8%
varijabiliteta u preferenciji brenda opisano preko če-
tiri nezavisne varijable (razumevanje, komparativna
prednost brenda, međuljudski odnosi i istorija), dok
je ostatak varijabiliteta pod uticajem drugih faktora. Rezultati istraživanja pokazali su da dva faktora ima-
ju statistički značajan uticaj na preferenciju brenda:
komparativna prednost brenda: ß = 0,622, t = 8,117,
p <0,01; i međuljudski odnosi: ß = 0,203, t = 2,757, p
<0,01; pri čemu, komparativna prednost brenda ima
jači uticaj. 3. Christodoulides, G. and Chernatony, L. (2010).
Consumer-based brand equitz conceptualization
and measurement. International Journal of Market
Research, 52 (1), 43-66.
4. Cobb-Walgren, C. J., Ruble, C. A. and Donthu, N.
(1995). Brand equity, brand preference, and purchase
intent. Journal of Advertising, 24 (3), 25–40. 3. REZULTATI ISTRAŽIVANJA Razumevanje i istorija kao dimenzije vred-
nosti brenda, nemaju statistički značajan uticaj na
preferenciju brenda. U sledećem koraku sprovedena je i regresiona
analiza kako bi se utvrdilo koje varijable imaju signi-
fikantan uticaj na preferenciju brenda. U tom cilju,
korišćena je prosta linearna regresija (tabela 5). Testi-
ran je pojedinačni uticaj nezavisnih varijabli na pre-
ferenciju brenda koja je zavisna varijabla. Analiza do-
bijenih rezultata pokazuje da sve nezavisne varijable Tabela 6. Višestruka regresiona analiza (Zavisna varijabla: Preferencija brenda) Tabela 6. Višestruka regresiona analiza (Zavisna varijabla: Preferencija brenda)
Varijable
β
t
Sig. VIF
Razumevanje
0,023
0,337
0,737
1,973
Komparativna prednost brenda
0,622
8,117
0,000**
2,467
Međuljudski odnosi
0,203
2,757
0,007**
2,284
Istorija
0,079
1,524
0,130
1,137
Napomena: p<0.01 (**); R2=0,678 134 Marija Vranješ, Dragoljub Jovičić, Dragana Drinić pravcu treba usmeriti napore. S tim u vezi, kompanije
moraju kontinuirano da rade na izgradnji komparativ-
ne prednosti kroz unapređenje reputacije brenda, pozi-
tivnog mišljenja, te ispunjavanje očekivanja potrošača. Pored toga, neophodno je punu pažnju posvetiti i me-
đuljudskim odnosima koji se ogledaju u inovativnosti,
prestižu i posvećenosti potrošačima. Ovaj rezultat je
u skladu sa stavovima autora Helgeson & Supphellen,
(2004.) prema kojima brend treba da omogući funk-
cionalne, iskustvene ali i simboličke koristi potrošaču. S druge strane, razumevanje i istorija nisu statistički
značajne za nivo preferencije brenda. Međutim, ni ove
dimenzije ne treba zanemariti, jer testiranjem poje-
dinačnog uticaja ovih varijabli, potvrđena je njihova
signifikantnost za nivo preferencije brenda. Na ovaj
način, menadžmentu je omogućeno da razume na-
čin izgradnje vrednosti brenda, da formira adekvatan
okvir za upravljanje brendom što bi u krajnjoj liniji
trebalo da dovede do rasta nivoa preferencije brenda i
poboljšanja konkurentske pozicije kompanije. Pre sprovođenja višestruke regresione analize,
utvrđen je faktor povećanja varijanse (VIF) kako bi se
utvrdila eventualna multikolinearnost. Problem mul-
tikolinearnosti se neće javiti, ukoliko vrednosti VIF
koeficijenta budu manje od 5 (Field, 2000). S obzirom
da su vrednosti navedenog koeficijenta u ovom istra-
živanju bile adekvatne i kretale se u rasponu 1,137-
2,467, može se zaključiti da je bilo opravdano sprove-
sti regresionu analizu. 1. Ariely, D. and Berns, G. S. (2010). Neuromarketing:
the hope and hype of neuroimaging in business.
Nature reviews Neuroscience, 11 (4), 284–292.
2. Ariely, D. and Norton, M. I. (2009). Conceptual
consumption. Annual Review of Psychology, 60 (1),
475–499. ZAKLJUČAK Poslovno okruženje u kom funkcionišu savremene
kompanije, postalo je izuzetno kompleksno. S jedne
strane, kompanije se suočavaju sa sve većim troškovi-
ma i sve intenzivnijom konkurencijom. S druge stra-
ne, potrošači postaju sve zahtevniji, dok je broj alter-
nativa koje im se nude sve veći. U takvim uslovima,
kada je neophodno razviti lojalnost, koncept brenda
dobija sve veći značaj. Samim tim, brendiranje sve
manje postaje stvar izbora i nameće se kao neminov-
nost. S ozirom na navedenu činjenicu, cilj ovog rada
je bio da utvrdi uticaj određenih dimenzija vrednosti
brenda na unapređenje nivoa preferencije brenda od
strane potrošača. Pored praktičnog doprinosa, sprovedeno istraživanje
pruža i doprinos postojećoj literaturi s obzirom na to
da identifikuje najznačajnije faktore koji utiču na nivo
preferencije brenda. Radovi u domaćoj literaturi bave se
merenjem vrednosti brenda, ali isključivo sa teorijskog
stanovišta u smislu objašnjavanja modela za merenje
performansi, ali bez empirijske analize i potvrde. Rezultati istraživanja primenom proste regresione
analize navode na zaključak da sve dimenzije vred-
nosti brenda imaju statistički signifikantan uticaj na
povećanje nivoa preferencije brenda, a to su: razume-
vanje, komparativna prednost brenda, međuljudski
odnosi i istorija. Međutim, primena višestruke re-
gresione analize, pokazuje da u slučaju zajedničkog
delovanja sve četiri nezavisne varijable, dve varijable:
međuljudski odnosi i komparativna prednost brenda
imaju statistički značajan uticaj na nivo preferencije, s
tim da komparativna prednost brenda ima jači uticaj. S druge strane, preostale dve dimenzije: razumevanje
i istorija nemaju signifikantan uticaj. Pored doprinosa, ovo istraživanje sa sobom nosi i
neka ograničenja koja bi se mogla prevazići u budu-
ćim istraživanjima. Jedno od ograničenja odnosi se na
veličinu uzorka. S tim u vezi, u narednom istraživanju
poželjno bi bilo da uzorak obuhvati veći broj ispitani-
ka. Pored toga, korisno bi bilo analizom obuhvatiti i
neke nove konstatacije kako bi se dobila sveobuhvat-
nija analiza. Takođe, buduća istraživanja bi mogla da
ispitaju dalji uticaj preferencije brenda, na nivo veza-
nosti potrošača za brend posmatranu kroz doslednost
potrošača pri korišćenju brenda, bez obzira na povre-
meni pad kvaliteta, kao i aktivnost potrošača vezane za
traganje za dodatnim informacijama o brendu i raz-
menjivanje iskustava sa prijateljima. Najzad, dimenzije
vrednosti brenda bi trebalo kontinuirano inovirati i
adaptirati u skladu sa novonastalim okolnostima. i
Sprovedeno istraživanje pruža korisne informa-
cije menadžmentu kompanija. Rezultati pokazuju da
međuljudski odnosi i komparativna prednost imaju
snažan uticaj na nivo preferencije. Pored toga, jačina
uticaja statistički značajnih dimenzija, sugeriše u kom Reference: 2. Ariely, D. and Norton, M. I. (2009). Conceptual
consumption. Annual Review of Psychology, 60 (1),
475–499. 1. Ariely, D. and Berns, G. S. (2010). Neuromarketing:
the hope and hype of neuroimaging in business.
Nature reviews Neuroscience, 11 (4), 284–292. Reference: Vrednovanje brenda iz perspektive potrošača 135 13. Kocić, M. i Senić, V. (2010). Ponuda kao kreativna
osnova direktnog marketinga. Marketing, 41 (2), 87-96. 5. Escalas, J. E. and Bettman, J. R. (2005). Self-Construal,
Reference Groups, and Brand Meaning. Journal of
Consumer Research, 32 (3), 378–389. g
g
g
14. Kocić, M., Šapić, S. i Kijevčanin V. (2013). Strategic
implications of Internet marketing plan, Proceedings
of the 2nd International scientistic conference:
Contemporary issues in economics, business and
management (p. 217-226.), Kragujevac: Ekonomski
fakultet, Univerzitet u Kragujevcu. 6. Field, A. (2000). Discovering statistics using SPSS for
Windows. Thousand Oaks, USA: Sage Publication.h h
g
7. Franzen, G. and Bouwman, M. (2001). The Mental
World of Brands: Mind, Memory and Brand Success. Oxfordshire: World Advertising Research Center. g
8. Helgeson, J. and Supphellen, M. (2004). A conceptual
and measurement comparison of self-congruity and
brand personality. The impact of socially desirable
responding. International Journal of Market Research,
46 (2), 205-233. 15. Kotler, P. and Keller, K. (2006). Marketing
Menadžment (12th edition). Beograd: Data status. g
16. Lehmann, D. R., Keller, K. L. and Farley, J. U. (2008). The Structure of Survey-Based Brand Metrics. Journal
of International Marketing, 16 (4), 29–56. 9. Hellier, P. K., Geursen, G. M., Carr, R. A. and Rickard,
J. A. (2003). Customer repurchase intention: A
general structural equation model. European Journal
of Marketing, 37 (11/12), 1762–1800.h 17. Myers, C. A. (2003). Managing brand equity: A look
at the impact of attributes. Journal of Product and
Brand Management, 12 (1), 39–51. g
18. Nunnally, J. C. (1978). Introduction to Psychological
Measurement. New York: McGraw-Hill. f
g
10. Jun, J. W., Cho, C. and Kwon, H. J. (2008). The Role
of Affect and Cognition in Consumer Evaluations
of Corporate Visual Identity: Perspectives from
the United States and Korea. Journal of Brand
Management, 15 (6), 382-398. 19. Predović, D. (2007). Vrednovanje marke, Od tržišne
vrednosti marke bazirane na potrošaču do finansijskog
vrednovanja. Zagreb: Mate. j
g
20. Veljković, S. i Đorđević, A. (2010). Vrednost brenda
za potrošače i preduzeća. Marketing, 41 (1), 3-16. g
11. Keegan, J. W. and Green, C. M. (2008). Global
Marketing. London: Pearson Education Ltd. g
21. Zeugner-Roth, K. P., Diamantopoulos, A. and
Montesinos, A. (2008). Home country image, country
brand equity and consumers product preferences: An
empirical study. Management International Review, 48
(5), 577-602. 12. Keller, L. K. (2011). How to Navigate the Future of
Brand Management. Reference: Marketing Management, 20 (2),
36-43. Evaluation of Brand from the
Perspective of Consumers Marija Vranješ, Dragoljub Jovičić, Dragana Drinić Brand is extremely important intellectual property of a
company. In order to attract new customers and keep as
many existing ones, companies have to work continuously
to improve the brand value, which ultimately leads to the
market share, revenues and profits growth. the level of preference - interpersonal relations and compara-
tive advantage of the brand. On the other hand, understand-
ing of brand and its history, doesn’t have statistically sig-
nificant effect. Results of these conducted studies may help
company’s management when formulating the appropriate
management’s framework of the brand in order to increase
brand preference, which ultimately may lead to improving
the competitive position of the company. i
The aim is to explore how certain aspect of brand value
influences the preference of a brand. In order to fulfill these
objectives, certain types of descriptive statistics measures
were used, together with reliability analysis, correlation and
regression analysis. The results show that statistically there
are two dimensions of brand value which effect increase of Keywords: dimensions of brand value, brand value mea-
surement, brand preference 136 Marija Vranješ, Dragoljub Jovičić, Dragana Drinić Kontakt: M. Sc. Marija Vranješ, asistent
vranjesmarija.vps@gmail.com
dr Dragoljub Jovičić, professor strukovnih studija
djovicic@uns.ac.rs
M.Sc. Dragana Drinić, asistent
dragana.vps@gmail.com
Visoka poslovna škola strukovnih studija u Novom Sadu,
Vladimira Perića Valtera 4, Novi Sad M. Sc. Marija Vranješ, asistent
vranjesmarija.vps@gmail.com
dr Dragoljub Jovičić, professor strukovnih studija
djovicic@uns.ac.rs
M.Sc. Dragana Drinić, asistent
dragana.vps@gmail.com
Visoka poslovna škola strukovnih studija u Novom Sadu,
Vladimira Perića Valtera 4, Novi Sad M. Sc. Marija Vranješ, asistent
vranjesmarija.vps@gmail.com
dr Dragoljub Jovičić, professor strukovnih studija
djovicic@uns.ac.rs M.Sc. Dragana Drinić, asistent
dragana.vps@gmail.com Visoka poslovna škola strukovnih studija u Novom Sadu,
Vladimira Perića Valtera 4, Novi Sad 136 Marija Vranješ, Dragoljub Jovičić, Dragana Drinić
|
https://openalex.org/W3015071352
|
https://europepmc.org/articles/pmc7145889?pdf=render
|
English
| null |
The Paneth Cell: The Curator and Defender of the Immature Small Intestine
|
Frontiers in immunology
| 2,020
|
cc-by
| 10,989
|
The Paneth Cell: The Curator and
Defender of the Immature Small
Intestine Shiloh R. Lueschow 1† and Steven J. McElroy 1,2*† 1 Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, United States, 2 Stead Family Department of
Pediatrics, University of Iowa, Iowa City, IA, United States Paneth cells were first described in the late 19th century by Gustav Schwalbe and
Josef Paneth as columnar epithelial cells possessing prominent eosinophilic granules
in their cytoplasm. Decades later there is continued interest in Paneth cells as they play
an integral role in maintaining intestinal homeostasis and modulating the physiology of
the small intestine and its associated microbial flora. Paneth cells are highly specialized
secretory epithelial cells located in the small intestinal crypts of Lieberkühn. The dense
granules produced by Paneth cells contain an abundance of antimicrobial peptides
and immunomodulating proteins that function to regulate the composition of the
intestinal flora. This in turn plays a significant role in secondary regulation of the host
microvasculature, the normal injury and repair mechanisms of the intestinal epithelial
layer, and the levels of intestinal inflammation. These critical functions may have even
more importance in the immature intestine of premature infants. While Paneth cells begin
to develop in the middle of human gestation, they do not become immune competent
or reach their adult density until closer to term gestation. This leaves preterm infants
deficient in normal Paneth cell biology during the greatest window of susceptibility to
develop intestinal pathology such as necrotizing enterocolitis (NEC). As 10% of infants
worldwide are currently born prematurely, there is a significant population of infants
contending with an inadequate cohort of Paneth cells. Infants who have developed NEC
have decreased Paneth cell numbers compared to age-matched controls, and ablation
of murine Paneth cells results in a NEC-like phenotype suggesting again that Paneth cell
function is critical to homeostasis to the immature intestine. This review will provide an up
to date and comprehensive look at Paneth cell ontogeny, the impact Paneth cells have
on the host-microbial axis in the immature intestine, and the repercussions of Paneth cell
dysfunction or loss on injury and repair mechanisms in the immature gut. †ORCID: Shiloh R. Lueschow
orcid.org/0000-0001-6185-7612
Steven J. McElroy
orcid.org/0000-0002-4321-723X Specialty section:
This article was submitted to
Mucosal Immunity,
a section of the journal
Frontiers in Immunology Received: 17 December 2019
Accepted: 13 March 2020
Published: 03 April 2020 Keywords: paneth cell, necrotizing enterocolitis, immature intestine, defensins, cathelicidin (LL37), cell death Edited by:
Tobias Strunk,
King Edward Memorial
Hospital, Australia Reviewed by:
Claudio Nicoletti,
University of Florence, Italy
Luisa Cervantes-Barragan,
Emory University, United States *Correspondence:
Steven J. McElroy
steven-mcelroy@uiowa.edu REVIEW
published: 03 April 2020
doi: 10.3389/fimmu.2020.00587 REVIEW published: 03 April 2020
doi: 10.3389/fimmu.2020.00587 THE ANATOMY OF THE PANETH CELL The human gastrointestinal surface is the largest surface area
of the body that is in contact with the external environment
(7, 8). This massive surface area is required to allow sufficient
nutrient absorption to support growth and health of the
host. The small intestine, where Paneth cells reside, has an
estimated surface area of 950 cm2 at birth, which grows and
expands to over 30 m2 by adulthood (7, 8). To achieve such a
massive surface area, the intestinal surface is clad by fingerlike
projections that stick out into the intestinal lumen creating
an expansive folding system. This systems’ entire surface is
covered by a single layer of columnar intestinal epithelial cells
(IECs). The intestinal epithelium is the most rapidly-renewing
tissue in the adult mammal (9) and undergoes continuous
turnover that is generated from Intestinal Stem cells (ISC). The ISC reside at or near the base of the pocket-like intestinal
crypts (10, 11) and continuously generate daughter cells
that differentiate near the top of the crypts before migrating
toward their final destinations. The differentiated cell types
are generally grouped by their function as belonging to either
the absorptive (enterocytes), or secretory (mucus-secreting
goblet,
antimicrobial-secreting
Paneth,
hormone-secreting
enteroendocrine cells, and chemosensing/immunomodulatory
cytokine-secreting tuft cells) lineage, with clear markers (e.g.,
hes1 expression of absorptive and sox9 for secretory) defining y
Paneth cells, like all other intestinal epithelial cell types,
are derived from ISCs. In the last decade, it has become
clear that ISCs are quite complex. Current models suggest
multiple, potentially interconvertible populations of stem cells
exist. The first is the crypt-base columnar (CBC) cells (20),
slender cells wedged at the very base of the crypt between the
Paneth cells. CBC cells carry the specific marker LGR5 and are
actively proliferating (21, 22). The second ISC population express
Bmi1, mTert, and Lrig1 markers, and have been hypothesized
to be quiescent stem cells until injury occurs, at which time
they actively proliferate and produce daughter progeny (23). Interconversion between the two compartments and overlap
between the populations has been demonstrated (24). Under
normal conditions, the LGR5+ ISCs proliferate to generate
daughter cells that move out of the crypt. These cells become
differentiated as they migrate, and both their differentiation
and the maintenance of the stem cells in their proper place is
driven through gradients and juxtracrine signaling of Bmp, Wnt,
Notch, and growth factor pathways (25, 26). Citation: Lueschow SR and McElroy SJ (2020)
The Paneth Cell: The Curator and
Defender of the Immature Small
Intestine. Front. Immunol. 11:587. doi: 10.3389/fimmu.2020.00587 In the small intestine, intestinal epithelial cells form an important physical and biochemical barrier
that prevents the microbial communities contained within the lumen from accessing the rest of
the body and causing infection (1). One particular type of intestinal epithelial cell, the Paneth
cell, was first discovered by Gustav Schwalbe in the late 19th century based on the eosinophilic April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org Paneth Cell Lueschow and McElroy granules evident in their cytoplasm. A few years later, Paneth cells
were described in depth by their namesake, Joseph Paneth (2, 3). They are now well-recognized as pyramidal shaped, columnar,
secretory cells situated at the base of the crypts of Lieberkühn,
which are small depressions in the mucosal surface along the
small intestine (4). While Paneth cells have occasionally also
been found patchily dispersed in the stomach and colon, this is
generally associated with mucosal inflammation as opposed to
homeostasis (4). commitment to one or the other arm (12). The typical pattern
for these cells is to migrate upwards toward the villus tip in a
conveyor-belt-type fashion until they are sloughed offthe upper
villus into the lumen. However, a unique aspect of Paneth cell
biology compared to the other intestinal epithelial cell types is
that instead of flowing upward out of the crypt, Paneth cells move
downwards further into the crypt as they mature. In addition,
while most epithelial cells are rapidly turned over in a few days,
Paneth cells can persist for just under 1 month (13). Paneth
cell presence is an intestinal priority and their density is rapidly
repopulated following their depletion (14). Following their
descent into the crypts, Paneth cells are interspersed between the
ISCs and can be distinguished by their columnar to pyramidal
shape and by the presence of eosinophilic granules within their
cytoplasm (Figure 1). Although Paneth cells were first discovered and described in
humans, they are not specific to humans. Paneth cells can be
found in many other vertebrates including primates, rodents,
horses, sheep, certain fish, and chickens (5, 6). Citation: While Paneth
cells have been found in this wide variety of other organisms
aside from humans, the ontogeny and function are not well-
understood for most of them aside from the well-studied and
characterized rodents as well as humans. Today, Paneth cells still
capture the attention of researchers as they serve an essential
role in modulating the microbiome, playing a key part of the
innate immune response, and aiding in the proliferation and
differentiation of the intestinal epithelium. While Paneth cells
have been shown to play important roles in the healthy gut
of adults, the development and role of Paneth cells in the
immature gut of the preterm infant remains an understudied, but
crucial avenue of research that could aid in the understanding
of the development of intestinal diseases such as necrotizing
enterocolitis (NEC). This review sets out to unveil some of the
mystery surrounding Paneth cells in the context of the preterm
infant gut and how it relates to NEC. PANETH CELL ONTOGENY AND
DIFFERENTIATION Paneth cells first appear in the small intestine of humans at
13.5 weeks gestational age (15, 16). Paneth cell density in the
developing fetal intestine is relatively low, but gradually increases
throughout gestation, with significant increases in the third
trimester after 29 weeks completed gestation (17, 18). Paneth cell
levels do not reach quantities similar to adult levels until term
gestation or later (17). Because Paneth cells are located primarily
in the distal small intestine, studies using human tissues have
been challenging. Thus, much of our understanding of in vivo
Paneth cell biology has been generated using animal models,
predominantly in mice. It is therefore important to note that
not all mammals develop Paneth cells prenatally, but instead
develop them mid-way through intestinal development after
villus development, but before intestinal maturity according to a
normal developmental pattern. For example, the commonly used
C57Bl/6 mouse strain does not develop Paneth cells until 7–10
days after birth (18, 19). Frontiers in Immunology | www.frontiersin.org THE ANATOMY OF THE PANETH CELL Furthermore, while
the exact sources of ligands for these pathways are not fully April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 2 Paneth Cell Lueschow and McElroy FIGURE 1 | The intestinal epithelium. (Left) H&E stained ileum from P14 C57Bl6 mouse with vilus, crypt, and lamina propria labeled. (Right) Schematic of the intestinal
epithelium, associated microbial flora, epithelial cell types (goblet cells, Paneth cells, enterocytes, and stem cells) intestinal microvasculature, and mucus layer. Corresponding labels for vilus, crypt, and lamina propria labeled are placed on the schematic to compare to the H&E stained section. FIGURE 1 | The intestinal epithelium. (Left) H&E stained ileum from P14 C57Bl6 mouse with vilus, crypt, and lamina propria labeled. (Right) Schematic of the intestinal
epithelium, associated microbial flora, epithelial cell types (goblet cells, Paneth cells, enterocytes, and stem cells) intestinal microvasculature, and mucus layer. Corresponding labels for vilus, crypt, and lamina propria labeled are placed on the schematic to compare to the H&E stained section. C57Bl/6 mice normally develop Paneth cells by day 10 of life
(19). It is however important to note that modifications to
Atoh1 signal pathways also affect goblet cell differentiation (36),
so understanding of signal pathways that distinguish goblet
cell from Paneth cell differentiation downstream of Atoh1 is
still incomplete. understood, it is important to note that Paneth cells produce EGF,
Notch, and Wnt, which in turn promote stem cell proliferation
and maintenance (27). In fact, Paneth cells can support LGR5+
cell growth and survival in vitro, and have been proposed as a key
nurse cell for the actively dividing stem population (27). Several biochemical pathways have been implicated in the
development of Paneth cells (Figure 2). Naïve daughter cells
are driven to either an absorptive enterocyte phenotype by
Notch signaling, or to a secretory phenotype through Wnt
signal pathways. The Wnt/β-catenin pathway is an important
stimulator of Paneth cell differentiation (28, 29). However,
the Wnt signal pathway and its relationship to Paneth cell
development is complex and still not completely elucidated. Genetic knockout of LGR-5, a downstream target of Wnt
signaling has been shown to produce precocious Paneth cell
differentiation in fetal intestine (29, 30). This contradictory data
may be due to alterations in negative feedback mediators in the
Wnt pathway. THE ANATOMY OF THE PANETH CELL Following differentiation into a secretory lineage,
activation of the transcription factors Atoh1 (also known as
Math1) induces differentiation into a combined goblet/Paneth
cell precursor cell lineage (31–35), while genetic ablation of
Atoh1 in transgenic mice has been shown to result in loss
of Paneth cell lineages (35, 36). Atoh1 has also been shown
to be affected by ErbB3, a Receptor Tyrosine Kinase also
known as neuregulin (37). Genetic loss of ErbB3 in mice results
in unchecked activity of the transcription factor Atoh1 and
induces precocious appearance of Paneth cells (37). In addition,
activation of ErbB3 can delay normal Paneth cell development. Frontiers in Immunology | www.frontiersin.org PANETH CELL ROLE IN THE SMALL
INTESTINE After their migration to the crypt base and subsequent
maturation, Paneth cells can be easily distinguished by their
prominent acidophilic granules. The granules hold many of
the proteins and peptides that Paneth cells secrete to both
modulate the microbiome and mediate the inflammatory
response. These include: α-defensins (cryptdins in mice),
lysozyme, secretory phospholipase A2 (sPLa2), TNF, RegIII,
angiogenin-4, MMP-7, CD15, CD95 ligand, xanthine oxidase,
IgA, CRIP, metallothionine, adipokines, serum amyloid A, α-
1-antitrypsin IL-17A, IL-1β and lipokines (3, 38, 39). These
granular components are assembled and packaged by an
extensive endoplasmic reticulum (ER) and Golgi apparatus
network into dense core granules. (13, 39–43) It is important
to note that it is possible that some components of the
granules may be produced elsewhere before being collected
and added to the granules. IgA is one such component which
may be produced by plasma cells in the lamina propria before April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 3 Paneth Cell Lueschow and McElroy communicates with the external environment (7, 52), protection
of the host from injury or bacterial invasion from the intestinal
flora (53) requires a complex system of defense mechanisms. In
the small intestine, a key component of host defense is epithelial
derived antimicrobial peptides (AMPs). AMPs are small peptides
generally >5 kDa in length, cationic at a neutral pH, and have
broad spectrum microbicidal activities at low concentrations
(45). These peptides are the main product contained in Paneth
cell granules. FIGURE 2 | Intestinal epithelial cell differentiation pathways. The intestinal
stem cell (ISC) differentiates into absorptive (enterocyte) or secretory
precursors through Wnt/Notch signaling. While enterocytes further differentiate
through HES-1 signaling, secretory lineages can differentiate into different cell
types depending on conditions. Wnt signal pathways drive ISC differentiation
into secretory precursor cells. Secretory precursors then develop either into
enteroendocrine cells through Neurog3 signaling, or into goblet and Paneth
cells following activation of Atoh1. Differentiation signal pathways to separate
development of goblet cells and Paneth cells are still unknown. It is also
important to note that recent data has shown that activation of ErbB3 acts as
a suppressor of Atoh1, while genetic deletion of ErbB3 induces precocious
development of Paneth cells. In humans, there are two major classes of AMPs: cathelicidins
and defensins. Cathelicidins are antimicrobial peptides with
broad antibacterial (54), anti-fungal (55), and anti-viral activity
(56), and are characterized by a highly conserved N-terminal
domain. PANETH CELL ROLE IN THE SMALL
INTESTINE Only after cleavage of the AMP does the protein exert
its myriad activities (57). Humans express only one cathelicidin,
LL-37 (originally hCAP-18) (58) and it is expressed in various
cells of the body including those of the intestinal epithelium
(59–62). However, in the small intestine, cathelicidin expression
is restricted to the neonatal period (63, 64) before markedly
decreasing and disappearing. The timing of this decrease is
important as it coincides with the appearance of Paneth cells
(19, 65) and the onset of expression of Paneth cell AMPs such
as α-Defensins (18). This “switch” from one AMP to another
occurs at roughly the mid-point of development of the small
intestine (66). It is important to note that mid-development of
the small intestine is also around the time when NEC often occurs
in infants born extremely prematurely (67) (Figure 3). FIGURE 2 | Intestinal epithelial cell differentiation pathways. The intestinal
stem cell (ISC) differentiates into absorptive (enterocyte) or secretory
precursors through Wnt/Notch signaling. While enterocytes further differentiate
through HES-1 signaling, secretory lineages can differentiate into different cell
types depending on conditions. Wnt signal pathways drive ISC differentiation
into secretory precursor cells. Secretory precursors then develop either into
enteroendocrine cells through Neurog3 signaling, or into goblet and Paneth
cells following activation of Atoh1. Differentiation signal pathways to separate
development of goblet cells and Paneth cells are still unknown. It is also
important to note that recent data has shown that activation of ErbB3 acts as
a suppressor of Atoh1, while genetic deletion of ErbB3 induces precocious
development of Paneth cells. FIGURE 2 | Intestinal epithelial cell differentiation pathways. The intestinal
stem cell (ISC) differentiates into absorptive (enterocyte) or secretory
precursors through Wnt/Notch signaling. While enterocytes further differentiate
through HES-1 signaling, secretory lineages can differentiate into different cell
types depending on conditions. Wnt signal pathways drive ISC differentiation
into secretory precursor cells. Secretory precursors then develop either into
enteroendocrine cells through Neurog3 signaling, or into goblet and Paneth
cells following activation of Atoh1. Differentiation signal pathways to separate
development of goblet cells and Paneth cells are still unknown. It is also
important to note that recent data has shown that activation of ErbB3 acts as
a suppressor of Atoh1, while genetic deletion of ErbB3 induces precocious
development of Paneth cells. The second class of AMP found in the small intestine are
defensins. PANETH CELL ROLE IN THE SMALL
INTESTINE Defensins are abundant in human cells and tissues
that are involved in host defense and have two main subtypes:
α-defensins, which are found in granule containing cells such as
neutrophils and Paneth cells (also known as cryptdins in mice),
and β-defensins which are produced by epithelial cells (68–71). Human Paneth cells produce two main α-defensins known as
HD-5 and HD-6 (72). In mice, loss of matrilysin (the proteolytic
enzyme needed to activate cryptdins) have altered microbiomes
and are more susceptible to Salmonella infections (73–75). In
addition, mice that have been genetically modified to express
HD-5 have enhanced resistance to bacterial invasion (74). accumulating and associating in Paneth cell granules (44). Since
Paneth cells are not currently able to be cultured without
other epithelial and stem cells, most of the data we have on
granular contents is from immunohistochemistry techniques. The granules are then released at the apical surface of the cell into
the lumen of the intestine where they serve a variety of biological
functions, primarily as microbiocidal agents against bacteria,
fungi, spirochetes, protozoa, and enveloped viruses (45). Paneth
cell granules are secreted both constitutively and in response to
pathogenic exposure, with common stimuli including cholinergic
stimulation and exposure to bacterial antigens (45–47). This
secretion of Paneth cell granular components is under tight
regulatory control, as these mediators are vital for maintenance
of intestinal homeostasis (38, 48, 49). AMPs work by inserting themselves into the bacterial
membrane and forming pores, which result in the leakage of
bacterial cytoplasmic content (76–78). They can also degenerate
bacterial cytoplasmic structures and form extracellular net-like
structures, which result in bacterial trapping (79). In animal
models, AMPs have been shown to preferentially target non-
commensal bacteria while sparing commensal normal flora (47,
80). In addition to killing pathogens, AMPs can also influence
the immune system through white blood cell chemotaxis
(81), activation of dendritic cells (82), and downregulation of
immunomodulators such as cortisol (68, 71). Frontiers in Immunology | www.frontiersin.org PANETH CELLS AND MECHANISMS OF
CELLULAR DEATH Paneth cell health remains a critical priority to the homeostasis
of the small intestine. We and others have shown that
following dithizone-induced loss, the small intestine replenishes
Paneth cell populations within 72 h (14, 50, 51). Since the
mammalian intestinal tract represents the largest surface area that Cells of the body undergo death for a multitude of reasons
and through various mechanisms. The mechanisms of cell April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 4 Paneth Cell Lueschow and McElroy FIGURE 3 | Small intestinal AMP switch during intestinal development. During development, the immature intestine is protected by the AMP CRAMP (LL-37 in
humans). However, CRAMP expression decreases around mid-development, at roughly the time that Paneth cells begin to develop. This “switch” occurs during
mid-intestinal development which is around post-natal (P) day 10–21 in C57Bl6 mice and in the second trimester (between 20 and 28 weeks of gestation) in humans. In infants born prematurely, this switch is temporally similar to when extremely preterm infants are most susceptible to develop NEC (18). FIGURE 3 | Small intestinal AMP switch during intestinal development. During development, the immature intestine is protected by the AMP CRAMP (LL-37 in
humans). However, CRAMP expression decreases around mid-development, at roughly the time that Paneth cells begin to develop. This “switch” occurs during
mid-intestinal development which is around post-natal (P) day 10–21 in C57Bl6 mice and in the second trimester (between 20 and 28 weeks of gestation) in humans
In infants born prematurely, this switch is temporally similar to when extremely preterm infants are most susceptible to develop NEC (18). has been attached to the cryptdin-2 promoter of Paneth cells
(14, 65). When these mice are exposed to diphtheria toxin,
all Paneth cells expressing the construct are lysed through
apoptotic pathways (90). death include apoptosis, necrosis, necroptosis, pyroptosis, and
autophagy. While NEC is defined by necrosis of the intestinal
tissue, many of these different cellular death pathways have
been implicated in the pathogenesis of NEC. Importantly,
several of these pathways are also mechanistically tied to Paneth
cell biology. Another form of cell death directly related to Paneth cells is
autophagy, which is a self-degradative process thought to help
remove cells with misfolded or aggregated proteins or other
intracellular damage (91). Autophagy is characterized by creation
of an intracellular vacuole known as the autophagosome (83). PANETH CELLS AND MECHANISMS OF
CELLULAR DEATH The autophagosome is formed around damaged intracellular
organelles or other selected targets. The autophagosome is
then fused with a lysosome allowing for degradation of the
components within the autophagosome followed by chromatin
condensation (83). The morphologic changes that occur tend to
be relatively well-regulated similar to the degree of regulation of
apoptosis. Also similar to apoptosis, because the degradation of
the dying cell takes place within another cell, this process tends
to prevent inflammation (83). Autophagy is also an important
process for Paneth cells. Because Paneth cells tend to live longer
than most other cells of the gut and have many aggregated
proteins that could be recycled by other neighboring cells, as
damage and stressors to the cells occur, autophagy becomes
activated (92). When mutations occur in the autophagy pathway
such as in Atg16l1, Paneth cells can become dysfunctional
and ultimately trigger intestinal inflammation, which can have
implications for gut health such is suggested to be the case
with Crohn’s disease (92) and NEC (93). Our laboratory has
also shown that autophagy may play a role in development of Apoptosis is a normal part of intestinal health that results
in disassembly of the cell and, in general, tends to avoid
causing inflammation (83). During apoptosis, cells tend to retract
pseudopods, condense chromatin (pyknosis), undergo nuclear
fragmentation and then experience blebbing of the plasma
membrane (84). This contrasts with cellular necrosis where
cells experience organelle swelling, extensive vacuole formation,
condensation of nuclei, and release of inflammatory cytokines in
a passive or accidental manner (83, 84). One type of apoptosis
seen in the intestinal epithelial layer is when the epithelial
cells move upward from the crypt toward the tip of the villus. Once they reach the tip, cells are sloughed into the intestinal
lumen in a process called anoikis, which is a form of apoptosis
(84). There is evidence to show that apoptosis is also involved
in the cell death experienced by cells in the stem cell region
within the small intestinal crypts although the regulation of
the process is not well-understood (84). Apoptosis has been
shown by multiple investigators to be important in development
of NEC (85–89). Frontiers in Immunology | www.frontiersin.org Frontiers in Immunology | www.frontiersin.org PANETH CELLS AND MECHANISMS OF
CELLULAR DEATH Additionally, apoptosis is directly relevant to
Paneth cell biology and NEC as our lab has shown that NEC-
like injury can be induced in mice by delivering diphtheria
toxin to PC-DTR mice where a human diphtheria toxin receptor April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 5 Paneth Cell Lueschow and McElroy FIGURE 4 | Proposed role of the Paneth cell in development of NEC. As the immature intestine (A) is exposed to inflammation (B), oxygen radicals are produced
creating a selective advantage for Proteobacteria sp. over obligate anaerobes such as the Firmicutes. This creates a feedback loop for sustaining and increasing the
pro-inflammatory state in the immature intestine. Previous work from our lab has shown that intestinal inflammation can reduce intestinal mucus production and cause
loss of Paneth cells (112, 129). (C) Loss of these important chemical and physical aspects of innate immunity allows bacteria to move from the mucus layer of the
intestinal lumen and gain closer proximity to the epithelial surface, (D) followed eventually by attachment and invasion of the epithelium. (E) Once bacteria invade the
intestinal tissue, further inflammation occurs including recruitment of leukocytes including neutrophils, macrophages, and monocytes (133–135) which lead to eventual FIGURE 4 | Proposed role of the Paneth cell in development of NEC. As the immature intestine (A) is exposed to inflammation (B), oxygen radicals are produced
creating a selective advantage for Proteobacteria sp. over obligate anaerobes such as the Firmicutes. This creates a feedback loop for sustaining and increasing the
pro-inflammatory state in the immature intestine. Previous work from our lab has shown that intestinal inflammation can reduce intestinal mucus production and cause
loss of Paneth cells (112, 129). (C) Loss of these important chemical and physical aspects of innate immunity allows bacteria to move from the mucus layer of the
intestinal lumen and gain closer proximity to the epithelial surface, (D) followed eventually by attachment and invasion of the epithelium. (E) Once bacteria invade the
intestinal tissue, further inflammation occurs including recruitment of leukocytes including neutrophils, macrophages, and monocytes (133–135) which lead to eventual
death of the tissues. FIGURE 4 | Proposed role of the Paneth cell in development of NEC. As the immature intestine (A) is exposed to inflammation (B), oxygen radicals are produced
creating a selective advantage for Proteobacteria sp. over obligate anaerobes such as the Firmicutes. PANETH CELLS AND MECHANISMS OF
CELLULAR DEATH This creates a feedback loop for sustaining and increasing the
pro-inflammatory state in the immature intestine. Previous work from our lab has shown that intestinal inflammation can reduce intestinal mucus production and cause
loss of Paneth cells (112, 129). (C) Loss of these important chemical and physical aspects of innate immunity allows bacteria to move from the mucus layer of the
intestinal lumen and gain closer proximity to the epithelial surface, (D) followed eventually by attachment and invasion of the epithelium. (E) Once bacteria invade the
intestinal tissue, further inflammation occurs including recruitment of leukocytes including neutrophils, macrophages, and monocytes (133–135) which lead to eventual
death of the tissues. NEC. Lueschow et al. (14) showed that dithizone-induced Paneth
cell loss in an experimental murine NEC model resulted in
upregulation of autophagy pathways in Paneth cells (14). as induce necroptosis when RIP1 and RIP3 are recruited and
deubiquitinated (84). RIP1 and RIP3 are generally under the
control of caspase-8, but when an inactivation of the caspase-
8 gene occurs, induction of necroptotic cell death ensues
although the mechanism by which this occurs is not completely
understood (84). Necroptosis is an increasingly important
mechanism of cellular death in the intestinal epithelium. Studies have shown that necroptosis of intestinal epithelial cells
can result in intestinal inflammation and ultimately produce
pathophysiology similar to inflammatory bowel disease (IBD). This was done by creating conditional knockout mice with
deletion of FADD or caspase-8, the regulator of necroptosis, in
intestinal epithelial cells (54, 95, 96). Interestingly, in addition
to induction of necroptosis, this knockout also resulted in Lastly, a more newly described type of cellular death is
necroptosis which acts as an intermediate between necrosis and
apoptosis. Cells undergoing necroptotic death show features
more morphologically similar to necrosis and the immune
system creates a highly inflammatory response, but in contrast
to necrosis, necroptosis is a well-regulated process, similar to
apoptosis (84, 94). Along with this relationship, necroptosis,
and apoptosis have a great deal of overlap in their regulation. Apoptosis is promoted by TNFα binding and conversion of the
TNFR complex I to the TNFR complex II/alternative TNFR
complex (84). PANETH CELLS AND NECROTIZING
ENTEROCOLITIS (NEC) For preterm infants, one of the leading causes of morbidity
and mortality, and the most devastating intestinal complication,
is development of NEC (97). The incidence of NEC varies
widely among developed countries, ranging from 5 to 22%
in infants with birth weight <1,000 g (98), and in the US is
around 7% (97). Risk factors associated with development of
NEC in the preterm infant include degree of prematurity, low
birth weight, formula feeding, intestinal ischemia, prolonged
antibiotic use, and anemia (99–102). However, the exact etiologic
mechanisms and pathophysiology of NEC is still incomplete. In addition, the NEC phenotype may actually be the result
of a final common pathway starting from multiple inciting
events that results in an imbalance between mucosal injury and
epithelial defense and repair, with activation of an unchecked
pro-inflammatory cascade (103). As a disease process, NEC is
unique in the Neonatal Intensive Care Unit (NICU) population. While the incidence of NEC is directly correlated to the degree
of prematurity (the more premature, the more likely to develop
NEC), the onset of NEC doesn’t happen at birth, but rather
weeks after and this delay is longer in the more premature
infants. The result is that the incidence of NEC begins to
increase at 28 weeks corrected gestational age, peaks at 32
weeks corrected gestational age, and steadily decreases at older
corrected gestational ages (67). Theories have been suggested to
explain this delay including feeding practices, development of
microbial dysbiosis, or the accumulation of mesenteric hypoxic
events (99–102). However, there is currently no universally
accepted mechanistic explanation. We propose that another
plausible reason may be a disruption in the function or quantity
of Paneth cells (17, 67, 104). p
g
p
g
Studying Paneth cell mechanistic biology in the immature
intestine is challenging in humans due to the difficulty of
obtaining tissue specimens for preterm infants (115, 116). To
help understand the potential role of Paneth cell biology in
NEC, several laboratories have instead utilized animal models
(100). Interestingly, when Paneth cells are disrupted in neonatal
rats followed by enteral exposure to E. coli, there is not only
an increase in bacterial translocation, but also a development
of NEC-like injury to the small intestinal tract (105). PANETH CELLS AND MECHANISMS OF
CELLULAR DEATH Necroptosis has also been recently shown to play a role
in development of NEC (94). In preterm infants who develop
NEC, there is a higher degree of expression in genes related
to necroptosis such as RIPK1, RIPK2, and MLKL compared
to preterm infants who do not develop NEC (94). Moreover,
increased expression of these three necroptosis related genes was
correlated with a greater degree of NEC severity (94). This trend
was also observed in murine experimental NEC conditions (94). Overall, these studies highlight the importance of necroptosis as
well as Paneth cells in NEC. PANETH CELLS AND MECHANISMS OF
CELLULAR DEATH Also, the TNFR complex II can regulate as well April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 6 Paneth Cell Lueschow and McElroy until approximately halfway through intestinal development and
maturation (22–24 weeks of human gestation and P7-10 or
mouse age—normal intestinal development in the mouse occurs
following birth while in the human it occurs in utero) (19, 65,
110). It is also important to note that these early Paneth cells
do not possess all the constituents contained in mature granules
(65), and it takes weeks in mice and months in humans before
the Paneth cell cohort reaches its optimal density and before
it becomes fully functional (17). Because of this developmental
pattern, premature infants are thus born before they can develop
a full complement of functional Paneth cells. As Paneth cells
help regulate the intestinal bacterial flora, and NEC requires
bacteria to induce intestinal injury, disruption of normal Paneth
cell function, especially in the immature intestine could very well
be involved in development of the NEC phenotype. Supporting
this theory, decreased numbers of lysozyme positive Paneth cells
were documented in infants with surgical NEC compared to
similar aged surgical controls in two separate studies (111, 112). These data would suggest that Paneth cells are either lost or
degranulated during or prior to development of NEC. However,
not all studies have shown decreases in Paneth cell function or
biology. A study looking at mRNA levels of Human defensin 5
and 6 found that they were increased in infants who developed
NEC compared to controls (113). This discrepancy may be
explained by timing of surgical resection following the initial
Paneth cell disruption. In mouse models, when Paneth cells are
disrupted using the heavy metal chelator dithizone, there is an
initial decrease in defensin expression followed by a significant
increase starting 72 h after treatment (14). In addition, a recent
article that examined presence of HD-6 showed a significant
decrease following development of NEC (114). Thus, timing of
the surgical collection may play a critical role in determining
Paneth cell-specific gene expression following NEC. spontaneous inflammation and an absence of Paneth cells (84,
95, 96). On further examination, the authors discovered that
Paneth cells were uniquely sensitive to necroptosis. This is now
thought to be due to the high expression of RIP3, a key modulator
of necroptosis, in Paneth cells of humans and mice (84, 95,
96). Frontiers in Immunology | www.frontiersin.org PANETH CELLS AND NECROTIZING
ENTEROCOLITIS (NEC) Lastly, it is
important to remember that Paneth cell loss may also impact the
stem cell niche. A healthy stem cell cohort is critical to induce
epithelial restitution following injury as Paneth cells support
the stem cell niche through the production of EGF, Notch, and
Wnt (27, 88, 104, 138). instead is a general chelator of heavy metals. To help resolve
this issue, we developed the PC-DTR mouse (14, 119, 120). The PC-DTR mouse has a human diphtheria toxin receptor
(DTR) inserted into mouse Paneth cells targeting the cryptdin-
2 promotor (65). Treatment with diphtheria toxin induces
apoptosis of any cells possessing DTR while sparing all other
cells. In this model, treatment with diphtheria toxin followed by
Klebsiella pneumoniae exposure also produces intestinal injury
that is equivalent to human NEC (14, 119). These data provide
further evidence that it is a disruption of, and not an absence of
Paneth cells that contributes to development of NEC-like injury
in the immature small intestine. While these studies show a strong association for Paneth
cell disfunction or loss with human NEC as well as a
mechanistic relationship in mice, questions about how Paneth
cell dysfunction may result in NEC remain (104, 121). It
is well-established that prior to the development of NEC
there is a dysbiotic change to the microbiome that is
marked by a bloom of Proteobacteria, more specifically
Enterobacteriaceae species (122–124). This phenomenon has
also been replicated in our Paneth cell disruption model of
NEC (14). In the normal homeostatic state, the microbiome
acts to suppress inflammation through several mechanisms
including induction of anti-inflammatory mediators such as IL-
10, suppression of pro-inflammatory mediators such as IL-17,
and by breaking down and fermenting complex, non-digestible
complex polysaccharides into short-chain fatty acids, which
possess anti-inflammatory properties (125–127). However, a
result of inflammation is increased production of nitric oxide
(NO) and superoxide radicals (O2−), which can then react
to form nitrates (NO3−). These nitrates can be fermented by
facultative anaerobic bacteria such as Enterobacteriaceae sp. that belong to the Proteobacteria phyla by utilizing anaerobic
respiration with host-derived nitrates as alternative electron
acceptors. Since obligate anaerobes cannot use nitrates as a
growth substrate, Proteobacteria are able to use this selective
pressure to out-compete the obligate anaerobic Firmicutes and
Bacteroidetes that rely on fermentation for growth (128). PANETH CELLS AND NECROTIZING
ENTEROCOLITIS (NEC) In
adapting this model to mice, our laboratory and others have
shown that selective ablation of Paneth cells followed by enteric
gavage of Klebsiella pneumoniae in 14-days old mice results in
grossly necrotic intestines (89, 117–119), an increase in serum
inflammatory markers (119), and alterations in the microbiome
(14) that are consistent with human NEC. The use of 2-weeks
old mice in this model is potentially advantageous as well as
they possess a gene expression profile of epithelial cell genes that
matches the expression profile seen in preterm human infants
during the window when they are most susceptible to develop
NEC (18, 67). Interestingly, disruption of Paneth cell biology
via administration of the heavy metal chelator dithizone prior
to normal Paneth cell development (5 days old mice) does
not result in a NEC-like phenotype (117). One critique of this
methodology is that dithizone is not specific to Paneth cells but As discussed above, Paneth cells play a key role in the
homeostasis of the small intestinal epithelial lining, and loss
or disruption of these cells has been shown to have significant
adverse consequences including a reduction in clearance of
bacterial pathogens (105, 106), disruption of normal stem cell
function (3, 107), and the development of inflammatory bowel
disease (108, 109). Paneth cells do not appear in the intestine April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 7 Paneth Cell Lueschow and McElroy This creates a more aerobic state leading to a competitive
advantage for Proteobacteria, such as Enterobacteriaceae species. As the microbiome becomes more dysbiotic, it suppresses
anti-inflammatory mechanisms creating a cycle of increasing
intestinal inflammation (136). This increasing inflammation can
then impact Paneth cell biology leading to a loss in Paneth
cells (14, 129, 137). In an already dysbiotic environment, this
combination is exactly the milieu that is modeled in our
animal model and predisposes to development of injury. This
is further compounded because the Paneth cells present in
the immature intestine are not fully mature or functional at
a baseline (18). This limited Paneth cell cohort also means
that there is a limited capacity for protection via AMPs (40). As Paneth cells are lost, AMP levels will further fall, likely
reaching a critical threshold under which bacterial invasion
of the epithelial tissue can begin to occur (105). FUNDING Funding was provided by the Stead Family Department of
Pediatrics and the Carver College of Medicine at the University
of Iowa. Thus, we think that as the premature infant is exposed to
foreign antigens such as formula feedings (132), there is an
increase in production of inflammatory cytokines (Figure 4). PANETH CELLS AND NECROTIZING
ENTEROCOLITIS (NEC) As
the proportion of commensal bacteria such as Firmicutes and
Bacteroidetes decrease, the production of anti-inflammatory
mediators also decreases which further facilitates increased
inflammation and dysbiosis. Our laboratory has previously
shown that in the immature murine small intestine, exposure to
inflammation can significantly decrease the density and function
of Paneth cells (129–131). In summary, the Paneth cell plays a critical role in many
facets of intestinal homeostasis, from regulating the microbiota
that closely associate with the epithelium, to maintaining
the health of the stem cell niche, to helping to regulate
levels of inflammation. Disruption of these secretory cells
can have an important effect on the ability of the intestinal
epithelium to not only protect itself from foreign invaders,
but to promote growth and development of the intestine. These functions are especially critical in the immature intestine
of premature infants who have a developing intestinal tract
associated with a dysbiotic microbiome. Thus, it is reasonable
that Paneth cell disruption has been linked mechanistically
to development of NEC-like injury. As mortality rates for
NEC remain static, a greater understanding of Paneth cell
biology may provide a critical novel pathway to understand the
development of NEC. AUTHOR CONTRIBUTIONS SM and SL contributed equally to the drafting and editing of
the work. 4. Bevins CL, Salzman NH. Paneth cells, antimicrobial peptides and
maintenance of intestinal homeostasis. Nat Rev Microbiol. (2011) 9:356–68.
doi: 10.1038/nrmicro2546 REFERENCES 3. Clevers
HC,
Bevins
CL. Paneth
cells:
maestros
of
the
small
intestinal
crypts. Annu
Rev
Physiol. (2013)
75:289–311. doi:
10.1146/annurev-physiol-030212-1
83744 1. Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier
function and immune homeostasis. Nat Rev Immunol. (2014) 14:141–53. doi: 10.1038/nri3608 1. Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier
function and immune homeostasis. Nat Rev Immunol. (2014) 14:141–53. doi: 10.1038/nri3608 4. Bevins CL, Salzman NH. Paneth cells, antimicrobial peptides and
maintenance of intestinal homeostasis. Nat Rev Microbiol. (2011) 9:356–68. doi: 10.1038/nrmicro2546 2. Paneth J. Ueber die secernirenden Zellen des Dünndarm-Epithels. Arc
Mikrosk Anat. (1887) 31:113–91. doi: 10.1007/BF02955706 April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 8 Paneth Cell Lueschow and McElroy intestinal stem cell niche factors. Proc Natl Acad Sci USA. (2007) 104:15418–
23. doi: 10.1073/pnas.0707210104 intestinal stem cell niche factors. Proc Natl Acad Sci USA. (2007) 104:15418–
23. doi: 10.1073/pnas.0707210104 5. Paulsen SM, Engstad RE, Robertsen B. Enhanced lysozyme production in
Atlantic salmon. (Salmo salar L.) macrophages treated with yeast beta-glucan
and bacterial lipopolysaccharide. Fish Shellfish Immunol. (2001) 11:23–37. doi: 10.1006/fsim.2000.0291 5. Paulsen SM, Engstad RE, Robertsen B. Enhanced lysozyme production in
Atlantic salmon. (Salmo salar L.) macrophages treated with yeast beta-glucan
and bacterial lipopolysaccharide. Fish Shellfish Immunol. (2001) 11:23–37. doi: 10.1006/fsim.2000.0291 26. Miyazono K, Kamiya Y, Morikawa M. Bone morphogenetic protein
receptors
and
signal
transduction. J
Biochem. (2010)
147:35–51. doi: 10.1093/jb/mvp148 6. Poindexter BJ, Klein GL, Milner SM, Bick RJ. Upregulation of defensins in
burn sheep small intestine. Eplasty. (2009) 10:e6. 27. Sato T, Van Es JH, Snippert HJ, Stange DE, Vries RG, Van Den Born M,
et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature. (2011) 469:415–8. doi: 10.1038/nature09637 7. Helander HF, Fandriks L. Surface area of the digestive tract - revisited. Scand
J Gastroenterol. (2014) 49:681–9. doi: 10.3109/00365521.2014.898326 28. Van Es JH, Jay P, GregorieffA, Van Gijn ME, Jonkheer S, Hatzis P, et al. Wnt
signalling induces maturation of Paneth cells in intestinal crypts. Nat Cell
Biol. (2005) 7:381–6. doi: 10.1038/ncb1240 8. Martin RJ, FanaroffAA, Walsh MC. Fanaroffand Martin’s Neonatal-
Perinatal Medicine: Diseases of the Fetus and Infant. Philadelphia, PA:
Elsevier/Saunders (2015). 9. Cheng H, Leblond CP. Origin, differentiation and renewal of the four main
epithelial cell types in the mouse small intestine. I Columnar cell Am J Anat. (1974) 141:461–79. doi: 10.1002/aja.1001410403 29. REFERENCES Andreu P, Peignon G, Slomianny C, Taketo MM, Colnot S, Robine
S,
et
al. A
genetic
study
of
the
role
of
the
Wnt/beta-catenin
signalling in Paneth cell differentiation. Dev Biol. (2008) 324:288–96. doi: 10.1016/j.ydbio.2008.09.027 10. Crosnier C, Stamataki D, Lewis J. Organizing cell renewal in the intestine:
stem cells, signals and combinatorial control. Nat Rev Genet. (2006) 7:349–
59. doi: 10.1038/nrg1840 30. Garcia MI, Ghiani M, Lefort A, Libert F, Strollo S, Vassart G. LGR5
deficiency deregulates Wnt signaling and leads to precocious Paneth
cell differentiation in the fetal intestine. Dev Biol. (2009) 331:58–67. doi: 10.1016/j.ydbio.2009.04.020 g
11. Van Der Flier LG, Clevers H. Stem cells, self-renewal, and differentiation
in the intestinal
epithelium. Annu
Rev
Physiol. (2009) 71:241–60. doi: 10.1146/annurev.physiol.010908.163145 31. Ben-Arie N, Bellen HJ, Armstrong DL, Mccall AE, Gordadze PR, Guo Q,
et al. Math1 is essential for genesis of cerebellar granule neurons. Nature. (1997) 390:169–72. doi: 10.1038/36579 12. Gerbe F, Van Es JH, Makrini L, Brulin B, Mellitzer G, Robine S, et al. Distinct ATOH1 and Neurog3 requirements define tuft cells as a new
secretory cell type in the intestinal epithelium. J Cell Biol. (2011) 192:767–80. doi: 10.1083/jcb.201010127 32. Bermingham NA, Hassan BA, Price SD, Vollrath MA, Ben-Arie N, Eatock
RA, et al. Math1: an essential gene for the generation of inner ear hair cells. Science. (1999) 284:1837–41. doi: 10.1126/science.284.5421.1837 13. Porter EM, Bevins CL, Ghosh D, Ganz T. The multifaceted Paneth cell. Cell
Mol Life Sci. (2002) 59:156–70. doi: 10.1007/s00018-002-8412-z 33. Jensen P, Smeyne R, Goldowitz D. Analysis of cerebellar development
in math1 null embryos and chimeras. J Neurosci. (2004) 24:2202–11. doi: 10.1523/JNEUROSCI.3427-03.2004 14. Lueschow SR, Stumphy J, Gong H, Kern SL, Elgin TG, Underwood MA,
et al. Loss of murine Paneth cell function alters the immature intestinal
microbiome and mimics changes seen in neonatal necrotizing enterocolitis. PLoS ONE. (2018) 13:e0204967. doi: 10.1371/journal.pone.0204967 34. Shroyer NF, Helmrath MA, Wang VY, Antalffy B, Henning SJ, Zoghbi
HY. Intestine-specific ablation of mouse atonal homolog 1 (Math1)
reveals a role in cellular homeostasis. Gastroenterology. (2007) 132:2478–88. doi: 10.1053/j.gastro.2007.03.047 15. Rumbo M, Schiffrin EJ. Ontogeny of intestinal epithelium immune
functions: developmental and environmental regulation. Cell Mol Life Sci. (2005) 62:1288–96. doi: 10.1007/s00018-005-5033-3 35. Nakanishi Y, Reina-Campos M, Nakanishi N, Llado V, Elmen L,
Peterson S, et al. Control of paneth cell fate, intestinal inflammation,
and tumorigenesis by PKCLAMBDA/iota. Cell Rep. (2016) 16:3297–310. doi: 10.1016/j.celrep.2016.08.054 16. REFERENCES Kandasamy J, Huda S, Ambalavanan N, Jilling T. Inflammatory signals
that regulate intestinal epithelial renewal, differentiation, migration and cell
death: implications for necrotizing enterocolitis. Pathophysiology. (2014)
21:67–80. doi: 10.1016/j.pathophys.2014.01.001 36. Durand A, Donahue B, Peignon G, Letourneur F, Cagnard N, Slomianny C,
et al. Functional intestinal stem cells after Paneth cell ablation induced by the
loss of transcription factor Math1. (Atoh1). Proc Natl Acad Sci USA. (2012)
109:8965–70. doi: 10.1073/pnas.1201652109 17. Heida FH, Beyduz G, Bulthuis ML, Kooi EM, Bos AF, Timmer A, et al. Paneth
cells in the developing gut: when do they arise and when are they immune
competent? Pediatr Res. (2016) 80:306–10. doi: 10.1038/pr.2016.67 37. Almohazey D, Lo YH, Vossler CV, Simmons AJ, Hsieh JJ, Bucar EB, et al. The ErbB3 receptor tyrosine kinase negatively regulates Paneth cells by
PI3K-dependent suppression of Atoh1. Cell Death Differ. (2017) 24:855–65. doi: 10.1038/cdd.2017.27 18. Stanford
AH,
Gong
H,
Noonan
M,
Lewis
AN,
Gong
Q,
Lanik
WE, et al. A direct comparison of mouse and human intestinal
development using epithelial gene expression patterns. Pediatr Res. (2019). doi: 10.1038/s41390-019-0472-y. [Epub ahead of print]. 19. Bry L, Falk P, Huttner K, Ouellette A, Midtvedt T, Gordon JI. Paneth cell
differentiation in the developing intestine of normal and transgenic mice. Proc Natl Acad Sci USA. (1994) 91:10335–9. doi: 10.1073/pnas.91.22.10335 38. Ouellette AJ. Paneth cells and innate mucosal immunity. Curr Opin
Gastroenterol. (2010) 26:547–53. doi: 10.1097/MOG.0b013e32833dccde 39. Johnson
LR. Physiology
of
the
Gastrointestinal
Tract. Amsterdam:
Elsevier/AP (2012). 20. Cheng H, Leblond CP. Origin, differentiation and renewal of the four main
epithelial cell types in the mouse small intestine. V Unitarian Theory of
the origin of the four epithelial cell types. Am J Anat. (1974) 141:537–61. doi: 10.1002/aja.1001410407 40. Satoh Y. Effect of live and heat-killed bacteria on the secretory activity
of Paneth cells in germ-free mice. Cell Tissue Res. (1988) 251:87–93. doi: 10.1007/BF00215451 21. Barker N, Van Es JH, Kuipers J, Kujala P, Van Den Born M, Cozijnsen M,
et al. Identification of stem cells in small intestine and colon by marker gene
Lgr5. Nature. (2007) 449:1003–7. doi: 10.1038/nature06196 41. Porter EM, Liu L, Oren A, Anton PA, Ganz T. Localization of human
intestinal defensin 5 in Paneth cell granules. Infect Immun. (1997) 65:2389–
95. doi: 10.1128/IAI.65.6.2389-2395.1997 42. Takahashi N, Vanlaere I, De Rycke R, Cauwels A, Joosten LA, Lubberts E,
et al. IL-17 produced by Paneth cells drives TNF-induced shock. J Exp Med. REFERENCES Ulm H, Wilmes M, Shai Y, Sahl HG. Antimicrobial host defensins - specific
antibiotic activities and innate defense modulation. Front Immunol. (2012)
3:249. doi: 10.3389/fimmu.2012.00249 56. Barlow PG, Svoboda P, Mackellar A, Nash AA, York IA, Pohl J, et al. Antiviral activity and increased host defense against influenza infection
elicited by the human cathelicidin LL-37. PLoS ONE. (2011) 6:e25333. doi: 10.1371/journal.pone.0025333 77. Hemshekhar M, Anaparti V, Mookherjee N. Functions of Cationic
Host Defense Peptides in Immunity. Pharmaceuticals. (2016) 9:40. doi: 10.3390/ph9030040 57. Durr UH, Sudheendra US, Ramamoorthy A. LL-37, the only human member
of the cathelicidin family of antimicrobial peptides. Biochim Biophys Acta. (2006) 1758:1408–25. doi: 10.1016/j.bbamem.2006.03.030 78. Ho J, Zhang L, Liu X, Wong SH, Wang MHT, Lau BWM, et al. Pathological role and diagnostic value of endogenous host defense peptides
in adult and neonatal sepsis: a systematic review. Shock. (2017) 47:673–9. doi: 10.1097/SHK.0000000000000815 58. Larrick JW, Hirata M, Balint RF, Lee J, Zhong J, Wright SC. Human CAP18:
a novel antimicrobial lipopolysaccharide-binding protein. Infect Immun. (1995) 63:1291–7. doi: 10.1128/IAI.63.4.1291-1297.1995 79. Schroeder BO, Ehmann D, Precht JC, Castillo PA, Kuchler R, Berger J, et al. Paneth cell alpha-defensin 6 (HD-6) is an antimicrobial peptide. Mucosal
Immunol. (2015) 8:661–71. doi: 10.1038/mi.2014.100 59. Frohm M, Agerberth B, Ahangari G, Stahle-Backdahl M, Liden S, Wigzell H,
et al. The expression of the gene coding for the antibacterial peptide LL-37 is
induced in human keratinocytes during inflammatory disorders. J Biol Chem. (1997) 272:15258–63. doi: 10.1074/jbc.272.24.15258 80. Masuda K, Sakai N, Nakamura K, Yoshioka S, Ayabe T. Bactericidal activity
of mouse alpha-defensin cryptdin-4 predominantly affects noncommensal
bacteria. J Innate Immun. (2011) 3:315–26. doi: 10.1159/000322037 81. Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, et al. Beta-
defensins: linking innate and adaptive immunity through dendritic and T cell
CCR6. Science. (1999) 286:525–8. doi: 10.1126/science.286.5439.525 60. Bals R, Wang X, ZasloffM, Wilson JM. The peptide antibiotic LL-
37/hCAP-18 is expressed in epithelia of the human lung where it has broad
antimicrobial activity at the airway surface. Proc Natl Acad Sci USA. (1998)
95:9541–6. doi: 10.1073/pnas.95.16.9541 82. Yang D, Chen Q, Chertov O, Oppenheim JJ. Human neutrophil defensins
selectively chemoattract naive T and immature dendritic cells. J Leukoc Biol. (2000) 68:9–14. doi: 10.1189/jlb.68.1.9 61. Frohm Nilsson M, Sandstedt B, Sorensen O, Weber G, Borregaard N,
Stahle-Backdahl M. REFERENCES (2008) 205:1755–61. doi: 10.1084/jem.20080588 22. Barker N, Huch M, Kujala P, Van De Wetering M, Snippert HJ, Van
Es JH, et al. Lgr5(+ve) stem cells drive self-renewal in the stomach and
build long-lived gastric units in vitro. Cell Stem Cell. (2010) 6:25–36. doi: 10.1016/j.stem.2009.11.013 43. Park SW, Kim M, Brown KM, D’agati VD, Lee HT. Paneth cell-derived
interleukin-17A causes multiorgan dysfunction after hepatic ischemia and
reperfusion injury. Hepatology. (2011) 53:1662–75. doi: 10.1002/hep.24253 23. Yan KS, Chia LA, Li X, Ootani A, Su J, Lee JY, et al. The intestinal stem cell
markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc
Natl Acad Sci USA. (2012) 109:466–71. doi: 10.1073/pnas.1118857109 44. Satoh Y, Ishikawa K, Tanaka H, Ono K. Immunohistochemical observations
of immunoglobulin A in the Paneth cells of germ-free and formerly-
germ-free rats. Histochemistry. (1986) 85:197–201. doi: 10.1007/BF004
94804 24. Munoz J, Stange DE, Schepers AG, Van De Wetering M, Koo BK,
Itzkovitz S, et al. The Lgr5 intestinal stem cell signature: robust expression
of proposed quiescent ‘+4’ cell markers. EMBO J. (2012) 31:3079–91. doi: 10.1038/emboj.2012.166 45. Ouellette AJ. Paneth cell alpha-defensins: peptide mediators of innate
immunity in the small intestine. Springer Semin Immunopathol. (2005)
27:133–46. doi: 10.1007/s00281-005-0202-x 45. Ouellette AJ. Paneth cell alpha-defensins: peptide mediators of innate
immunity in the small intestine. Springer Semin Immunopathol. (2005)
27:133–46. doi: 10.1007/s00281-005-0202-x 25. Kosinski C, Li VS, Chan AS, Zhang J, Ho C, Tsui WY, et al. Gene expression
patterns of human colon tops and basal crypts and BMP antagonists as April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 9 Lueschow and McElroy Paneth Cell 46. Ayabe T, Satchell DP, Wilson CL, Parks WC, Selsted ME, Ouellette AJ. Secretion of microbicidal alpha-defensins by intestinal Paneth cells in
response to bacteria. Nat Immunol. (2000) 1:113–8. doi: 10.1038/77783 ablation
in
transgenic
mice. J
Biol
Chem. (1997)
272:23
729–40. doi: 10.1074/jbc.272.38.23729 ablation
in
transgenic
mice. J
Biol
Chem. (1997)
272:23
729–40. doi: 10.1074/jbc.272.38.23729 66. Mcelroy SJ, Weitkamp JH. Innate Immunity in the Small Intestine of the
Preterm Infant. NeoReviews. (2011) 12:e517–26. doi: 10.1542/neo.12-9-e517 47. Nakamura K, Sakuragi N, Takakuwa A, Ayabe T. Paneth cell alpha-defensins
and enteric microbiota in health and disease. Biosci Microbiota Food Health. (2016) 35:57–67. doi: 10.12938/bmfh.2015-019 67. Yee WH, Soraisham AS, Shah VS, Aziz K, Yoon W, Lee SK. Incidence
and timing of presentation of necrotizing enterocolitis in preterm infants. Pediatrics. (2012) 129:e298–304. doi: 10.1542/peds.2011-2022 48. REFERENCES Mizrahi A, Barlow O, Berdon W, Blanc WA, Silverman WA. Necrotizing
Enterocolitis
in
Premature
Infants. J
Pediatr. (1965)
66:697–705. doi: 10.1016/S0022-3476(65)80003-8 68. Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev
Immunol. (2003) 3:710–20. doi: 10.1038/nri1180 49. Salzman NH, Underwood MA, Bevins CL. Paneth cells, defensins,
and the commensal microbiota: a hypothesis on intimate interplay
at
the
intestinal
mucosa. Semin
Immunol. (2007)
19:70–83. doi: 10.1016/j.smim.2007.04.002 69. Wehkamp J, Schauber J, Stange EF. Defensins and cathelicidins in
gastrointestinal infections. Curr Opin Gastroenterol. (2007) 23:32–8. doi: 10.1097/MOG.0b013e32801182c2 70. Kolls
JK,
Mccray
PB
Jr,
Chan
YR. Cytokine-mediated
regulation
of
antimicrobial
proteins. Nat
Rev
Immunol. (2008)
8:829–35. doi: 10.1038/nri2433 50. Sawada M, Takahashi K, Sawada S, Midorikawa O. Selective killing of Paneth
cells by intravenous administration of dithizone in rats. Int J Exp Pathol. (1991) 72:407–21. 71. Ganz T. The role of antimicrobial peptides in innate immunity. Integr Comp
Biol. (2003) 43:300–4. doi: 10.1093/icb/43.2.300 51. Sawada
M,
Nishikawa
M,
Adachi
T,
Midorikawa
O,
Hiai
H. A
Paneth cell specific zinc-binding protein in the rat. Purification and
immunohistochemical localization. Lab Invest. (1993) 68:338–44. 72. De Smet K, Contreras R. Human antimicrobial peptides: defensins,
cathelicidins
and
histatins. Biotechnol
Lett. (2005)
27:1337–47. doi: 10.1007/s10529-005-0936-5 52. Hooper LV, Midtvedt T, Gordon JI. How host-microbial interactions
shape the nutrient environment of the mammalian intestine. Annu
Rev
Nutr. (2002)
22:283–307. doi:
10.1146/annurev.nutr.22.011602.0
92259 73. Wilson CL, Ouellette AJ, Satchell DP, Ayabe T, Lopez-Boado YS, Stratman
JL, et al. Regulation of intestinal alpha-defensin activation by the
metalloproteinase matrilysin in innate host defense. Science. (1999) 286:113–
7. doi: 10.1126/science.286.5437.113 53. Gilbert JA, Blaser MJ, Caporaso JG, Jansson JK, Lynch SV, Knight R. Current
understanding of the human microbiome. Nat Med. (2018) 24:392–400. doi: 10.1038/nm.4517 74. Salzman NH, Ghosh D, Huttner KM, Paterson Y, Bevins CL. Protection
against enteric salmonellosis in transgenic mice expressing a human
intestinal defensin. Nature. (2003) 422:522–6. doi: 10.1038/nature01520 54. Chromek M, Arvidsson I, Karpman D. The antimicrobial peptide
cathelicidin protects mice from Escherichia coli O157:H7-mediated disease. PLoS ONE. (2012) 7:e46476. doi: 10.1371/journal.pone.0046476 75. Salzman NH, Hung K, Haribhai D, Chu H, Karlsson-Sjoberg J, Amir E, et al. Enteric defensins are essential regulators of intestinal microbial ecology. Nat
Immunol. (2010) 11:76–83. doi: 10.1038/ni.1825 55. Wong JH, Ng TB, Legowska A, Rolka K, Hui M, Cho CH. Antifungal action
of human cathelicidin fragment. (LL13-37) on Candida albicans. Peptides. (2011) 32:1996–2002. doi: 10.1016/j.peptides.2011.08.018 76. REFERENCES Welz PS, Wullaert A, Vlantis K, Kondylis V, Fernandez-Majada V, Ermolaeva
M, et al. FADD prevents RIP3-mediated epithelial cell necrosis and chronic
intestinal inflammation. Nature. (2011) 477:330–4. doi: 10.1038/nature
10273 117. Zhang C, Sherman MP, Prince LS, Bader D, Weitkamp JH, Slaughter JC,
et al. Paneth cell ablation in the presence of Klebsiella pneumoniae induces
necrotizing enterocolitis (NEC)-like injury in the small intestine of immature
mice. Dis Model Mech. (2012) 5:522–32. doi: 10.1242/dmm.009001 97. Patel RM, Kandefer S, Walsh MC, Bell EF, Carlo WA, Laptook
AR,
et
al. Causes
and
timing
of
death
in
extremely
premature
infants from 2000 through 2011. N Engl J Med. (2015) 372:331–40. doi: 10.1056/NEJMoa1403489 118. Eckert J, Scott B, Lawrence SM, Ihnat M, Chaaban H. FLLL32, a curcumin
analog, ameliorates intestinal injury in necrotizing enterocolitis. J Inflamm
Res. (2017) 10:75–81. doi: 10.2147/JIR.S131051 98. Battersby
C,
Santhalingam
T,
Costeloe
K,
Modi
N. Incidence
of
neonatal
necrotising
enterocolitis
in
high-income
countries:
a
systematic review. Arch Dis Child Fetal Neonatal Ed. (2018) 103:F182–9. doi: 10.1136/archdischild-2017-313880 119. White JR, Gong H, Pope B, Schlievert P, Mcelroy SJ. Paneth-cell-disruption-
induced necrotizing enterocolitis in mice requires live bacteria and occurs
independently of TLR4 signaling. Dis Model Mech. (2017) 10:727–36. doi: 10.1242/dmm.028589 99. Lin PW, Nasr TR, Stoll BJ. Necrotizing enterocolitis: recent scientific
advances in pathophysiology and prevention. Semin Perinatol. (2008) 32:70–
82. doi: 10.1053/j.semperi.2008.01.004 120. Berger
JN,
Gong
H,
Good
M,
Mcelroy
SJ. Dithizone-induced
Paneth
cell
disruption
significantly
decreases
intestinal
perfusion
in
the
murine
small
intestine. J
Pediatr
Surg. (2019)
54:2402–7. doi: 10.1016/j.jpedsurg.2019.02.021 100. Ares GJ, Mcelroy SJ, Hunter CJ. The science and necessity of using animal
models in the study of necrotizing enterocolitis. Semin Pediatr Surg. (2018)
27:29–33. doi: 10.1053/j.sempedsurg.2017.11.006 121. Underwood MA. Paneth cells and necrotizing enterocolitis. Gut Microbes. (2012) 3:562–5. doi: 10.4161/gmic.21738 101. Hackam
D,
Caplan
M. Necrotizing
enterocolitis:
pathophysiology
from
a
historical
context. Semin
Pediatr
Surg. (2018)
27:11–8. doi: 10.1053/j.sempedsurg.2017.11.003 122. Gritz EC, Bhandari V. The human neonatal gut microbiome: a brief review. Front Pediatr. (2015) 3:17. doi: 10.3389/fped.2015.00060 102. Hackam DJ, Sodhi CP, Good M. New insights into necrotizing enterocolitis:
from laboratory observation to personalized prevention and treatment. J
Pediatr Surg. (2019) 54:398–404. doi: 10.1016/j.jpedsurg.2018.06.012 123. Lu CY, Ni YH. Gut microbiota and the development of pediatric diseases. J
Gastroenterol. (2015) 50:720–6. doi: 10.1007/s00535-015-1082-z 124. Niemarkt HJ, De Meij TG, Van De Velde ME, Van Der Schee MP, Van
Goudoever JB, Kramer BW, et al. REFERENCES Secretory autophagy holds the key to
lysozyme secretion during bacterial infection of the intestine. Autophagy. (2018) 14:1–3. doi: 10.1080/15548627.2017.1401425 110. Moxey PC, Trier JS. Specialized cell types in the human fetal small intestine. Anat Rec. (1978) 191:269–85. doi: 10.1002/ar.1091910302 89. Mcelroy
SJ,
Castle
SL,
Bernard
JK,
Almohazey
D,
Hunter
CJ,
Bell
BA,
et
al. The
ErbB4
ligand
neuregulin-4
protects
against
experimental necrotizing enterocolitis. Am J Pathol. (2014) 184:2768–78. doi: 10.1016/j.ajpath.2014.06.015 111. Coutinho HB, Da Mota HC, Coutinho VB, Robalinho TI, Furtado AF,
Walker E, et al. Absence of lysozyme (muramidase) in the intestinal Paneth
cells of newborn infants with necrotising enterocolitis. J Clin Pathol. (1998)
51:512–4. doi: 10.1136/jcp.51.7.512 90. Alouf JE, PopoffMR. The comprehensive sourcebook of bacterial protein
toxins. Amsterdam; Boston: Elsevier (2006). 91. Glick D, Barth S, Macleod KF. Autophagy: cellular and molecular
mechanisms. J Pathol. (2010) 221:3–12. doi: 10.1002/path.2697 112. Mcelroy SJ, Prince LS, Weitkamp JH, Reese J, Slaughter JC, Polk
DB. Tumor necrosis factor receptor 1-dependent depletion of mucus
in immature small intestine: a potential role in neonatal necrotizing
enterocolitis. Am J Physiol Gastrointest Liver Physiol. (2011) 301:G656–66. doi: 10.1152/ajpgi.00550.2010 92. Van Es JH, Clevers H. Paneth cells. Curr Biol. (2014) 24:R547–8. doi: 10.1016/j.cub.2014.04.049 93. Sampath V, Bhandari V, Berger J, Merchant D, Zhang L, Ladd M,
et
al. A
functional
ATG16L1
(T300A)
variant
is
associated
with
necrotizing enterocolitis in premature infants. Pediatr Res. (2017) 81:582–8. doi: 10.1038/pr.2016.260 113. Salzman NH, Polin RA, Harris MC, Ruchelli E, Hebra A, Zirin-Butler S, et al. Enteric defensin expression in necrotizing enterocolitis. Pediatr Res. (1998)
44:20–6. doi: 10.1203/00006450-199807000-00003 94. Werts AD, Fulton WB, Ladd MR, Saad-Eldin A, Chen YX, Kovler
ML,
et
al. A
novel
role
for
necroptosis
in
the
pathogenesis
of
necrotizing enterocolitis. Cell Mol Gastroenterol Hepatol. (2019) 9:403–23. doi: 10.1016/j.jcmgh.2019.11.002 114. Markasz L, Wanders A, Szekely L, Lilja HE. Diminished DEFA6 expression
in paneth cells is associated with necrotizing enterocolitis. Gastroenterol Res
Pract. (2018) 2018:7345426. doi: 10.1155/2018/7345426 115. Ralls MW, Gadepalli SK, Sylvester KG, Good M. Development of the
necrotizing enterocolitis society registry and biorepository. Semin Pediatr
Surg. (2018) 27:25–8. doi: 10.1053/j.sempedsurg.2017.11.005 95. Gunther C, Martini E, Wittkopf N, Amann K, Weigmann B, Neumann
H, et al. Caspase-8 regulates TNF-alpha-induced epithelial necroptosis and
terminal ileitis. Nature. (2011) 477:335–9. doi: 10.1038/nature10400 116. Chaaban
H,
Markel
TA,
Canvasser
J,
Good
M. Biobanking
for
necrotizing enterocolitis: Needs and standards. J Pediatr Surg. (2019). doi: 10.1016/j.jpedsurg.2019.05.002. [Epub ahead of print]. 96. REFERENCES The human cationic antimicrobial protein (hCAP18),
a peptide antibiotic, is widely expressed in human squamous epithelia
and colocalizes with interleukin-6. Infect Immun. (1999) 67:2561–6. doi: 10.1128/IAI.67.5.2561-2566.1999 83. Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: mechanistic
description of dead and dying eukaryotic cells. Infect Immun. (2005)
73:1907–16. doi: 10.1128/IAI.73.4.1907-1916.2005 62. Hase K, Eckmann L, Leopard JD, Varki N, KagnoffMF. Cell differentiation
is a key determinant of cathelicidin LL-37/human cationic antimicrobial
protein 18 expression by human colon epithelium. Infect Immun. (2002)
70:953–63. doi: 10.1128/IAI.70.2.953-963.2002 84. Gunther C, Neumann H, Neurath MF, Becker C. Apoptosis, necrosis and
necroptosis: cell death regulation in the intestinal epithelium. Gut. (2013)
62:1062–71. doi: 10.1136/gutjnl-2011-301364 85. Jilling T, Lu J, Jackson M, Caplan MS. Intestinal epithelial apoptosis
initiates
gross
bowel
necrosis
in
an
experimental
rat
model
of
neonatal
necrotizing
enterocolitis. Pediatr
Res. (2004)
55:622–9. doi: 10.1203/01.PDR.0000113463.70435.74 63. Kai-Larsen
Y,
Bergsson
G,
Gudmundsson
GH,
Printz
G,
Jornvall
H, Marchini G, et al. Antimicrobial components of the neonatal
gut
affected
upon
colonization. Pediatr
Res. (2007)
61:530–6. doi: 10.1203/pdr.0b013e318045be83 86. Clark JA, Lane RH, Maclennan NK, Holubec H, Dvorakova K, Halpern MD,
et al. Epidermal growth factor reduces intestinal apoptosis in an experimental
model of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol. (2005) 288:G755–62. doi: 10.1152/ajpgi.00172.2004 64. Menard S, Forster V, Lotz M, Gutle D, Duerr CU, Gallo RL, et al. Developmental switch of intestinal antimicrobial peptide expression. J Exp
Med. (2008) 205:183–93. doi: 10.1084/jem.20071022 65. Garabedian EM, Roberts LJ, Mcnevin MS, Gordon JI. Examining
the
role
of
Paneth
cells
in
the
small
intestine
by
lineage 87. Feng JX, Besner GE. Heparin-binding epidermal growth factor-like
growth factor promotes enterocyte migration and proliferation in neonatal April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 10 Lueschow and McElroy Paneth Cell rats with necrotizing enterocolitis. J Pediatr Surg. (2007) 42:214–20. doi: 10.1016/j.jpedsurg.2006.09.055 108. Bel S, Pendse M, Wang Y, Li Y, Ruhn KA, Hassell B, et al. Paneth cells secrete
lysozyme via secretory autophagy during bacterial infection of the intestine. Science. (2017) 357:1047–52. doi: 10.1126/science.aal4677 rats with necrotizing enterocolitis. J Pediatr Surg. (2007) 42:214–20. doi: 10.1016/j.jpedsurg.2006.09.055 88. Neal MD, Sodhi CP, Jia H, Dyer M, Egan C, Yazji I, et al. Toll like receptor
4 is expressed on intestinal stem cells and regulates their proliferation and
apoptosis via the p53-upregulated modulator of apoptosis. J Biol Chem. (2012) 287:37296–308. doi: 10.1074/jbc.M112.375881 109. Delorme-Axford E, Klionsky DJ. REFERENCES Necrotizing enterocolitis: a clinical
review on diagnostic biomarkers and the role of the intestinal microbiota. Inflamm Bowel Dis. (2015) 21:436–44. doi: 10.1097/MIB.00000000000
00184 103. Caplan MS, Underwood MA, Modi N, Patel R, Gordon PV, Sylvester
KG, et al. Necrotizing enterocolitis: using regulatory science and drug
development to improve outcomes. J Pediatr. (2019) 212:208–15 e201. doi: 10.1016/j.jpeds.2019.05.032 125. Mazmanian SK, Round JL, Kasper DL. A microbial symbiosis factor
prevents
intestinal
inflammatory
disease. Nature. (2008)
453:620–5. doi: 10.1038/nature07008 104. Mcelroy SJ, Underwood MA, Sherman MP. Paneth cells and necrotizing
enterocolitis: a novel hypothesis for disease pathogenesis. Neonatology. (2013) 103:10–20. doi: 10.1159/000342340 126. Spees AM, Lopez CA, Kingsbury DD, Winter SE, Baumler AJ. Colonization
resistance: battle of the bugs or Menage a Trois with the host? PLoS Pathog. (2013) 9:e1003730. doi: 10.1371/journal.ppat.1003730 105. Sherman MP, Bennett SH, Hwang FF, Sherman J, Bevins CL. Paneth cells and
antibacterial host defense in neonatal small intestine. Infect Immun. (2005)
73:6143–6. doi: 10.1128/IAI.73.9.6143-6146.2005 127. Shin NR, Whon TW, Bae JW. Proteobacteria: microbial signature
of dysbiosis in gut microbiota. Trends Biotechnol. (2015) 33:496–503. doi: 10.1016/j.tibtech.2015.06.011 106. Vaishnava S, Behrendt CL, Ismail AS, Eckmann L, Hooper LV. Paneth cells
directly sense gut commensals and maintain homeostasis at the intestinal
host-microbial interface. Proc Natl Acad Sci USA. (2008) 105:20858–63. doi: 10.1073/pnas.0808723105 128. Winter SE, Winter MG, Xavier MN, Thiennimitr P, Poon V, Keestra AM,
et al. Host-derived nitrate boosts growth of E. coli in the inflamed gut. Science. (2013) 339:708–11. doi: 10.1126/science.1232467 107. Clevers H. Stem cells: a unifying theory for the crypt. Nature. (2013) 495:53–
4. doi: 10.1038/nature11958 April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 11 Lueschow and McElroy Paneth Cell 129. Brown KS, Gong H, Frey MR, Pope B, Golden M, Martin K, et al. Tumor
necrosis factor induces developmental stage-dependent structural changes
in the immature small intestine. Mediators Inflamm. (2014) 2014:852378. doi: 10.1155/2014/852378 135. Mohankumar K, Namachivayam K, Song T, Jake Cha B, Slate A, Hendrickson
JE, et al. A murine neonatal model of necrotizing enterocolitis caused
by anemia and red blood cell transfusions. Nat Commun. (2019) 10:3494. doi: 10.1038/s41467-019-11199-5 130. Fricke EM, Elgin TG, Gong H, Reese J, Gibson-Corley KN, Weiss RM, et al. Lipopolysaccharide-induced maternal inflammation induces direct placental
injury without alteration in placental blood flow and induces a secondary
fetal intestinal injury that persists into adulthood. Am J Reprod Immunol. (2018) 79:e12816. doi: 10.1111/aji.12816 136. Frontiers in Immunology | www.frontiersin.org April 2020 | Volume 11 | Article 587 REFERENCES Elgin TG, Kern SL, Mcelroy SJ. Development of the neonatal intestinal
microbiome and its association with necrotizing enterocolitis. Clin Ther. (2016) 38:706–15. doi: 10.1016/j.clinthera.2016.01.005 137. Fung
CM,
White
JR,
Brown
AS,
Gong
H,
Weitkamp
JH,
Frey
MR,
et
al. Intrauterine
growth
restriction
alters
mouse
intestinal
architecture
during
development. PLoS
ONE. (2016)
11:e0146542. doi: 10.1371/journal.pone.0146542 131. Elgin TG, Fricke EM, Gong H, Reese J, Mills DA, Kalantera KM,
et al. Fetal exposure to maternal inflammation
interrupts murine
intestinal development and increases susceptibility to neonatal intestinal
injury. Dis Model Mech. (2019) 12:dmm040808. doi: 10.1242/dmm.0
40808 138. Li B, Lee C, Cadete M, Zhu H, Koike Y, Hock A, et al. Impaired
Wnt/beta-catenin pathway leads to dysfunction of intestinal regeneration
during
necrotizing
enterocolitis. Cell
Death
Dis. (2019)
10:743. doi: 10.1038/s41419-019-1987-1 132. Liu Y, Zhu L, Fatheree NY, Liu X, Pacheco SE, Tatevian N, et al. Changes in intestinal Toll-like receptors and cytokines precede histological
injury in a rat model of
necrotizing
enterocolitis. Am
J
Physiol
Gastrointest Liver Physiol. (2009) 297:G442–50. doi: 10.1152/ajpgi.0018
2.2009 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. 133. Anand RJ, Gribar SC, Li J, Kohler JW, Branca MF, Dubowski T,
et al. Hypoxia causes an increase in phagocytosis by macrophages
in a HIF-1alpha-dependent manner. J Leukoc Biol. (2007) 82:1257–65. doi: 10.1189/jlb.0307195 Copyright © 2020 Lueschow and McElroy. 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. 134. Egan CE, Sodhi CP, Good M, Lin J, Jia H, Yamaguchi Y, et al. Toll-
like receptor 4-mediated lymphocyte influx induces neonatal necrotizing
enterocolitis. J Clin Invest. (2016) 126:495–508. doi: 10.1172/JCI83356 April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 12
|
https://openalex.org/W4327843799
|
https://hal.science/hal-03979335/document
|
French
| null |
The ecological footprint of the French-German border (1871-1914)
|
HAL (Le Centre pour la Communication Scientifique Directe)
| 2,021
|
cc-by-sa
| 9,574
|
L’empreinte écologique de la frontière franco-allemande
(1871-1914)
Benoît Vaillot Benoît Vaillot To cite this version: Benoît Vaillot. L’empreinte écologique de la frontière franco-allemande (1871-1914). Revue du Rhin
Supérieur, 2021, 3, pp.21-40. 10.57086/rrs.183. hal-03979335 Benoît Vaillot. L’empreinte écologique de la frontière franco-allemande (1871-1914). Revue du Rhin
Supérieur, 2021, 3, pp.21-40. 10.57086/rrs.183. hal-03979335 HAL Id: hal-03979335
https://hal.science/hal-03979335v1
Submitted on 8 Feb 2023 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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 - ShareAlike 4.0 International License BENOIT VAILLOT
L’empreinte écologique
de la frontière franco-allemande
(1871-1914) Benoit Vaillot est docteur en histoire de l’Institut Universitaire Européen, et chercheur
associé à l’Université de Strasbourg et au Centre Marc-Bloch. Ses recherches portent sur
la frontière franco-allemande entre 1871 et 1914, lorsque la frontière a été déplacée après
l’annexion de l’Alsace-Lorraine par l’Empire allemand. Il s’intéresse à la construction de la
souveraineté et des identités nationales par en bas, dans une perspective transnationale. Il a enseigné l’histoire contemporaine et la culture générale à l’université, ainsi que l’his-
toire-géographie en lycée et collège. La frontière constitue un poste d’observation privilégié de l’empreinte éco-
logique des sociétés humaines, dans la mesure où s’y confrontent deux
conceptions différentes de la gestion de l’environnement. Les espaces forestiers
sont particulièrement intéressants, parce que l’on peut y analyser finement les
relations de pouvoir écologiques. Il s’agit dans cet article d’analyser sur une
période relativement courte, entre 1871 et 1914, comment la frontière franco-al-
lemande transforme l’environnement, et l’influence que ce dernier peut avoir sur
la construction de la souveraineté et des identités nationales. 21 The border is a privileged observation post for the ecological footprint of human
societes, insofar as two different conceptions of environemental management
are confronted. Forest areas are particularly interesting because they allow an
analysis of ecological power relations. This article analyses, over a relatively
short period of time, between 1871 and 1914, how the French-German border
transformed the environment, and the influence that the latter could have on the
construction of sovereignty and national identities. 22 À
ll À
l’issue de la guerre de 1870, la France se voit contrainte de céder à
l’Empire allemand les territoires qui formeront l’Alsace-Lorraine,
par le traité de Francfort du 10 mai 1871. La nouvelle frontière fran-
co-allemande suit alors en grande partie la ligne de crête des Vosges, et
traverse principalement des espaces forestiers en altitude. Les forêts ont
même été au cœur des négociations relatives à son tracé, car elles sont non
seulement essentielles pour la défense du territoire, mais aussi utiles à bien
des égards : on y exploite le bois, on y chasse les animaux et l’on commence
à s’y rendre à des fins récréatives en cette fin de xixe siècle. Fig. 1. Profil altimétrique de la frontière franco-allemande (1871-1914) Fig. 1. 1. Sophie Houdart et Olivier Thiery (dir.), Humains, non-humains : comment repeupler les
sciences sociales, Paris, La Découverte, 2011 ; Bruno Latour, Politiques de la nature. Comment
faire entrer les sciences en démocratie, Paris, Éditions la Découverte, 1999. q
faire entrer les sciences en démocratie, Paris, Éditions la Découverte, 1999. 7. Conrad J. Bahre et Charles F. Hutchinson, « Historic Vegetation Change in La Frontera West
of the Rio Grande », in G. L. Webster et C. J. Bahre (dir.), Changing Plant Life of La Frontera :
Observations on Vegetation in the U.S./Mexico Borderlands, Albuquerque, University of New
Mexico Press, 2001, p. 67-83. 6. A. R. Graybill, « Boundless Nature. Borders and the Environment in North America and
Beyond », art. cit. 2. Anne-Laure Amilhat Szary, « “Montagnes mobiles ?” », Journal of Alpine Research | Revue de
géographie alpine, 101-2 (2013) [En ligne : https://journals.openedition.org/rga/2135]. 5. Jean-Pierre Husson, La forêt lorraine. Étude de géographie humaine, thèse d’État, Université
de Metz, 1987. 3. Sylvain Guyot, « The Eco-Frontier Paradigm : Rethinking the Links between Space, Nature and
Politics », Geopolitics, 16-3 (2011), p. 675-706. 4. Andrew R. Graybill, « Boundless Nature. Borders and the Environment in North America and
Beyond », in A. C. Isenberg et A. R. Graybill (dir.), The Oxford Handbook of Environmental History,
Oxford / New York, Oxford University Press, 2014, p. 668-687. Mexico Press, 2001, p. 67-83. 2. Anne-Laure Amilhat Szary, « “Montagnes mobiles ?” », Journal of Alpine Research | Revue de
géographie alpine, 101-2 (2013) [En ligne : https://journals.openedition.org/rga/2135].
3. Sylvain Guyot, « The Eco-Frontier Paradigm : Rethinking the Links between Space, Nature and
Politics », Geopolitics, 16-3 (2011), p. 675-706.
4. Andrew R. Graybill, « Boundless Nature. Borders and the Environment in North America and
Beyond », in A. C. Isenberg et A. R. Graybill (dir.), The Oxford Handbook of Environmental History,
Oxford / New York, Oxford University Press, 2014, p. 668-687.
5. Jean-Pierre Husson, La forêt lorraine. Étude de géographie humaine, thèse d’État, Université
de Metz, 1987.
6. A. R. Graybill, « Boundless Nature. Borders and the Environment in North America and
Beyond », art. cit.
7. Conrad J. Bahre et Charles F. Hutchinson, « Historic Vegetation Change in La Frontera West
of the Rio Grande », in G. L. Webster et C. J. Bahre (dir.), Changing Plant Life of La Frontera :
Observations on Vegetation in the U.S./Mexico Borderlands, Albuquerque, University of New
Mexico Press, 2001, p. 67-83. BENOIT VAILLOT
L’empreinte écologique
de la frontière franco-allemande
(1871-1914) Profil altimétrique de la frontière franco-allemande (1871-1914) Une approche anthropocentrée nous fait oublier que les forêts sont
avant tout le cadre de vie de multiples acteurs « non humains », comme
les arbres, les plantes ou même les animaux1. Ces acteurs vivants « non humains » ne sont pas des données immuables, mais le produit de forces
historiques. Si l’on prête suffisamment attention à l’environnement, l’étude
des frontières révèle l’empreinte des sociétés humaines sur leurs écosys-
tèmes. Les forêts reflètent les sociétés humaines dans leurs actions et dans
leurs perceptions, et c’est pourquoi elles intéressent l’historien ; elles per-
mettent de penser les conditions de production des relations sociales dans
des espaces caractérisés par des spécificités environnementales, qu’elles
soient climatiques, topographiques ou culturelles2. Il s’agit de proposer
une approche environnementale des rapports de domination au sein des
sociétés dans leur relation aux acteurs vivants « non humains »3. La question environnementale reste un angle mort de l’historiogra-
phie des frontières. Il faut principalement se tourner vers la géographie,
la géohistoire en particulier, si l’on veut s’appuyer sur des études articu-
lant environnement et frontières4. L’œuvre de Jean-Pierre Husson, auteur
d’une histoire de la forêt lorraine dont la grande spécificité est précisément
d’avoir été séparée par la frontière franco-allemande entre 1871 et 1918, fait
exception5. Certains historiens nord-américains ont par ailleurs précoce-
ment étudié l’impact des sociétés humaines sur les écosystèmes en mettant
en avant la question des frontières6. Leurs études ont mis en évidence les
divergences environnementales qui se sont développées au fur et à mesure
de la formation des États. Conrad J. Bahre et Charles F. Hutchinson, par
exemple, ont montré en quoi les modifications intervenues dans la flore
au xxe siècle le long de la frontière mexicano-étatsunienne, ne sont pas
dues aux conditions climatiques, mais bel et bien aux sociétés humaines7. À partir d’une série de photographies des obélisques démarquant la frontière
à l’ouest du Rio Grande prises dans les années 1890 et de clichés réalisés
entre les années 1960 et 1990, ils retracent l’évolution de la végétation. Frontières et espaces transfrontaliers, une approche environnementale
24 24 Il n’est malheureusement pas possible d’adopter la même méthode,
ne disposant pas de photographies ou de descriptions suffisantes de la
frontière franco-allemande au début des années 1870. 8. La Revue des Eaux et Forêts rattachée à l’École nationale des Eaux et Forêts de Nancy, la
Forstwissenschaftliches Centralblatt et le Journal für das Forst-, Jagd- und Fischereiwesen qui
dépendent de la Höhere Forstlehranstalt d’Eberswalde, sont des revues scientifiques centrali-
sant les débats et l’actualité de la science forestière en France et en Allemagne, mais qui ne se
limitent pas au cadre national.
9. Henri Gerdolle, « Die Wälder Deutsch-Lothringen », Deutsche Geographische Blätter, 18 (1877),
p. 19-36 ; Id., L’administration forestière allemande en Alsace-Lorraine, Metz, Charles Thomas,
1877. 10. August Kahl, Der Buchenhochwald auf dem Vogesensandstein der Oberförsterei Bannstein
(Canton Bitsch, Lothringen) nach Standort und Art der Verjüngung, Munich, Johannes Falk, 1883 ;
Id., Forstgeschichtliche Skizzen aus den Staats- und Gemeindewaldungen von Rappoltsweiler und
Reichenweier aus der Zeit vom Ausgange des Mittelalters bis zu Anfang des xix. Jahrhunderts,
Strasbourg, Heitz J. H. E, 1894 ; Id., Handbuch für den Elsass-Lothringischen Förster, enthaltend
eine Zusammenstellung und Erlaüterung aller wichtigen Gesetze, Verordnungen, Vorschriften fuer
die Forstschutzbeamten in den Staats- und ungeteilten Waldungen und in den Gemeinde- und
Anstalts-Waldungen, Melsungen, Neudamm, 1900. 8. La Revue des Eaux et Forêts rattachée à l’École nationale des Eaux et Forêts de Nancy, la
Forstwissenschaftliches Centralblatt et le Journal für das Forst-, Jagd- und Fischereiwesen qui
dépendent de la Höhere Forstlehranstalt d’Eberswalde, sont des revues scientifiques centrali-
sant les débats et l’actualité de la science forestière en France et en Allemagne, mais qui ne se
limitent pas au cadre national.
9. Henri Gerdolle, « Die Wälder Deutsch-Lothringen », Deutsche Geographische Blätter, 18 (1877),
p. 19-36 ; Id., L’administration forestière allemande en Alsace-Lorraine, Metz, Charles Thomas,
1877.
10. August Kahl, Der Buchenhochwald auf dem Vogesensandstein der Oberförsterei Bannstein
(Canton Bitsch, Lothringen) nach Standort und Art der Verjüngung, Munich, Johannes Falk, 1883 ;
Id., Forstgeschichtliche Skizzen aus den Staats- und Gemeindewaldungen von Rappoltsweiler und
Reichenweier aus der Zeit vom Ausgange des Mittelalters bis zu Anfang des xix. Jahrhunderts,
Strasbourg, Heitz J. H. E, 1894 ; Id., Handbuch für den Elsass-Lothringischen Förster, enthaltend
eine Zusammenstellung und Erlaüterung aller wichtigen Gesetze, Verordnungen, Vorschriften fuer
die Forstschutzbeamten in den Staats- und ungeteilten Waldungen und in den Gemeinde- und
Anstalts-Waldungen, Melsungen, Neudamm, 1900.
11. Gustave Huffel, « Les forêts d’Alsace-Lorraine », Revue des Eaux et Forêts, 6 (1918), p. 265-280.
12. C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute
futaie », Revue des Eaux et Forêts, 1921, p. 33-39.
13. Robin Degron et Jean-Pierre Husson, « Les forêts d’Alsace-Lorraine (1871-1918) : les
contrastes de l’héritage allemand, reflets de l’annexion », Histoire, économie & société, 18-3
(1999), p. 593-611. 13. Robin Degron et Jean-Pierre Husson, « Les forêts d’Alsace-Lorraine (1871-1918) : les
contrastes de l’héritage allemand, reflets de l’annexion », Histoire, économie & société, 18-3
(1999), p. 593-611. 11. Gustave Huffel, « Les forêts d’Alsace-Lorraine », Revue des Eaux et Forêts, 6 (1918), p. 265-280.
12. C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute
futaie », Revue des Eaux et Forêts, 1921, p. 33-39. 11. Gustave Huffel, « Les forêts d’Alsace-Lorraine », Revue des Eaux et Forêts, 6 (1918), p. 265-280.
12. C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute
futaie », Revue des Eaux et Forêts, 1921, p. 33-39.
13. Robin Degron et Jean-Pierre Husson, « Les forêts d’Alsace-Lorraine (1871-1918) : les
d
l’hé i
ll
d
fl
d
l’
i
Hi
i
é
i
ié é 1877.
10. August Kahl, Der Buchenhochwald auf dem Vogesensandstein der Oberförsterei Bannstein
(Canton Bitsch, Lothringen) nach Standort und Art der Verjüngung, Munich, Johannes Falk, 1883 ;
Id., Forstgeschichtliche Skizzen aus den Staats- und Gemeindewaldungen von Rappoltsweiler und
Reichenweier aus der Zeit vom Ausgange des Mittelalters bis zu Anfang des xix. Jahrhunderts,
Strasbourg, Heitz J. H. E, 1894 ; Id., Handbuch für den Elsass-Lothringischen Förster, enthaltend
eine Zusammenstellung und Erlaüterung aller wichtigen Gesetze, Verordnungen, Vorschriften fuer
die Forstschutzbeamten in den Staats- und ungeteilten Waldungen und in den Gemeinde- und
Anstalts-Waldungen, Melsungen, Neudamm, 1900.
11. Gustave Huffel, « Les forêts d’Alsace-Lorraine », Revue des Eaux et Forêts, 6 (1918), p. 265-280.
12. C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute
futaie », Revue des Eaux et Forêts, 1921, p. 33-39.
13. Robin Degron et Jean-Pierre Husson, « Les forêts d’Alsace-Lorraine (1871-1918) : les
contrastes de l’héritage allemand, reflets de l’annexion », Histoire, économie & société, 18-3
(1999), p. 593-611. BENOIT VAILLOT
L’empreinte écologique
de la frontière franco-allemande
(1871-1914) Même les nombreuses
cartes postales réalisées entre les années 1880 et 1914 ne permettent pas
de pallier ce manque, car elles sont prises sous des angles trop différents
pour établir des comparaisons, et ne sont pas datées avec précision. C’est
pourquoi une grande attention a été prêtée à tous ceux qui ont été sensibles
à la transformation de l’environnement. À ce titre, les écrits de forestiers
publiés dans des revues spécialisées constituent des sources exception-
nelles8. Les témoignages des gardes forestiers restés en Alsace-Lorraine
après 1871, comme Henri Gerdolle9, et de ceux, formés en Allemagne, qui
s’y installent après l’annexion, comme August Kahl10, livrent quantité
d’informations sur les changements écologiques qu’introduisent les admi-
nistrations forestières. Les rapports de Gustave Huffel11 et C. Zwilling12, qui
établissent un bilan de l’administration forestière allemande lors du retour
de l’Alsace-Lorraine à la France après la Première Guerre mondiale, sont
aussi très précieux, bien qu’ils souffrent d’un parti-pris qu’il faut mettre
à distance13. La frontière franco-allemande tracée par le traité de Francfort
constitue un poste d’observation privilégié de l’empreinte écologique des
sociétés humaines, dans la mesure où s’y confrontent deux conceptions
différentes de la gestion de l’environnement entre 1871 et 1914. Les espaces
forestiers sont particulièrement intéressants, parce qu’adaptés à l’analyse du « schéma des relations de pouvoirs écologiques »14. Il s’agit d’analyser
sur une période très courte comment une frontière transforme l’environ-
nement, et l’influence que ce dernier peut avoir sur la construction de
la souveraineté et des identités nationales. Cette approche transnationale
permet par ailleurs d’éviter l’écueil d’une histoire environnementale dans
un cadre national15. 14. Richard Grove, Ecology, Climate and Empire. Colonialism and Global Environmental History,
1400-1940, Cambridge, White Horse Press, 1997, p. 183.
15. Richard White, « The Nationalization of Nature », The Journal of American History, 86-3 (1999),
p. 976-986.
16. François Reitel, « Le rôle de l’armée dans la conservation des forêts en France », Bulletin de
l’Association des Géographes Français, 501 (1984), p. 143-154.
17. Jean-Paul Amat, « Le rôle stratégique de la forêt (1871-1914). Exemples dans les forêts lor-
raines. », Revue historique des armées, 1 (1993), p. 62-69.
18. Michaël Séramour, Frontières d’acier. Histoire de la fortification permanente en Lorraine et en
Alsace 1871-1945, Saint-Avertin, Éditions Sutton, 2015. 14. Richard Grove, Ecology, Climate and Empire. Colonialism and Global Environmental History,
1400-1940, Cambridge, White Horse Press, 1997, p. 183.
15. Richard White, « The Nationalization of Nature », The Journal of American History, 86-3 (1999),
p. 976-986. L’AMÉNAGEMENT DE L’ENVIRONNEMENT
PAR LES AUTORITÉS MILITAIRES La mise en défense de la frontière franco-allemande a eu des consé-
quences majeures sur les forêts, car les autorités militaires assignent à ces
dernières un rôle stratégique majeur. Pour reprendre François Reitel,
« [l]es forêts permettent de couper les routes d’invasion, de couvrir les
places fortes, de canaliser un ennemi vers les forteresses, d’économiser
des hommes »16. Après la guerre de 1870, les états-majors des deux pays ont dû repenser
les défenses nationales pour s’adapter aux nouvelles réalités physiques
de la frontière17. La situation est très préoccupante pour la France : si la
citadelle de Belfort a finalement été conservée, Paris n’est plus qu’à 300
kilomètres de l’Allemagne. Inversement, le contrôle de la majeure partie du
massif vosgien et la possession des places fortes de Metz et Strasbourg per-
mettent à l’Empire allemand d’exclure toute menace d’offensive française
à court et moyen terme. Dès le début des années 1870, plusieurs chantiers
de fortification débutent de part et d’autre de la frontière, afin de consti-
tuer des obstacles partout où les hauteurs et les forêts peuvent manquer. Les forts allemands de type Biehler sont construits autour de Strasbourg
entre 1872 et 188518. L’extension des forts autour de Metz, débutée par les
autorités militaires françaises, est achevée au profit de l’Empire allemand. L’état-major français déploie de son côté le programme Séré de Rivières Frontières et espaces transfrontaliers, une approche environnementale
26 26 à partir de 1874, qui fait sortir de terre toute une série de forts autour
de Verdun, Toul et Épinal afin de former des camps retranchés, ceinturés
de forts détachés. La place forte de Belfort est renforcée afin de verrouiller
la trouée du même nom. Ces différents môles de résistance ont pour but de
contraindre l’ennemi à s’engager dans les trouées de Stenay et des Charmes,
densément boisées. Après 1885, l’état-major français se borne principalement à moderniser
les ouvrages principaux et à aménager les abords. Tout au contraire, la place
de Strasbourg est renforcée. L’état-major allemand entreprend, à partir des
années 1890, un programme de fortification de Metz qui en fait le pivot de
sa défense face à la France. Une ligne défensive allant de Thionville à Metz
– appelée Moselstellung – est édifiée à partir de 1899 et devient très vite
le plus grand ensemble fortifié au monde19. La trouée de Lorraine, quant
à elle, est défendue par les garnisons allemandes. 19. Clayton Donnell et Brian Delf, The German Fortress of Metz 1870-1944, Oxford/New York,
Osprey Publishing, 2008.
20. François Roth, « La frontière franco-allemande, 1871-1918 », Annales de l’Est, 1 (1992),
p. 35-52. 21. Archives du Territoire de Belfort [désormais AD90], 1 M 388, Courrier du chef de Génie de
Belfort à l’inspecteur adjoint des Eaux et Forêts de Belfort, 29 mars 1893.
22. AD90, 1 M 385, Courrier du Conservateur des Forêts de Besançon à l’Inspecteur adjoint
des Forêts de Belfort, 10 avril 1877 ; Archives départementales du Haut-Rhin [désormais AD68],
8 AL 1/7152, Construction de nouveaux chemins, routes et voies forestières près de la frontière,
1892-1914. 21. Archives du Territoire de Belfort [désormais AD90], 1 M 388, Courrier du chef de Génie de
Belfort à l’inspecteur adjoint des Eaux et Forêts de Belfort, 29 mars 1893. 22. AD90, 1 M 385, Courrier du Conservateur des Forêts de Besançon à l’Inspecteur adjoint
des Forêts de Belfort, 10 avril 1877 ; Archives départementales du Haut-Rhin [désormais AD68],
8 AL 1/7152, Construction de nouveaux chemins, routes et voies forestières près de la frontière,
1892-1914. L’AMÉNAGEMENT DE L’ENVIRONNEMENT
PAR LES AUTORITÉS MILITAIRES À la veille de la Première
Guerre mondiale, les départements frontaliers forment la première ligne
de défense française, tandis que l’Alsace-Lorraine remplit le rôle de glacis
militaire de l’Empire allemand. Les ouvrages militaires ne constituent pas
de lignes de défense continue comme le seront plus tard les lignes Maginot
ou le Westwall, ce qui explique pourquoi l’expérience sensible des habitants
de la frontière est plus marquée par le paysage forestier que par les forts
et forteresses : « À la frontière on ne voyait ni blockhaus ni barbelés mais
le calme étendu des champs et des forêts »20. Fig. 2. L’intégration des espaces forestiers frontaliers à la défense Fig. 2. L’intégration des espaces forestiers frontaliers à la défense L’absence ou la présence d’espaces forestiers à la frontière est fortement
corrélée aux nécessités de défense nationale. Les défrichements et aména-
gements forestiers sont soumis à l’autorisation d’une commission mixte des
travaux publics, au sein de laquelle le génie militaire a voix prépondérante21. Les travaux de voies de communication compris non loin des ouvrages
de défense doivent recevoir l’adhésion des autorités militaires22. Même le
dégagement de la frontière et la construction de chemins de schlittage
pour l’exploitation forestière en haute montagne ne sauraient s’en passer. En 1897, le colonel commandant du 152e régiment d’infanterie demande à ce
que la section vosgienne (Nancy) du Club alpin français renonce à baliser Frontières et espaces transfrontaliers, une approche environnementale
28 28 les sentiers qui mènent à la frontière23. L’autorité militaire empêche parti-
culièrement la construction de routes empierrées ou bitumées de plus de
quatre mètres de large, car les voitures militaires pourraient l’emprunter. q
g
p
p
Les autorités militaires ont donc non seulement assumé de fait un rôle
de conservation du couvert forestier, en raison du rôle défensif qu’elles lui
attribuent, mais elles participent aussi au reboisement. Les bois de la rive
droite de la Moselle, par exemple, sont propriété de l’armée allemande et
protégés en raison de leur rôle stratégique et ce malgré la forte pression
urbaine de l’agglomération de Metz à la fin du xixe siècle. En France,
planter des arbres à la frontière devient, après 1871, un acte patriotique, car
c’est préparer une « barrière » contre une éventuelle invasion allemande. 23. Archives départementales de Meurthe-et-Moselle [désormais AD54], 4 M 179, Courrier du
général commandant le 6e Corps d’armée au préfet de Meurthe-et-Moselle, 23 juillet 1897.
24. Jean-Pierre Husson, « Les paysages anciens de la forêt domaniale de Gérardmer : contribu-
tion patrimoniale à la révision d’aménagement », Revue Forestière Française, 5 (1997), p. 469-476.
25. J.-P. Husson, La forêt lorraine, op. cit, p. 314.
26. F. Reitel, « Le rôle de l’armée dans la conservation des forêts en France », art. cit.
27. Jean-Paul Amat, « Guerre et milieux naturels : les forêts meurtries de l’Est de la France,
70 ans après Verdun », L’Espace géographique, 16-3 (1987), p. 217-233. 27. Jean-Paul Amat, « Guerre et milieux naturels : les forêts meurtries de l’Est de la France,
70 ans après Verdun », L’Espace géographique, 16-3 (1987), p. 217-233. 26. F. Reitel, « Le rôle de l’armée dans la conservation des forêts en France », art. cit. L’AMÉNAGEMENT DE L’ENVIRONNEMENT
PAR LES AUTORITÉS MILITAIRES La
tempête de 1902, qui provoque de nombreux chablis (chutes d’arbres) sur
le versant français des Vosges, en particulier dans la forêt de Gérardmer,
est vécue comme un traumatisme par les populations et l’administration
des Eaux et Forêts, car elle découvre la frontière24. Le gouvernement d’Al-
sace-Lorraine ne dépense pas moins de 50 000 marks par an pour l’achat de
friches, afin de constituer des boisements de protection25. Jusqu’en 1914, les
surfaces forestières progressent tout le long de la frontière, à l’exception du
pays messin et thionvillois, en raison de la construction de la Moselstellung
qui nécessite le défrichement de plusieurs bois. L’empreinte militaire sur l’environnement est rendue visible à travers
les aménagements effectués dans les forêts26. Les peuplements forestiers
frontaliers sont percés de trouées qui n’ont pas pour objet de régénérer les
bois, mais de les rendre praticables par des hommes en armes27. Inversement,
des rideaux d’arbres sont plantés pour constituer des barrières naturelles
censées empêcher toute progression ennemie. Les forêts à l’est de Verdun,
entre les forts de Douaumont et de Vaux, sont ainsi aménagées pour servir
d’écran de protection. L’édification d’ouvrages militaires nécessite parfois le
déboisement afin de dégager des vues, d’ouvrir des axes de tir, de construire
des redoutes, de creuser des retranchements, de percer des lignes de
chemin de fer, etc. Les forêts domaniales, communales et particulières qui entourent Épinal en 1883 sont ainsi déboisées28. À la frontière, l’expropria-
tion des bois pour raison d’utilité publique est courante29. La commune de
Chavelot, par exemple, est contrainte de se dessaisir de ses forêts en 1887
pour permettre la modernisation des forts d’Uxegney et de Grande-Haye. La progression des surfaces forestières à la frontière sur la période
est en grande partie due à l’intervention des états-majors. La qualité et la
diversité des forêts sont quant à elles profondément modifiées par les admi-
nistrations forestières françaises et allemandes, qui adoptent des méthodes
sylvicoles différentes. entourent Épinal en 1883 sont ainsi déboisées28. À la frontière, l’expropria-
tion des bois pour raison d’utilité publique est courante29. La commune de
Chavelot, par exemple, est contrainte de se dessaisir de ses forêts en 1887
pour permettre la modernisation des forts d’Uxegney et de Grande-Haye. La progression des surfaces forestières à la frontière sur la période
est en grande partie due à l’intervention des états-majors. L’AMÉNAGEMENT DE L’ENVIRONNEMENT
PAR LES AUTORITÉS MILITAIRES La qualité et la
diversité des forêts sont quant à elles profondément modifiées par les admi-
nistrations forestières françaises et allemandes, qui adoptent des méthodes
sylvicoles différentes. 33. Thaddeus Sunseri, « Reinterpreting a Colonial Rebellion : Forestry and Social Control in
German East Africa, 1874-1915 », Environmental History, 8-3 (2003), p. 430-451 : Id., « Exploiting
the Urwald : German Post-Colonial Forestry in Poland and Central Africa, 1900–1960 », Past &
Present, 214-1 (2012), p. 305-342 ; Jawad Daheur, « La forêt : espace de légitimation du projet
colonial allemand en Chine (1898-1914) », Recherches germaniques, 43 (2013), p. 95-110. 31. Michael Imort, « Wilhelmine Forestry and the Forest as a Symbol of Germandom », in T. Zelle
et T. Lekan (dir.), Germany’s Nature: Cultural Landscapes and Environmental History, New
Brunswick, Rutgers University Press, 2005, p. 55-60. 28. Archives départementales des Vosges [désormais AD88], 38 R 12, Arrêté préfectoral concer-
nant les travaux de défense, 12 avril 1883.
29. F. Reitel, « Le rôle de l’armée dans la conservation des forêts en France », art. cit.
30. Heinrich Rubner, « Geistige Strömungen in der Forstpolitik und ihre Auswirkungen auf die
Forstwirtschaft des neuzeitlichen Frankreich », Allgemeine Forst und Jagdzeitung, 1 (1993),
p. 12-17.
31. Michael Imort, « Wilhelmine Forestry and the Forest as a Symbol of Germandom », in T. Zeller
et T. Lekan (dir.), Germany’s Nature: Cultural Landscapes and Environmental History, New
Brunswick, Rutgers University Press, 2005, p. 55-60.
32. Jawad Daheur, Le Parc à bois de l’Allemagne. Course aux ressources et hégémonie commer-
ciale dans les bassins de la Vistule et de la Warta (1840-1914), thèse de doctorat, Université de
Strasbourg, 2016 ; Jeffrey K. Wilson, « Environmental Chauvinism in the Prussian East : Forestry
as a Civilizing Mission on the Ethnic Frontier, 1871-1914 », Central European History, 41-1 (2008),
p. 27-70.
33. Thaddeus Sunseri, « Reinterpreting a Colonial Rebellion : Forestry and Social Control in
German East Africa, 1874-1915 », Environmental History, 8-3 (2003), p. 430-451 : Id., « Exploiting
the Urwald : German Post-Colonial Forestry in Poland and Central Africa, 1900–1960 », Past &
Present, 214-1 (2012), p. 305-342 ; Jawad Daheur, « La forêt : espace de légitimation du projet
colonial allemand en Chine (1898-1914) », Recherches germaniques, 43 (2013), p. 95-110. 28. Archives départementales des Vosges [désormais AD88], 38 R 12, Arrêté préfectoral concer-
nant les travaux de défense, 12 avril 1883. 30. Heinrich Rubner, « Geistige Strömungen in der Forstpolitik und ihre Auswirkungen auf die
Forstwirtschaft des neuzeitlichen Frankreich », Allgemeine Forst und Jagdzeitung, 1 (1993),
p. 12-17. 29. F. Reitel, « Le rôle de l’armée dans la conservation des forêts en France », art. cit. 32. Jawad Daheur, Le Parc à bois de l’Allemagne. Course aux ressources et hégémonie commer-
ciale dans les bassins de la Vistule et de la Warta (1840-1914), thèse de doctorat, Université de
Strasbourg, 2016 ; Jeffrey K. Wilson, « Environmental Chauvinism in the Prussian East : Forestry
as a Civilizing Mission on the Ethnic Frontier, 1871-1914 », Central European History, 41-1 (2008),
p. 27-70. LA CONFRONTATION DE DEUX SYLVICULTURES L’historien doit maintenant tâcher de pénétrer à l’intérieur de ces
forêts, traversées par la frontière du traité de Francfort, soumises à deux
sylvicultures différentes30. L’analyse des rapports de pouvoir liés à l’ex-
ploitation des forêts en Alsace-Lorraine, mis en perspective avec ce qui
est pratiqué dans l’Est de la France à la même époque, révèle un « impé-
rialisme écologique » allemand31. À travers l’introduction de nouvelles
méthodes sylvicoles, les autorités allemandes exercent leur domination
sur les populations d’Alsace-Lorraine, d’une manière comparable à celle
rencontrée dans les provinces polonaises de Prusse32 et dans les territoires
coloniaux allemands33. 33. Thaddeus Sunseri, « Reinterpreting a Colonial Rebellion : Forestry and Social Control in
German East Africa, 1874-1915 », Environmental History, 8-3 (2003), p. 430-451 : Id., « Exploiting
the Urwald : German Post-Colonial Forestry in Poland and Central Africa, 1900–1960 », Past &
Present, 214-1 (2012), p. 305-342 ; Jawad Daheur, « La forêt : espace de légitimation du projet
colonial allemand en Chine (1898-1914) », Recherches germaniques, 43 (2013), p. 95-110. Frontières et espaces transfrontaliers, une approche environnementale
30 30 La science forestière allemande au xixe siècle est pionnière à bien des
égards et jouit d’une grande renommée internationale34. Le pivot de cette
expertise repose sur la Höhere Forstlehranstalt (établissement supérieur de
foresterie) d’Eberswalde, où sont formés les gardes forestiers de Prusse
depuis 183035. La création concomitante de l’École nationale des Eaux et
Forêts à Nancy n’a pas d’autre origine que la volonté de rivaliser avec l’Alle-
magne dans ce domaine. La rivalité franco-allemande s’exprime avec éclat
à la frontière, où des expériences sylvicoles sont effectuées pour démontrer
sa prétendue supériorité. Ainsi, Hermann Bertog, professeur à la Höhere
Forstlehranstalt, assiste en 1897 aux expériences sylvicoles effectuées dans
la forêt de Nancy36. Les sciences forestières française et prussienne ne
se distinguent pas tant par leurs objectifs sylvicoles – le reboisement,
la conversion des taillis en futaies, l’enrésinement, etc. – que par les
méthodes mises en œuvre pour y parvenir, méthodes censées refléter le
génie national37. L’annexion de quelque 135 000 hectares de forêts doma-
niales et de 200 000 hectares de forêts communales ou appartenant à des
établissements publics offrent aux autorités allemandes l’occasion d’appli-
quer à l’échelle de l’Alsace-Lorraine, les méthodes de la science forestière
prussienne et de mettre fin à une gestion française jugée calamiteuse38. 34. Henry E. Lowood, « The Calculating Forester: Quantification, Cameral Science, and the
Emergence of Scientific Forestry Management in Germany », in T. Frängsmyr, J. L. Heilbron et
R. E. Rider (dir.), The quantifying spirit in the 18th century, Berkeley, University of California Press,
1990, p. 315-342. 35. H. Rubner, « Geistige Strömungen in der Forstpolitik und ihre Auswirkungen auf die
Forstwirtschaft des neuzeitlichen Frankreich », art. cit. 38. F. von Etzel, Aus dem Reichslande, Berlin, Julius Springer, 1876 ; et plus tard, Karl Eduard Ney,
Zur xix. Versammlung des Elsaß-Lothringischen Forstvereins, 1897. 36. Archives diplomatiques [désormais Arch. Dipl.], 131 CPCOM 131, Demande de Mr. Bertog,
Visite de l’école forestière de Nancy. 37. Jean-Pierre Husson, Les forêts françaises, Nancy, Presses universitaires de Nancy, 1995,
p. 156. 39. H. Gerdolle, L’administration forestière allemande en Alsace-Lorraine, op. cit. 40. Archives départementales du Bas-Rhin [désormais AD67], 27 AL 1064 et 1065, Fonctionnaires
de l’administration forestière, 1879-1916. 41. Alexandre Barte de Sainte-Fare, « Le régime forestier en Alsace-Lorraine », Revue des Eaux et
Forêts, 1879, p. 5-13 et p. 97-115. 38. F. von Etzel, Aus dem Reichslande, Berlin, Julius Springer, 1876 ; e
Zur xix. Versammlung des Elsaß-Lothringischen Forstvereins, 1897. LA CONFRONTATION DE DEUX SYLVICULTURES Avant même la signature du traité de Francfort, un Oberforstbeamter
en charge de l’administration forestière est détaché auprès du commis-
saire civil pour l’Alsace-Lorraine39. Très vite, l’administration forestière est
réorganisée sur le modèle prussien40. Les Oberförstereien ont une superficie
de moyenne de 5 600 hectares contre 6 000 à 7 000 pour les cantonne-
ments français41. En revanche, les triages sont plus vastes et s’étendent en
moyenne sur 600 hectares. Si les prérogatives de police des espaces naturels et des forêts des gardes forestiers français et allemands sont similaires,
leur rôle de gestion et de protection des forêts est extrêmement différent. La hiérarchie de la nouvelle administration forestière d’Alsace-Lorraine est
simplifiée, et ses agents disposent de plus de responsabilités. Les gardes
forestiers allemands disposent d’une plus grande autonomie que leurs
homologues français ; ils sont chargés de réviser les limites parcellaires,
de dresser les états d’assiette et de travail d’amélioration, de marteler les
coupes, de les exploiter, de les recevoir et de les vendre. L’exploitation des forêts communales et domaniales du massif des
Vosges diffère ainsi profondément selon le côté de la frontière où l’on se
situe42. Sur le versant français, le bois est vendu sur pied après estimation
du volume et c’est à l’acheteur d’en organiser la coupe. La montagne est
parsemée de scieries communales ou domaniales, attribuées par adjudi-
cation. Les ouvriers qui y travaillent, appelés sagards, logent sur place et
sont rémunérés par l’adjudicataire du marché public43. C’est un tout autre
système qui prévaut sur le versant allemand, où l’exploitation se fait en
régie. L’administration des forêts prend à sa charge l’abattage des arbres et
vend le bois coupé en petits lots. Ce mode d’exploitation est plus coûteux
en personnel, mais permet d’augmenter considérablement les revenus de
la gestion forestière44. Les scieries communales et domaniales n’ont ainsi
plus aucune utilité, et sont donc vendues ou louées, et parfois transformées
en maisons forestières. Sur toute la période, les gardes forestiers allemands améliorent les
dessertes forestières dans les Vosges et n’hésitent pas à créer 100 kilomètres
de nouvelles pistes alors qu’en France, le génie militaire s’oppose à tous
travaux d’envergure45. Au tournant du siècle, un petit réseau de chemins
de fer à usage forestier voit même le jour, et permet notamment l’exploi-
tation du massif du Donon. 42. H. Gerdolle, L’administration forestière allemande en Alsace-Lorraine, op. cit.
43. Marc Brignon et Jean-Louis Boithias, Les scieries et les anciens sagards des Vosges.
Bûcherons, schlitteurs, voituriers et voileurs, Nonette, Créer, 1985.
44. Charles Grad, Considérations sur les finances et l’administration de l’Alsace-Lorraine sous la
régime allemand, Paris, Germer-Baillère, 1877.
45. R. Degron et J.-P. Husson, « Les forêts d’Alsace-Lorraine (1871-1918) », art. cit. LA CONFRONTATION DE DEUX SYLVICULTURES Sur le versant allemand des Vosges, les coûts
d’exploitation sont sensiblement plus bas que sur le versant français, où
l’on débarde encore le bois par schlittage, flottage et traction animale. Les
travaux d’aménagement de l’administration des forêts d’Alsace-Lorraine
favorisent l’augmentation des investissements forestiers au moment même
où s’accroît la demande de l’industrie locale. Frontières et espaces transfrontaliers, une approche environnementale
32 Frontières et espaces transfrontaliers, une approche environnementale
32 Les forêts du versant allemand des Vosges ne sont pas seulement
exploitées avec plus d’efficacité que celles du versant français, elles sont
aussi aménagées pour correspondre à la représentation que l’imaginaire
national allemand s’en fait au xixe siècle46. L’administration des forêts
d’Alsace-Lorraine accorde très tôt une place aux activités récréatives dans
l’aménagement des forêts, particulièrement près du Donon, ce qui est inédit
de l’autre côté de la frontière. Les Oberförster sont d’ailleurs souvent à la tête
des sections locales du Vogesenclub, association de montagne fondée par les
autorités allemandes47. L’exploitation des forêts communales et domaniales
d’Alsace-Lorraine est tellement efficace qu’elle représente 11 % des revenus
du Reichsland en 1913, contre seulement 0,7 % en France48. La principale question qui occupe les forestiers français et allemands
au xixe siècle est celle de la conversion des taillis en futaie, c’est-à-dire
la transformation de parcelles forestières combinant plusieurs niveaux de
végétations différents (taillis-sous-futaie) en forêts de grands arbres aux
fûts dégagés (futaie)49. Cette dernière est entreprise de façon radicalement
différente de part et d’autre de la frontière. La surexploitation des forêts
du versant allemand a conduit l’administration des forêts d’Alsace-Lor-
raine à recourir à la régénération artificielle des parcelles forestières,
chaque fois que la conversion en essences nobles et délicates était lente
ou difficile. Cette régénération consiste à réaliser des coupes rases suivies
de plantations, et s’oppose à la régénération naturelle privilégiée sur le
versant français. En effet, les gardes forestiers français opèrent progressi-
vement et prudemment la conversion des taillis en futaie. Les forêts sont
régénérées par la réalisation d’éclaircies successives – c’est-à-dire par des
coupes d’amélioration – destinées à favoriser la croissance du peuplement. Les arbres porte-graines sont ensuite dégagés par des coupes de régénéra-
tion qui laissent aux jeunes semis la lumière dont ils ont besoin. Sur un
même massif forestier, coexistent deux conceptions différentes de la forêt :
peu aménagée et « naturelle » sur le versant français ; ordonnée et « artifi-
cielle » sur le versant allemand. 46. Jeffrey K. Wilson, The German Forest: Nature, Identity, and the Contestation of a National
Symbol, 1871-1914, Toronto/Buffalo/Londres, University of Toronto Press, 2012.
47. L’association prend le nom de Club Vosgien lors de la réunion de l’Alsace-Lorraine à la France
en 1918.
48. G. Huffel, « Les forêts d’Alsace-Lorraine », art. cit.
49. L. Badre, « Évolution des aménagements des futaies résineuses dans l’Est de la France et
plus spécialement dans le Haut-Rhin », Revue Forestière Française, 7-8 (1952), p. 500-506. 49. L. Badre, « Évolution des aménagements des futaies résineuses dans l’Est de la France et
plus spécialement dans le Haut-Rhin », Revue Forestière Française, 7-8 (1952), p. 500-506. 47. L’association prend le nom de Club Vosgien lors de la réunion de l’Alsace-Lorraine à la France
en 1918.f 50. Jeffrey K. Wilson, « Environmental Chauvinism in the Prussian East : Forestry as a Civilizing
Mission on the Ethnic Frontier, 1871-1914 », Central European History, 41-1 (2008), p. 27-70.
51. La forêt de Dabo est l’exemple le plus connu d’échec d’extinction des droits ruraux. Jean-
Pierre Husson, « L’héritage forestier prussien en Moselle et son évolution récente », Bulletin de
l’Association de Géographes Français, 65-3 (1988), p. 217-225.
52. G. Huffel, « Les forêts d’Alsace-Lorraine », art. cit.
53. Michel Devèze, « Forêts françaises et forêts allemandes. Étude historique comparée », Revue
historique, 235 (1966), p. 347-380 et 236 (1966), p. 47-68.
54. Simon Schama, Le paysage et la mémoire, Paris, Seuil, 1999. LA CONFRONTATION DE DEUX SYLVICULTURES La conversion des taillis en futaie met en miroir deux sociétés dif-
férentes, et deux méthodes de gestion dont les habitants ont pleinement
conscience. La mise en place d’une politique sylvicole moderne et rationnelle sur le modèle prussien en Alsace-Lorraine est animée par un chauvinisme
environnemental qui ne laisse pas de place aux acteurs locaux50. L’abandon
brutal des taillis-sous-futaie, qui procuraient du bois de chauffage aux
populations rurales, par exemple, n’est rendu possible que par l’existence
d’un pouvoir autoritaire. Cette politique a par ailleurs rencontré des échecs
dans les forêts domaniales51. Inversement, en France, le conservatisme de
l’administration des Eaux et Forêts, qui prône une conversion progressive
des taillis en futaie, correspond à une plus grande prise en compte des
populations rurales. Sur les deux versants des Vosges, les États cherchent à
éteindre les droits ruraux comme l’affouage, qui réserve une partie des bois
des forêts communales à l’usage domestique des habitants, mais les consé-
quences ne sont pas les mêmes. Sous la iiie République, les habitants des
campagnes sont des citoyens qui ont des moyens juridiques et politiques
pour défendre leurs droits, là où il est difficile d’aller contre la puissance
publique dans l’Empire allemand. La conversion des taillis en futaie et
l’extinction des droits ruraux sont donc loin d’être achevées sur le versant
français des Vosges à la veille de la Première guerre mondiale, et quasiment
réalisée sur le versant allemand52. Dans un même massif forestier, deux types de sylviculture coexistent
et s’affrontent : intensive du côté allemand, extensive du côté français. Elles
reflètent des rapports à l’environnement différents53. La frontière est ainsi
productrice de différentiation spatiale jusque dans les espaces forestiers les
plus reculés, ce qui n’est pas sans conséquence sur les paysages. 55. Louis Rudault, « Comparaison entre les méthodes forestières françaises et allemandes appli-
quées en Alsace », Revue des Eaux et Forêts, 1935, p. 831-844 et p. 908-921. 57. F. von Etzel, Aus dem Reichslande, op. cit. 58. Adolphe Tétreau, Commentaire de la loi du 4 avril 1882 sur la restauration et la conservation
des terrains en montagne, Paul Dupont, 1883. 59. C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute
futaie », art. cit. 55. Louis Rudault, « Comparaison entre les méthodes forestières françaises et allemandes appli-
quées en Alsace », Revue des Eaux et Forêts, 1935, p. 831-844 et p. 908-921.
56. Ibid.
57. F. von Etzel, Aus dem Reichslande, op. cit.
58. Adolphe Tétreau, Commentaire de la loi du 4 avril 1882 sur la restauration et la conservation
des terrains en montagne, Paul Dupont, 1883.
59. C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute
futaie », art. cit. 58. Adolphe Tétreau, Commentaire de la loi du 4 avril 1882 sur la restauration et la conservation
des terrains en montagne, Paul Dupont, 1883.
59. C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute
f
i
i 8. Adolphe Tétreau, Commentaire de la loi du
es terrains en montagne, Paul Dupont, 1883. 60. Vincent Moriniaux, « Les Français face à l’enrésinement, xvie-xxe siècles », Annales de géo-
graphie, 609-610 (1999), p. 660-663.
61. R. Degron et J.-P. Husson, « Les forêts d’Alsace-Lorraine (1871-1918) », art. cit.
62. C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute
futaie », art. cit.
63. Gérard Leser, « Le sapin dans les rites, les croyances populaires et l’imaginaire alsacien »,
in A. Corvol (dir.), Le sapin. Enjeux anciens, enjeux actuels, Paris, L’Harmattan, 2001, p. 349-370.
64. Philippe Guinier, « Trois conifères de la flore vosgienne », Bulletin de la Société Botanique de
France, 106 (1959), p. 168-183.
65. Jean-Pierre Husson, « Forêts sommitales et chaumes vosgiennes confrontées à l’essor touris-
tique », Bulletin de l’Association de Géographes Français, 74-3 (1997), p. 320-328. LA TRANSFORMATION DES PAYSAGES FORESTIERS L’administration des forêts allemande a particulièrement modifié le
paysage en introduisant un grand nombre d’essences d’arbres. Les paysages
que nous croyons les plus épargnés par l’intervention humaine se révèlent
parfois, à y regarder de plus près, ceux qui en sont le plus pétris54. À la
veille de la Première Guerre mondiale, le garde forestier Louis Rudault Frontières et espaces transfrontaliers, une approche environnementale
34 34 est frappé de constater qu’en n’importe quel point, l’observateur même le
moins averti est frappé par l’aspect différent des sapinières selon le côté de
la frontière55. Alors que sur le côté français, les futaies régulières sont quasi
absentes, sur le versant allemand des Vosges, on aperçoit des futaies régu-
lières avec des peuplements équiennes (composés d’arbres ayant le même
âge). C’est également vrai ailleurs, par exemple dans les bois entre Réchésy
et Pfetterhouse qui jouxtent la frontière de la Suisse, où un « médiocre tail-
lis-sous-futaie » en territoire français rencontre un « magnifique perchis
de hêtre et de chêne » en territoire allemand56.f Les différences des cycles de coupe et de régénération des forêts l’ex-
pliquent en grande partie57. En France, les pins sont abattus à l’âge de
80 ans, les sapins et les hêtres lorsqu’ils sont centenaires, et les chênes
seulement après 120 ans. L’administration des forêts d’Alsace-Lorraine,
pour sa part, ne coupe les sapins, les pins et les hêtres qu’une fois âgés
de 120 ans et les chênes après 160 ans. À mesure que l’on avance dans la
période, les classes d’âge et les essences des arbres cohabitant sur une même
parcelle boisée tendent à être plus homogènes sur le territoire allemand. Les gardes forestiers ont beau, par exemple, planter le même chêne rouge
de part et d’autre de la frontière, les forêts françaises ont une plus grande
diversité de classes d’âge et d’essences d’arbres en raison de la méthode de
régénération naturelle employée par l’administration des Eaux et Forêts. En territoire français, les coupes d’arbres sont nombreuses mais sélectives,
ce qui favorise une certaine permanence du paysage, d’autant plus que la
loi du 4 avril 1882 élève la forêt au rang de patrimoine national à aménager
avec prudence58. Les agents forestiers allemands réalisent quant à eux des
coupes rases pour régénérer les parcelles, ce qui modifie radicalement
le paysage59. Fig. 3. LA TRANSFORMATION DES PAYSAGES FORESTIERS Les différentes formes de reboisement du massif vosgien (début xxe siècle) Benoit Vaillot | L’empreinte écologique de la frontière franco-allemande
35 Fig. 3. Les différentes formes de reboisement du massif vosgien (début xxe siècle) La transformation des paysages forestiers au xixe siècle est princi-
palement due au reboisement qui s’est réalisé à travers l’enrésinement60. Après 1871, la substitution des feuillus par des résineux à la croissance plus
rapide est mise en œuvre de façon tout à fait différente de part et d’autre
de la frontière. L’enrésinement devient systématique en Alsace-Lorraine à
mesure que le taillis-sous-futaie est abandonné, même si, dans la partie la
plus occidentale de la frontière, la rupture avec l’administration française
est moins nette61. Les gardes forestiers allemands privilégient les pins
et les épicéas pour réaliser rapidement une futaie et combler les vides62. Il faut tout de même mentionner l’introduction du sapin de Nordmann
dans le massif du Champ du Feu à partir de 189063. L’administration des
Eaux et Forêts fait preuve, quant à elle, d’un conservatisme affirmé en favo-
risant le sapin blanc, que l’on trouve à l’état naturel dans les Vosges, et le
pin sylvestre64. L’administration des forêts d’Alsace-Lorraine favorise également le
pin noir et le pin blanc, dont l’introduction dans les Vosges remonte au
début du xixe siècle, et tente d’acclimater de nouvelles espèces, en parti-
culier le pin gris65. Le pin de montagne est la manifestation la plus nette
de l’impérialisme écologique allemand. En effet, il est systématiquement Frontières et espaces transfrontaliers, une approche environnementale
36 36 planté du Lac noir jusqu’à la Tête des Faux, dominant le col du Bonhomme,
entre 800 et 1 200 mètres d’altitude66. Les gardes forestiers allemands pri-
vilégient une unité paysagère avec la Forêt-Noire, où cette essence est très
présente ; ce faisant, ils recomposent un paysage familier et homogène de
part et d’autre du Rhin67. Même lorsque l’épicéa – quasiment absent des Vosges avant 1871 – est
utilisé des deux côtés de la frontière, l’enrésinement diffère profondément. Les gardes forestiers allemands emploient l’épicéa commun de façon systé-
matique pour le reboisement après les violentes tempêtes de 1898 et 1902, et
introduisent l’épinette blanche et l’épinette de Sitka. Plus de 1 000 hectares
d’épicéas sont plantés pour maintenir le potentiel productif du versant
allemand des Vosges, ce qui modifie le paysage de la vallée de la Bruche,
par exemple68. 69. A. Bérard, « Les reboisements artificiels dans les forêts vosgiennes au xixe siècle », Revue
Forestière Française, 5 (1959), p. 371-375. 70. Andrée Corvol, Le sapin. Enjeux anciens, enjeux actuels, Paris, L’Harmattan, 2001 ; Bernard
Debarbieux, « Imaginaires nationaux et post-nationaux du lieu », Communications, 87 (2012),
p. 27-41. 66. P. Guinier, « Trois conifères de la flore vosgienne », art. cit.
67. D. Doll, « L’évolution des essences forestières dans le canton de Munster (1850-1950) »,
Revue Forestière Française, 6 (1985), p. 491-500.
68. Le pays welche s’étend sur la vallée de la Bruche, le val d’Argent et le val d’Orbey, et situé
non loin de la frontière. Éric Maire et Sylvie Guillerme, « L’évolution d’un paysage pastoral
identitaire : la moyenne montagne du Pays Welche, xviiie-xxe siècle », in A. Corvol, C. Dereix,
P. Gresser et F. Lormant (dir.), Forêt et montagne, Paris, L’Harmattan, 2015, p. 143-166.
69. A. Bérard, « Les reboisements artificiels dans les forêts vosgiennes au xixe siècle », Revue
Forestière Française, 5 (1959), p. 371-375.
70. Andrée Corvol, Le sapin. Enjeux anciens, enjeux actuels, Paris, L’Harmattan, 2001 ; Bernard
Debarbieux, « Imaginaires nationaux et post-nationaux du lieu », Communications, 87 (2012),
p. 27-41.
71. AD88, 109 P 3, Courrier du préfet des Vosges au directeur des douanes d’Épinal, 3 mars 1903. 68. Le pays welche s’étend sur la vallée de la Bruche, le val d’Argent et le val d’Orbey, et situé
non loin de la frontière. Éric Maire et Sylvie Guillerme, « L’évolution d’un paysage pastoral
dentitaire : la moyenne montagne du Pays Welche, xviiie-xxe siècle », in A. Corvol, C. Dereix,
P. Gresser et F. Lormant (dir.), Forêt et montagne, Paris, L’Harmattan, 2015, p. 143-166.i 71. AD88, 109 P 3, Courrier du préfet des Vosges au directeur des douanes d’Épinal, 3 mars 1903. 67. D. Doll, « L’évolution des essences forestières dans le canton de Munster (1850-1950) »,
Revue Forestière Française, 6 (1985), p. 491-500. 66. P. Guinier, « Trois conifères de la flore vosgienne », art. cit. LA TRANSFORMATION DES PAYSAGES FORESTIERS Leurs collègues français ne jurent que par le sapin blanc et
n’utilisent l’épicéa commun qu’à défaut de mieux pour reconstituer le sol
forestier lors de la reconquête d’espaces agricoles69. Le sapin des Vosges
est devenu, dans la France de la fin du xixe siècle, un véritable géo-sym-
bole70. Les propriétaires privés importent néanmoins des milliers de
plants d’épicéa au début du xxe siècle pour reboiser les communes fran-
çaises voisines de la frontière. Pas moins de 5 000 plants d’épicéa sont
ainsi repiqués à Laveline en 190371. Les gardes forestiers d’Alsace-Lor-
raine formés à Eberswalde introduisent des espèces de pin et d’épicéa qui
composent près des deux tiers des forêts de Prusse ; a contrario, le sapin est
absent d’Allemagne du Nord. Fig. 4. Les différences de paysage dans le massif vosgien (début xxe siècle) Benoit Vaillot | L’empreinte écologique de la frontière franco-allemande
37 Fig. 4. Les différences de paysage dans le massif vosgien (début xxe siècle) À la veille de la Première Guerre mondiale, l’introduction de plusieurs
espèces de pins a tellement modifié le paysage forestier du côté allemand
que les espèces de sapins indigènes reculent et deviennent minoritaires
en certains endroits. L’impérialisme écologique allemand a aussi eu des
conséquences indirectes sur l’environnement. Les conditions de gestion
et d’exploitation des forêts ont profondément modifié les milieux. Partout
où les landes et forêts clairières, composées de pins et de bouleaux, ont été
brutalement remplacées par des plantations denses de pins et de hêtres,
l’anémone de printemps a disparu et d’autres plantes, comme le daphné
camélée, le lycopode en massue et le lycopode petit-cyprès, sont devenues
de plus en plus rares72. Les aménagements forestiers sur le versant alsacien,
exposé au soleil levant, ont provoqué un recul des tourbières et donc la
raréfaction de la lycopodiella inundata et du sedum. Les gardes forestiers allemands plantent aussi, de leur propre initia-
tive, des arbres exotiques à partir des années 1880, au contraire de leurs
collègues français. Ils cherchent à diversifier les essences en introduisant
de nouvelles espèces. C’est grâce à leurs initiatives que l’on trouve dans les
Vosges, à la veille de la Première Guerre mondiale, quelques plants de cèdre
blanc, de genévrier de Virginie, de mélèze du Japon, de thuya géant, de
thuya occidental, de pruche du Canada, de carya tomentosa, de noyer cendré,
de noyer noir et de chêne chevelu73. 72. Émile Walter, « Modifications survenues dans la flore d’Alsace et de Lorraine depuis 1870 »,
Bulletin de la Société Botanique de France, 73-6 (1926), p. 5-61.
73. Ibid. 75. Franz von Oppenau, Die Pflege und Verbesserung der Hochweiden und Bergwiesen.
Vortrag, gehalten den 15. Januar 1893 in der Generalversammlung des landwirtschaftlichen
Kantonalvereins zu Münster, 1893 ; Id., Die Hochweiden und Bergwiesen der Vogesen, op. cit. 76. Albrecht Lehmann et Klaus Schriewer, Der Wald, ein deutscher Mythos ? Perspektiven eines
Kulturthemas, Berlin, Dietrich Reimer, 2000. 74. Franz von Oppenau, Die Hochweiden und Bergwiesen der Vogesen, ihr dermaliger Zustand,
sowie Vorschläge zur Verbesserung des Pflanzenbestandes auf denselben. Vortrag, gehalten zu
Strasburg am 12. Oktober 1899 in der Sitzung der Gesellschaft zur Förderung der Wissenschaften,
des Ackerbaues und der Künste im Unter-Elsaß, 1899. 79. François Walter, Les figures paysagères de la nation. Territoire et paysage en Europe
(16e-20e siècle), Paris, Éditions de l’EHESS, 2004, p. 147. 78. Albrecht Lehmann, « Waldbewußtsein. Zur Analyse eines Kulturthemas in der Gegenwart »,
Forstwissenschaftliches Centralblatt, 120 (2001), p. 38-49.i 77. J. K. Wilson, The German Forest, op. cit. 78. Albrecht Lehmann, « Waldbewußtsein. Zur Analyse eines Kulturthemas in der Gegenwart »,
Forstwissenschaftliches Centralblatt, 120 (2001), p. 38-49.
79. François Walter, Les figures paysagères de la nation. Territoire et paysage en Europe
(16e-20e siècle), Paris, Éditions de l’EHESS, 2004, p. 147. LA TRANSFORMATION DES PAYSAGES FORESTIERS Enfin, des acteurs indépendants de
l’État ont aussi participé activement à modifier le paysage par l’introduction
de nouvelles espèces botaniques. L’ingénieur agronome Franz von Oppenau
acclimate par exemple des plantes alpines tout le long de la frontière,
afin d’améliorer les herbages, pâturages et prairies. En 1894, il établit ses Frontières et espaces transfrontaliers, une approche environnementale
38 38 premières cultures à Munster, puis crée sa pépinière à Metzeral74. Oppenau
distribue les graines de sa pépinière aux fermiers locaux et les encourage
à les cultiver, afin de diversifier le fourrage des animaux et améliorer la
qualité du lait. Il est ainsi à l’origine directe de la présence du pâturin des
Alpes, de la fléole des Alpes, de méons, de la renouée vivipare, du trèfle
alpin, du plantain des Alpes et de la crépide dorée sur le versant alsacien
des Vosges75. Tous ces changements paysagers sont perceptibles par les populations. Lorsque l’on traverse la frontière à la veille de la Première Guerre mondiale,
l’on franchit aussi une frontière sensible. Sur le versant allemand, les
essences ont tendance à être moins mélangées et les arbres sont en
moyenne plus petits que sur le versant français, en raison des choix sylvi-
coles effectués par l’administration des forêts d’Alsace-Lorraine. Les cônes
et les aiguilles en territoire français tendent à joncher plus couramment
le sol et dégagent une forte odeur de résine qui indique déjà que l’on entre
dans un autre territoire. Néanmoins, les fleurs semblent plus nombreuses
et diverses en territoire allemand. Les autorités allemandes investissent aussi le paysage par un discours
« esthético-patriotique » qui articule Heimat et Vaterland76. Les forêts ne
sont pas seulement un lieu d’exploitation de ressources, mais aussi un lieu
central de la construction de l’identité nationale allemande77. Dans l’imagi-
naire national allemand, les forêts représentant plus que tout autre paysage
la « nature », même si elles sont très aménagées78. Les individus expéri-
mentent ainsi le sentiment national par le paysage forestier modelé par
l’administration des forêts d’Alsace-Lorraine79. C’est la raison pour laquelle
la science forestière allemande accorde une grande importance aux activités
récréatives. Les lieux de randonnée sont d’ailleurs agrémentés d’arbres appréciés des touristes, comme le cerisier noir introduit à Kaysersberg à la
fin du xixe siècle80. On ne retrouve pas en France le même investissement de l’imaginaire
national dans les forêts. 80. É. Walter, « Modifications survenues dans la flore d’Alsace et de Lorraine depuis 1870 », art. cit.
81. AD88, 7 T 20, Situation des sociétés scolaires forestières de l’arrondissement de Saint-Dié,
1901-1902.
82. Émile Cardot, Manuel de l’arbre : pour l’enseignement sylvo-pastoral dans les écoles. L’arbre,
la forêt et les pâturages de montagne, Paris, Touring-Club de France, 1907. Cet ouvrage a été un
grand succès de librairie.
83. Vincent Moriniaux, « Le mythe de la ligne bleue des Vosges et la vision nationaliste de la
sapinière », in A. Corvol (dir.), Le sapin. Enjeux anciens, enjeux actuels, op. cit., p. 371-383.
84. Michel Pastoureau, Bleu. Histoire d’une couleur, Paris, Seuil, 2000.
85. AD88, 40 J 127 : « Je désire reposer dans la même tombe que mon père et ma sœur, en face
de cette ligne bleue des Vosges, d’où monte jusqu’à mon cœur fidèle la plainte touchante des
vaincus » ; Aude Dontenwille-Gerbaud, « La ligne bleue des Vosges », Mots. Les langages du poli-
tique, 105 (2014), p. 13-26. LA TRANSFORMATION DES PAYSAGES FORESTIERS Cependant, la connaissance des arbres et de la
sylviculture entre dans la pédagogie des écoles primaires françaises au
tournant du siècle. Les instituteurs soutiennent l’administration des Eaux
et Forêts en assumant le rôle de vulgarisateurs sylvicoles. Comme l’écra-
sante majorité des communes situées non loin de la frontière disposent de
bois communaux, les enseignants sont à l’origine de « sociétés scolaires
forestières »81. Leurs réalisations concrètes sont limitées ; elles ont principa-
lement aidé au reboisement des terrains en friche. Mais elles ont mobilisé
de nombreux acteurs locaux du monde associatif et de l’enseignement dans
l’Est de la France. En 1902, on en compte au moins une à Arnould, Ban de
Sapt, Denipaire, Frapelle, Le Beulay, Lesseux, Petite-Fosse et Saulcy. Les
instituteurs réalisent l’apprentissage de la nature aux enfants, car « aimer
les arbres c’est aimer la patrie »82. Le paysage associé à la frontière franco-allemande est néanmoins une
forêt dans l’imaginaire national français83. Pour les Français, cette frontière
est nécessairement bleue, en raison de la réverbération du soleil sur les
arbres qui se détachent sur le ciel, mais aussi – et peut-être surtout – parce
que cette couleur est associée à la France depuis le xiiie siècle84. Maurice
Barrès évoque régulièrement les « montagnes bleues des Vosges » ou encore
la « dentelure bleue des crêtes, la nouvelle frontière, si distincte les veilles
de pluie ». Dans son testament, Jules Ferry forge une expression vouée à une
grande postérité en France : celle de « ligne bleue des Vosges »85. Les surfaces forestières à la frontière franco-allemande entre 1871 et
1914 progressent, en grande partie grâce aux états-majors qui les intègrent
aux systèmes défensifs. La souveraineté que les États exercent sur les Frontières et espaces transfrontaliers, une approche environnementale
40 40 populations vivant à la frontière concerne aussi les arbres, les plantes et
les fleurs qui les environnent. L’administration forestière allemande fait
montre d’un véritable impérialisme écologique en rompant radicalement
avec les méthodes sylvicoles françaises employées jusqu’en 1871. Ainsi, deux
sylvicultures obéissant à des imaginaires nationaux différents s’opposent
dans le massif des Vosges, avec la ligne de crête pour front écologique ;
comme une préfiguration des terribles combats que connaitra la région
pendant la Première Guerre mondiale. 86. Béatrice von Hirschhausen, « Leçon des frontières fantômes : les traces du passé nous
viennent (aussi) du futur », L’Espace géographique, 46-2 (2017), p. 97-105. LA TRANSFORMATION DES PAYSAGES FORESTIERS Les paysages en ressortent profon-
dément modifiés, si bien qu’encore aujourd’hui, après plus d’un siècle la
composition et le peuplement des forêts selon le versant des Vosges restent
différents : on peut parler de « frontière écologique fantôme »86.
|
https://openalex.org/W4285253522
|
https://scindeks-clanci.ceon.rs/data/pdf/0040-2389/2022/0040-23892201004M.pdf
|
Bosnian
| null |
Efficiency analysis of textile companies in Serbia
|
Tekstilna industrija
| 2,022
|
cc-by
| 5,912
|
TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 EFFICIENCY ANALYSIS OF TEXTILE
COMPANIES IN SERBIA Abstract: The purpose of the research is to use the ratio analysis to determine the level of effi ciency of companies
engaged in the production of textiles in the Republic of Serbia in the period from 2017 to 2019. The research was
conducted on a sample of three companies engaged in the production of textiles, whose shares are listed on the
Belgrade Stock Exchange. The paper observes their management effi ciency: total assets, fi xed assets, current as-
sets, inventories, trade receivables and trade payables. The research determined that fl uctuations in the values of
the used effi ciency indicators were present during the observed period. Further, it was determined that there is a
signifi cant space for improving the effi ciency of the observed textile companies. The results of the research can be
important for the management of the company, owners (shareholders) of the company, investors and creditors. eywords: textile industry, effi ciency, ratio indicators, Belgrade Stock Exchange, Republic of Serbia Marko Milašinović1, Marko Špiler2, Snežana Knežević2*, Aleksandra Mitrović1 1 Univerzitet u Kragujevcu, Fakultet za hotelijerstvo i turizam u Vrnjačkoj Banji
2 Univerzitet u Beogradu, Fakultet organizacionih nauka
* e-mail: snezana.knezevic@fon.bg.ac.rs Professional paper
UDC: 657: 658:005.0:677:338.4
DOI: 10.5937/TEKSTIND2201004M Professional paper
UDC: 657: 658:005.0:677:338.4
DOI: 10.5937/TEKSTIND2201004M Apstrakt: Svrha istraživanja u ovom radu je da se primenom racio analize utvrdi nivo efi kasnosti preduzeća
koja se bave proizvodnjom tekstila u Republici Srbiji u periodu od 2017. do 2019. godine. Istraživanje je spro-
vedeno na uzorku od tri preduzeća koja se bave proizvodnjom tekstila, a čije su akcije listirane na Beogradskoj
berzi. U radu je posmatrana njihova efi kasnost upravljanja: ukupnom imovinom, stalnom imovinom, obrtnom
imovinom, zalihama, potraživanjima od kupaca i obavezama prema dobavljačima. Istraživanjem je utvrđeno
da su tokom posmatranog perioda prisutne fl uktuacije vrednosti korišćenih pokazatelja efi kasnosti. Takođe,
utvrđeno je da postoji značajan prostor za unapređenje efi kasnosti posmatranih tekstilnih preduzeća. Rezul-
tati istraživanja mogu biti od značaja za menadžment preduzeća, vlasnike (akcionare) preduzeća, investitore i
poverioce. 1. UVOD Naglašava se da ima dovoljno
uslova i prostora za podsticanje oživljavanja tekstilne
industrije u Srbiji uprkos sporom oporavku nastalom
usled zastoja u privredi i nestašice kapitala [6, p. 138]. Imajući u vidu da je tekstilna industrija i dalje važna
u smislu zapošljavanja i shodno tome uravnoteženog
regionalnog razvoja, ističe se da postoji potreba za re-
strukturisanjem ove grane iz radno intenzivne u kapi-
talno intenzivnu [7, p. 7]. Ključni proizvod računovodstvenih informacija
i sistema izveštavanja su fi nansijski izveštaji, a među
njima kao bazični izdvajaju se bilans stanja i bilans
uspeha, a pored njih, radi integrisanog pogleda na
fi nansijske performanse preduzeć a, važno je pažljivo
sagledati i izveštaj o novčanim tokovima za ocenu
kvaliteta zarade preduzeć a [15, p. 14]. Kao značajan
izvor fi nansijskih informacija o poslovanju preduzeća
javljaju se fi nansijski izveštaji, koji su namenjeni kako
eksternim, tako i internim korisnicima [16], [17]. Kako
isti sadrže veliki broj podataka, potrebno je istaći ne-
ophodnost primene neke od tehnika fi nansijske anali-
ze (horizontalne, vertikalne i racio analize) radi sticanja
korisnih informacija za fi nansijske odluke [18]. Kvalitet
računovodstvenih informacija preduzeć a može uticati
na efi kasnost njegovog odlučivanja po pitanju investi-
cija [19]. Prevarno fi nansijsko izveštavanje je već dugo
prisutno u računovodstvenoj praksi [20, p. 262], i takva
vrsta izveštavanja ograničava upotrebljivost fi nansij-
skih informacija za poslovno odlučivanje [21]. Finansij-
ska racija se koriste za komparaciju fi nansijskih perfor-
mansi preduzeć a za posmatrani period, komparaciju
između preduzeć a, fokusirajuć i se na sličnosti, razlike i
trendove [22], a široko ih koriste menadžeri, kreditori,
regulatori i investitori. Ovi pokazatelji mogu da budu
koristan analitički alat za razne interesne grupe [23, p. 54]. Finansijska racija su se dugo analizirala kao mere
fi nansijskog stanja preduzeć a za predviđanje korpora-
tivnog neuspeha [24, p. 306]. Glavni razlog za merenje poslovnih performansi
je podrška donosiocima odluka na različitim hijerar-
hijskim nivoima [8]. Evaluacija performansi preduze-
ć a sastavni je deo upravljanja svakom kompanijom,
što omoguć ava utvrđivanje uticaja odluka poslovnog
upravljanja na ostvarene performanse, kao i smer re-
zultata i odluka koji su potrebni da bi ih poboljšali. Analiza fi nansijskih izveštaja primenjuje analitičke ala-
te i tehnike koristeći podatke iz fi nansijskih izveštaja
radi izvođenja merenja i racija koji su značajni i kori-
sni za donošenje odluka [9]. 1. UVOD ni sektori industrije tekstila i odeć e specifi čni po tome
što zahtevaju obilje radne snage sa vrlo malo kapital-
nih investicija, a to je vrsta industrije koja je potrebna
zemljama u razvoju i ekonomijama [3, p. 302]. U pro-
izvodnji odeć e pružaoci usluga s niskim platama (ce-
nom rada) imaju apsolutnu prednost. Strani konku-
renti u takozvanim „zemljama s niskim platama“ imaju
niže troškove rada sa sličnim kvalitetom snadbevanja
uslugama a, pored toga, imaju i apsolutne i relativne Tekstilna industrija kao grana prerađivačke indu-
strije zapošljava veliki broj jeftine i niskokvalifi kovane
radne snage, pri čemu nju najviše čine žene [1, p. 98]. Odevna industrija je radno intenzivna i nudi zanima-
nja na početnom nivou za nekvalifi kovane radnike u
razvijenim zemljama i zemljama u razvoju [2, p. 292]. Zemlje koje su u procesu razvoja svojih ekonomija, fo-
kusirane su na industriju tekstila i odeće jer su određe- 4 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 ekonomske prednosti [3, p. 302]. Globalna konkuren-
cija je poveć ala pritis ak na proizvodne organizacije da
se poboljšaju njihove aktivnosti [4]. Preduzeća koja
pripadaju tekstilnoj i odevnoj industriji usko su među-
sobno povezana, ali svaka industrija pokazuje razlike
u poslovnom okruženju, proizvodima, proizvodnji i
sklonosti kupaca [5, p. 132]. koji se odnosi na kapacitet preduzeć a da obezbedi
adekvatan novčani tok koji je namenjen ažurnom iz-
mirenju njegovih tekuć ih (kratkoročnih) i dugoročnih
obaveza [11, p. 117]. Evaluacija performansi kompani-
je je primarni cilj analize fi nansijskih izveštaja [12, p. 252]. Merenje performansi može biti defi nisano kao
proces kvantifi kovanja poslovnih aktivnosti [13]. Racio
analiza je verovatno jedna od najpoznatijih i najšire
korišćenih alata u analizi fi nansijskih izveštaja i ima
važnu ulogu u proceni učinka preduzeća i njegovog
položaja [14, p. 20]. Tekstilna i odevna industrija bile su među vodećim
izvoznim industrijama u Srbiji duži vremenski period i
zapošljavale su veliki broj radnika. Tekstilna industrija
Srbije ima dugu tradiciju i bila je jedna od najuspeš-
nijih grana prerađivačke industrije, nezavisno od toga
što nije imala status prioritetne privredne grane [1, p. 102]. Danas je situacija mnogo drugačija, primarna
proizvodnja još nije revitalizovana, veliki deo sirovina
se uvozi, što sve zajedno ima negativne implikacije na
konkurentnost domaćih preduzeća u okviru tekstil-
ne i odevne industrije. 1. UVOD Finansijska analiza koja
obuhvata horizontalnu, vertikalnu i racio analizu, pre
svega služi u dve svrhe: (a) za upoređivanje sa stan-
dardom, i (b) da predvidi buduć e perspektive posma-
tranog preduzeća, čime bi se zadovoljili informacioni
zahtevi raznih adresata. Tradicionalno, performanse
preduzeć a se ocenjuju primenom fi nansijske racio
analize, ali se primeć uje da je u današnjem dinamič-
nom i veoma konkurentnom poslovnom okruženju
pravovremena kontrola performansi od ključne važ-
nosti za održivo poslovanje, te stoga postoji sve već a
pažnja posveć ena pokazateljima nefi nansijskog učin-
ka [10, pp. 125–160]. Predmet ovog rada je efi kasnost proizvođača tek-
stila u Republici Srbiji. Cilj rada je da se sagleda da li su
posmatrana tekstilna preduzeća efi kasno upravljalala
svojim resursima u periodu od 2017. do 2019. godi-
ne. Efi kasnost je merena koefi cijentom obrta ukupne
imovine, koefi cijentom obrta stalne imovine, koefi ci-
jentom obrta obrtne imovine, koefi cijentom obrta ku-
paca, koefi cijentom obrta zaliha i koefi cijentom obrta
dobavljača. Akcionari, poverioci i druge zainteresovane strane
evaluiraju fi nansijski položaj preduzeć a radi procene
njegove sposobnosti za efi kasno obavljanje poslovne
aktivnosti. Povezan s tim, važan deo poslovanja je onaj Razvijeno je nekoliko pitanja koja se odnose na
tretiranu problematiku. Istraživačka pitanja su: 5 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 IP1. Kako bi pokazatelji efi kasnosti mogli uticati
na fi nansijske performanse kompanija? IP1. Kako bi pokazatelji efi kasnosti mogli uticati
na fi nansijske performanse kompanija? Koefi cijent obrta stalne imovine meri efi kasnost
korišćenja stalne imovine preduzeća u generisanju
prihoda od prodaje [30]. Usled činjenice da su stalna
sredstva skupa za kupovinu i održavanje, neophodno
je da budu što efi kasnija u generisanju prodaje [26]. Vrednost ovog pokazatelja u velikoj meri zavisi i od in-
dustrije kojoj preduzeće pripada [31]. Niska vrednost
ovog pokazatelja ukazuje da preduzeće nedovoljno
koristi svoje kapacitete ili da su oni predimenzionirani. IP2. Koji su ključni aspekti koji se odnose na efi ka-
snost a ukazuju na negativne tendencije kompanija? IP2. Koji su ključni aspekti koji se odnose na efi ka-
snost a ukazuju na negativne tendencije kompanija? Rad je podeljen u pet delova. Nakon uvodnog
dela sledi prikaz najčešće korišćenih pokazatelja efi ka-
snosti (fi nansijskih racija) koji se javljaju u akademskoj
i stručnoj literaturi. Slede podaci o uzorku u trećem
delu. Rezultati istraživanja prikazani su u četvrtom
delu. 1. UVOD U poslednjem delu izneta su zaključna razmatra-
nja, kao i ograničenja u sprovedenom istraživanju iz
kojih proističu dalji pravci istraživanja. Koefi cijent obrta obrtne imovine, kao odnos pri-
hoda od prodaje i prosečne obrtne imovine, pokazu-
je koliko se puta, u proseku, obrtna imovine obrne u
toku godine kroz prihode od prodaje [26]. Drugim re-
čima, ovaj pokazatelj meri sposobnost preduzeća da
generiše prihode od prodaje kroz svoja obrtna sred-
stva. Preduzeće treba nastojati da ovaj pokazatelj ima
višu vrednost, uz trend rasta, jer to znači da preduzeće
radi na doslednom poboljšanju svojih politika u obla-
sti zaliha, potraživanja, gotovine i drugih obrtnih sred-
stava [26]. 2. MERE EFIKASNOSTI POSLOVANJA
PREDUZEĆA Pokazatelji efi kasnosti mere uspešnost upravlja-
nja različitim poslovnim resursima preduzeća [25],
odnosno mere sposobnost preduzeća da konvertu-
je sopstvena sredstva u gotovinu ili prihode [26]. Za
ovu grupu pokazatelja nisu zainteresovani samo me-
nadžeri preduzeća, kao lica kojima je povereno uprav-
ljanje sredstvima, već i poverioci (kreditori, dobavljači
i dr.); [27]. Ova grupa racija omogućava sagledavanje
broja obrta pojedinih kategorija (sredstava) u poslov-
nom ciklusu, odnosno podataka o vremenu njihovog
vezivanja [28]. Pokazatelji efi kasnosti povezani su sa
pokazateljima profi tabilnosti, odnosno ukoliko je pre-
duzeće efi kasnije u korišćenju sredstava, ono postaje
profi tabilno [26]. Prilikom utvrđivanja racio pokaza-
telja efi kasnosti, koriste se podaci iz bilansa stanja i
bilansa uspeha. Kao najčešće korišćeni pokazatelji efi -
kasnosti javljaju se: Koefi cijent obrta potraživanja od kupaca, kao od-
nos prihoda od prodaje i prosečnih potraživanja od
kupaca, meri efi kasnost kojom preduzeće naplaćuje
svoja potraživanja od kupaca [32]. Ovaj pokazatelj
ukazuje koliko je puta preduzeće bilo u mogućnosti
da pretvori potraživanja od kupaca u gotovinu [32]. Za preduzeće je bolje da vrednost ovog pokazatelja
bude viša, jer to ukazuje da brže naplaćuje svoja po-
traživanja a, samim tim, brže dolazi do gotovine (kao
najlikvidnijeg oblika sredstava preduzeća). Adekvatna
naplata potraživanja je, pored profi tabilnosti, druga
najznačajnija mera uspešnosti poslovanja preduzeća
[33]. U situacija kada preduzeće vodi izuzetno strogu
kreditnu politiku, vrednost ovog pokazatelja će biti
izuzetno visoka [34]. Preterana prodaja na kredit, ne-
mogućnost kupaca da plate obaveze ili slaba naplata
potraživanja imaće za posledicu nisku vrednost koe-
fi cijenta. •
Koefi cijent obrta ukupne imovine. •
Koefi cijent obrta stalne imovine. •
Koefi cijent obrta obrtne imovine. •
Koefi cijent obrta kupaca. Koefi cijent obrta zaliha ukazuje koliko efi kasno
preduzeće upravlja zalihama, odnosno koliko se puta
u toku godine, u proseku, zalihe obrnu [32]. Za razliku
od prethodno korišćenih pokazatelja aktivnosti, kod
kojih se u brojiocu nalaze prihodi od prodaje, kod ko-
efi cijenta obrta zaliha u brojiocu se nalazi cena košta-
nja prodatih proizvoda. Konkretno, ovaj pokazatelj se
utvrđuje stavljanjem u odnos cene koštanja prodatih
proizvoda i prosečne vrednosti zaliha [32]. Zbog ne-
mogućnosti da se iz bilansa uspeha utvrdi cena košta-
nja prodatih proizvoda, u brojiocu se nalaze troškovi
proizvodnje uvećani za povećanje vrednosti zaliha,
odnosno umanjeni za smanjenje vrednosti zaliha [35]. Viša vrednost koefi cijenta znači efi kasnije upravljanje
zalihama u preduzeću, odnosno zalihe se kraće zadr- •
Koefi cijent obrta zaliha. 2. MERE EFIKASNOSTI POSLOVANJA
PREDUZEĆA •
Koefi cijent obrta dobavljača. Koefi cijent obrta ukupne poslovne imovine pred-
stavlja najuopšteniji pokazatelj aktivnosti preduzeća,
budući da predstavlja odnos prihoda od prodaje i
prosečnog stanja ukupnih poslovnih sredstava [29]. On pokazuje koliko se puta u toku godine vrednost
ukupno angažovane poslovne imovine preduzeća ge-
nerisala u prodaji. Na njegovu visinu se može uticati
povećanjem koefi cijenta obrta pojedinih sredstava
(npr. stalnih sredstava, obrtnih sredstva i njenih obli-
ka). Što je vrednost ovog koefi cijneta viša, to je i veća
sposobnost preduzeća da koristi svoju ukupnu imovi-
nu u stvaranju prihoda od prodaje [26]. 6 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 žavaju u skladištu. To može ukazivati da preduzeće
manje ulaže u zalihe, kao i na postojanje ubrzanog
poslovnog ciklusa pretvaranja zaliha u gotovinu i po-
stojanje manje verovatnoće nestanka zaliha [36]. Kako
Mitrović & Knežević [37, p. 64] ističu, niska vrednost
koefi cijenta obrta zaliha može se javiti kao rezultat
niskog kvaliteta proizvodnje, vrednovanja zaliha po
visokim cenama, uključivanja bezvrednih i starih proi-
zvoda u zalihe i drugo. žavaju u skladištu. To može ukazivati da preduzeće
manje ulaže u zalihe, kao i na postojanje ubrzanog
poslovnog ciklusa pretvaranja zaliha u gotovinu i po-
stojanje manje verovatnoće nestanka zaliha [36]. Kako
Mitrović & Knežević [37, p. 64] ističu, niska vrednost
koefi cijenta obrta zaliha može se javiti kao rezultat
niskog kvaliteta proizvodnje, vrednovanja zaliha po
visokim cenama, uključivanja bezvrednih i starih proi-
zvoda u zalihe i drugo. 13 – Proizvodnja tekstila), a analizom je obuhvaćeno
njihovo poslovanje od 2017. do 2019. godine na osno-
vu javno dostupnih fi nansijskih izveštaja [40]. Od pet
preduzeća, koja pripadaju pomenutoj oblasti a čije
su akcije na dan 14. 07. 2021. godine listirane na Beo-
gradskoj berzi, iz daljeg istraživanja isključena su dva
preduzeća [39]. Razlog njihovog isključivanja nalazi se
u činjenici da se jedno preduzeće nalazi u stečaju, dok
drugo preduzeće nema javno dostupne fi nansijske
izveštaje [40]. Na problem javne nedostupnosti fi nan-
sijskih izveštaja preduzeća koja su listirana na Beo-
gradskoj berzi ukazuju i [41], [42]. Tri posmatrana pre-
duzeća u daljem tekstu su označena kao preduzeće A
(Dunav A.D. Grocka), B (Kluz Padobrani A.D. Beograd) i
C (Yumco, A.D. Vranje). Koefi cijent obrta dobavljača predstavlja odnos
nabavki na kredit i prosečnih obaveza prema dobav-
ljačima [34]. 4. REZULTATI ISTRAŽIVANJA Radi odgovora na defi nisana istraživačka pitanja,
primenjena su selektovana fi nansijska racija za testira-
nje efi kasnosti preduzeća. U nastavku su predstavljeni
dobijeni rezultati. Vrednost navedenih pokazatelja efi -
kasnosti posmatranih tekstilnih preduzeća u periodu
od 2017. do 2019. godine prikazana je u sledećoj ta-
beli. Tokom posmatranog perioda prisutne su fl uktu-
acije vrednosti svih šest korišćenih pokazatelja efi ka-
snosti. Tako je kod preduzeća A, tokom posmatranog
perioda, došlo do pada vrednosti koefi cijenta obrta
ukupne imovine. U prvoj posmatranoj godini, predu-
zeće A je na svaki dinar prosečno angažovane imovi-
ne ostvarilo 0,55 dinara prihoda od prodaje, da bi u
poslednoj posmatranoj godini ostvarivalo 0,45 dinara. Pad vrednosti ovog pokazatelja efi kasnosti kod pre-
duzeća A javlja se usled većeg pada vrednosti prihoda
od prodaje, u odnosu na zabeleženo smanjenje vred-
nosti ukupne imovine. Međutim, i pored navedenih
oscilacija, preduzeće A je u sve tri posmatrane godine 2. MERE EFIKASNOSTI POSLOVANJA
PREDUZEĆA Usled nedostatka podataka o nabavkama
na kredit u fi nansijskim izveštajima, prilikom utvrđi-
vanja ovog pokazatelja vrši se aproksimacija nabavki
na kredit sa vrednošću cene koštanja prodatih goto-
vih proizvoda, uvećane za povećanje vrednosti zaliha,
odnosno umanjene za smanjenje zaliha [34]. Pokazuje
koliko se puta prosečno u toku godine izvrši isplata
obaveza prema dobavljačima. Niža vrednost pokaza-
telja ukazuje na postojanje dužeg perioda između dve
isplate dobavljačima. Stavljajući u odnos broj dana
u godini i koefi cijent obrta dobavljača dobija se pro-
sečno vreme plaćanja obaveza prema dobavljačima. Pokazuje koliko u proseku protekne dana od trenutka
nabavke sirovina, materijala i usluga od dobavljača na
kredit do trenutka isplate tih obaveza. U slučaju da se
upoređivanjem utvrdi viša vrednost pokazatelja u od-
nosu na ugovoreni rok plaćanja prema dobavljačima,
to može ukazivati da preduzeće ima problem s nedo-
statkom gotovine [38]. 3. PODACI O UZORKU Istraživanje u radu je sprovedeno na uzorku pre-
duzeća čije su akcije listirane na Beogradskoj berzi u
okviru Sektora C-Prerađivačka industrija [39], koja se
bave proizvodnjom tekstila (odnosno preduzeća čija
je šifra oblasti prema Uredbi o sektorskoj klasifi kaciji Tabela: Efi kasnost tekstilnih preduzeća u periodu od 2017. do 2019. godine
A
B
C
2017
2018
2019
2017
2018
2019
2017
2018
2019
Koefi cijent obrta
ukupne imovine
0,5538
0,4938
0,4503
0,0085
0,0027
0,0000
0,2263
0,3697
0,3366
Koefi cijent obrta
stalne imovine
0,8227
0,7177
0,6723
0,0113
0,0036
0,0000
0,3776
0,4831
0,4654
Koefi cijent obrta
obrtne imovine
16,942
15,823
13,639
0,0350
0,0107
0,0000
0,5657
15,745
12,166
Koefi cijent obrta
kupaca
132,906
171,546
160,754
0,4326
0,1376
0,0000
26,212
239,944
60,827
Koefi cijent obrta
zaliha
14,721
13,748
11,090
0,0381
0,0117
0,0003
11,247
24,651
22,895
Koefi cijent obrta
dobavljača
26,648
37,247
44,919
0,2078
0,2538
0,2807
0,4396
0,9316
10,210 Tabela: Efi kasnost tekstilnih preduzeća u periodu od 2017. do 2019. godine 7 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 zabeležilo najvišu vrednost ovog pokazatelja. Sma-
njenje vrednosti koefi cijenta obrta ukupne imovine
zabeleženo je i kod preduzeća B, da bi u poslednjoj
posmatranoj godini usled neostvarivanja prihoda od
prodaje, vrednost ovog pokazatelja bila 0. Preduzeće
C je u 2018. godini, usled rasta prihoda od prodaje
i smanjenja vrednosti prosečne imovine u odnosu
na 2017. godinu, ostvarilo rast koefi cijenta ukupne
imovine, odnosno na svaki dinar prosečno uložene
imovine ostvarilo je 0,37 dinara prihoda od prodaje. Međutim, u 2019. godini je usledio pad vrednosti koe-
fi cijenta obrta ukupne imovine u odnosu na 2018. go-
dinu. Tako je preduzeće C u 2019. godini ostvarilo 0,34
dinara prihoda od prodaje na svaki dinar prosečno
angažovane ukupne imovine. Razlog za smanjenje se
nalazi u činjenici da je zabeleženo povećanje vredno-
sti ukupne imovine, ali i smanjenje vrednosti prihoda
od prodaje. U sve tri posmatrane godine, preduzeće A
je ostvarilo najvišu vrednost efi kasnosti ukupne imo-
vine. dinara prihoda od prodaje. U 2018. godini ta vrednost
iznosi 1,58, odnosno 1,36 u 2019. godini. Preduzeće
B je u 2017. godini na svaki dinar prosečno uložene
obrtne imovine ostvarilo 0,03 dinara prihoda od pro-
daje, da bi u 2018. godini, ostvarilo 0,01 dinar. Usled
već navedenih činjenica (da preduzeće B u 2019. go-
dini nije imalo prihode od prodaje), u poslednjoj po-
smatranoj godini vrednost koefi cijenta obrta obrtne
imovine iznosio je 0. 3. PODACI O UZORKU Usled rasta prihoda od prodaje i
smanjenja vrednosti prosečnih potraživanja, preduze-
će C je u 2018. godini zabeležilo rast koefi cijenta obrta
obrtne imovine u odnosu na 2017. godinu. Međutim,
u 2019. godini zabeleženo je smanjenje efi kasnosti
obrtne imovine. Kao i u slučaju prethodna dva kori-
šćena pokazatelja, preduzeće A je u sve tri posmatra-
ne godine ostvarilo najviše vrednosti koefi cijenta obr-
ta obrtne imovine. Iz tabele se može videti da je tokom posmatranog
perioda vrednost koefi cijenta obrta kupaca zabeležila
fl uktuacije. Preduzeća A i C su u 2018. godini zabele-
žila rast vrednosti ovog pokazatelja efi kasnosti u od-
nosu na 2017. godinu, da bi u 2019. godini usledilo
smanjenje vrednosti (pri čemu je ta vrednost viša u
odnosu na 2017. godinu). Dakle, u 2018. godini pre-
duzeća A i C su skratila period naplate potraživanja
od kupaca u odnosu na 2017. godinu, da bi u 2019. godini došlo do dužeg perioda naplate potraživanja
od kupaca. Preduzeće A je u 2017. godini 13,29 puta
naplaćivalo prosečna potraživanja od kupaca, u 2018. godini 17,15 puta, a 16,07 puta u 2019. godini. Predu-
zeće B beleži trend konstantnog smanjenja vrednosti
koefi cijenta obrta kupaca, da bi u poslednjoj posma-
tranoj godini ta vrednost bila 0 (usled odsustva priho-
da od prodaje). To ukazuje da preduzeće ima velikih
problema sa prodajom i naplatom svojih potraživanja. Kao što je navedeno, preduzeće C je u 2018. godini
(vrednost koefi cijent obrta kupaca iznosi 23,99) pove-
ćalo svoju efi kasnost naplate potraživanja od kupaca
u odnosu na 2017. godinu (kada je vrednost koefi ci-
jenta iznosila 2,62), da bi u 2019. godini usledilo sma-
njenje efi kasnosti. U 2017. i 2019. godini, preduzeće A
je zabeležilo najvišu vrednost koefi cijenta obrta kupa-
ca, dok je u 2018. godini to bilo preduzeće C. Preduzeće A usled smanjena vrednosti prihoda
od prodaje i smanjenja vrednosti prosečne stalne
imovine beleži smanjenje vrednosti koefi cijenta obrta
stalne imovine tokom posmatranog perioda. Tako je
preduzeće A u 2017. godini na svaki dinar prosečno
angažovane stalne imovine stvorilo 0,82 dinara priho-
da od prodaje, da bi u 2019. godini, stvorilo 0,67 di-
nara. Preduzeće B takođe beleži trend smanjenja efi -
kasnosti stalne imovine u periodu od 2017. do 2019. godine. Ovo preduzeće je u 2017. godini na svaki
dinar prosečno angažovane stalne imovine ostvarilo
0,01 dinara prihoda od prodaje. 3. PODACI O UZORKU U poslednjoj posma-
tranoj godini, usled nepostojanja prihoda od prodaje,
vrednost koefi cijenta obrta stalne imovine iznosi 0. Usled rasta vrednosti prihoda od prodaje i smanjenja
prosečne vrednosti stalne imovine, preduzeće C je u
2018. godini ostvarilo višu vrednost efi kasnosti stalne
imovine u odnosu na 2017. godinu. Preduzeće C je u
2018. godini na svaki dinar prosečno uložene stalne
imovine ostvarilo 0,48 dinara, dok je u 2017. godini to
bilo 0,38 dinara. U 2019. godini je usledio pad efi ka-
snosti stalne imovine preduzeća C. Tako je, na kraju
poslednje posmatrane godine, preduzeće C na svaki
dinar prosečno angažovane stalne imovine ostvarilo
0,46 dinara prihoda od prodaje. Kako je preduzeće A u
sve tri posmatrane godine zabeležilo najvišu vrednost
koefi cijenta stalne imovine, može se reći da je najefi -
kasnije koristilo stalnu imovinu u generisanju prihoda
od prodaje. U posmatranom periodu preduzeća A i B su sma-
njila efi kasnost upravljanja zalihama, odnosno prisu-
tan je trend smanjenja vrednosti koefi cijenta obrta
zaliha. U preduzeću A, zalihe se obrnu 1,47 puta u
2017. godini, 1,37 puta u 2018. i 1,11 puta u 2019. Pre-
duzeće C je u 2018. godini povećalo efi kasnost zaliha
u odnosu na 2017. godinu, da bi u 2019. godini došlo
do pada efi kasnosti zaliha. U 2017. godini preduzeće
A je najefi kasnije upravljalo zalihama, dok je u 2018. i
2019. godini to činilo preduzeće C. Preduzeća A i B su zabeležila trend smanjenja
efi kasnosti obrtne imovine u generisanju prihoda
od prodaje. Na svaki dinar prosečno uložene obrtne
imovine, preduzeće A je u 2017. godini ostvarilo 1,69 8 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 Koefi cijent obrta dobavljača preduzeća A u 2017. godini iznosio je 2,66, što znači da se 2,66 puta godiš-
nje isplate prosečne obaveze prema dobavljačima. U
2018. godini vrednost ovog pokazatelja je porasla na
3,72, da bi trend rasta bio nastavljen i 2019. godine
(kada je koefi cijent obrta dobavljača 4,49). Vrednost
koefi cijenta obrta dobavljača kod preduzeća B u sve
tri posmatrane godine, kao i kod preduzeća C u prve
dve posmatrane godine, niža je od 1. To ukazuje da iz-
među dve isplate obaveza prema dobavljačima prođe
više od godinu dana, što može biti jedan od signala
da je sposobnost preduzeća za izvršenje obaveza do-
vedena u pitanje. U sve tri posmatrane godine, predu-
zeće A je ostvarilo najvišu vrednost koefi cijenta obrta
dobavljača. 3. PODACI O UZORKU njiće se troškovi skladištenja istih. Takođe, preduzeća
na taj način slobodna novčana sredstva (koja bi bila
“zarobljena” u zalihama) mogu uložiti u druge aktiv-
nosti koje će doneti određenu zaradu. Kroz adekvatnu
analizu poslovanja (postojećih i potencijalnih) kupa-
ca, preduzeća treba da sagledaju realne mogućnosti
za naplatu svojih potraživanja kako bi uskladila prilive
(po osnovu naplate potraživanja) sa odlivima gotovi-
ne (plaćanja prema dobavljačima, kreditorima i dr.). U radu se javlja nekoliko ograničenja. Kako su po-
smatrana samo akcionarska tekstilna preduzeća sa
Beogradske berze, naredna istraživanja treba usme-
riti i na tekstilna preduzeća koja nisu registrovana
kao akcionarska društva. Takođe, treba posmatrati
poslovanje tekstilnih preduzeća u dužem vremen-
skom periodu. Povećanjem veličine uzorka i vremena
posmatranja može se steći preciznija i potpunija sli-
ka o poslovanju preduzeća. Kako istraživanjem nije
obuhvaćeno poslovanje tekstilnih preduzeća u 2020. godini, godini pandemije COVID-19, u narednim istra-
živanjima treba sagledati i uticaj pandemije na efi ka-
snost tekstilnih preduzeća. Da bi se stekla potpuna
slika o poslovanju preduzeća, pored pokazatelja efi -
kasnosti, budućim istraživanjima bi trebalo obuhvatiti
i pokazatelje likvidnosti, solventnosti i profi tabilnosti
[43]. Kako se zadnjih godina značajna pažnja posve-
ćuje zaštiti životne sredine [44], [45], [46], naredna
istraživanja treba usmeriti i na ispitivanje efi kasnosti
tekstilnih preduzeća upravljanjem otpadom, kao ne-
fi nansijskom performansom u poslovanju preduzeća. 5. ZAKLJUČAK Kako se efi kasnost pozitivno odražava na profi ta-
bilnost preduzeća, neophodno je Praćenje efi kasnosti
preduzeća predstavlja važan zadatak koji se stavlja
pred menadžment preduzeća, posebno kada se uzme
u obzir činjenica da efi kasno korišćenje sredstava do-
vodi i do profi tabilnog poslovanja preduzeća. Spro-
vedenim istraživanjem je utvrđeno da postoji znača-
jan prostor za unapređenje efi kasnosti posmatranih
tekstilnih preduzeća, što je potvrđeno rezultatima
istraživanja da određena preduzeća nisu ostvarila pri-
hode od prodaje, ili su ih ostvarila na veoma niskom
nivou, čime nastavak poslovanja može biti doveden u
pitanje. Takođe, utvrđeno je da određena preduzeća
nemaju javno dostupne fi nansijske izveštaje, čime je
blagovremeno donošenja poslovnih odluka dovede-
no u pitanje. LITERATURA [1] Urošević, S., Đorđević, D., Cvijanović, J. M. (2009). Značaj doradnih poslova za razvoj tekstilne i odev-
ne industrija Srbije u procesu tranzicije, Industrija, 2,
97–125. Koristeć i informacije koje su predstavljene kao
rezultat primenjene fi nansijske analiize, mogu se dati
preporuke koje ć e omoguć iti preduzećima da poprave
(ili maksimizuju) svoje performanse u segmentu efi ka-
snosti. To je posebno korisno kao signal menadžerima
koje aktivnosti bi trebalo da preduzmu za efi kasnije
upravljanje preduzećem i maksimizaciju njegove za-
rade. Kako bi unapredila svoju efi kasnost, posmatrana
preduzeća treba da pokrenu aktivnosti koje će dovesti
do povećanja prihoda od prodaje kroz ulaska na nova
tržišta ili unapređenja prodaje postojećim kupcima. Takođe, preduzeća treba da nastoje da se oslobode
“zarobljenog” kapitala u vidu zastarelih postrojenja
i opreme i ulažu u savremenu opremu. To se može
odraziti na povećanje proizvodnje, kao i na sniženje
troškova iste (smanjenje troškova amortizacije, sma-
njenje troškova radne snage, smanjenje škartova i sl.). Kada je reč o zalima, preduzeća treba da nastoje da
drže optimalan nivo istih. Na taj način neće biti ugro-
ženo normalno odvijanje procesa proizvodnje i sma- [2] Samo, A. H., Murad, H. (2019). Impact of liquidity
and fi nancial leverage on fi rm’s profi tability – an
empirical analysis of the textile industry of Pakistan,
Research Journal of Textile and Apparel, 23(4), 291–
305. doi:10.1108/rjta-09-2018-0055. [3] Adler, U. (2004). Structural change: The dominant
feature in the economic development of the German
textile and clothing industries, Journal of Fashion
Marketing and Management: An International Journal,
8(3), 300–319. doi:10.1108/13612020410547815. [4] Pavlović, N., Čelić, I. (2020). The analysis of compet-
itive strategies from the perspective of small and
medium enterprises, Hotel and Tourism Manage-
ment, 8(1), 101–110. doi: 10.5937/menhottur2001101P. 9 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 tu pružanja računovodstvenih informacija, Ekonom-
ija u digitalnom dobu, Banja Luka 26. Maj 2018, pp. 246–257. [5] Hyunju, J., Choonsup, H. (2011). Financial Ratio
Analysis of the Textile and Apparel Industries, Jour-
nal of Fashion Business, 15(3), 125–141. [6] Urošević, S., Fedajev A., Nikolić, R. (2011). Signifi -
cance and perspectives of textile industry in Repub-
lic of Serbia, in tran sitional environment, Industria
textila, 62(3), 134–140. [17] Vasilev, D., Cvetković, D., Grgur, A. (2019). De-
tection of fraudulent actions in the fi nancial state-
ments with particular emphasis on hotel compa-
nies, Hotel and Tourism Management, 7(1), 115–125. doi:10.5937/menhottur1901115V. [7] Bukvić, R. M., Petrović, D. LITERATURA R. (2020). Current status
and perspectives of the textile industry in Serbia,
10th International Conference “Economics and Man-
agement-Based on New Technologies” EMoNT-2020,
Vrnjačka Banja 21-24 June 2020, 1–7. [18] Milašinović, M., Mitrović, A., Milojević, S. (2020). Fi-
nancial performance measuring of a hotel company
- case study. Third International Scientifi c Conference
‘ISCTBL 2020’ CHALLENGES OF TOURISM AND BUSI-
NESS LOGISTICS IN THE 21ST CENTURY, Stip 13 Novem-
ber 2020, pp. 228–234. [18] Milašinović, M., Mitrović, A., Milojević, S. (2020). Fi-
nancial performance measuring of a hotel company i - case study. Third International Scientifi c Conference
‘ISCTBL 2020’ CHALLENGES OF TOURISM AND BUSI-
NESS LOGISTICS IN THE 21ST CENTURY, Stip 13 Novem-
ber 2020, pp. 228–234. [8] Martins, R. A., Mergulhão, R. C. (2006). Financial and
Non-fi nancial and Non-fi nancial Performance Meas-
ures Financial Performance Measures in Six Sigma
Initiatives, Third International Conference on Produc-
tion Research- Americas’ Region 2006 (ICPR-AM06),
1–14. [19] Alhadi, A., Habib, A., Taylor, G., Hasa, M. M. (2020). Financial statement comparability and corporate
investment effi ciency, Meditari Accountancy Re-
search, ahead-of-print (ahead-of-print). doi:10.1108/
MEDAR-12-2019-0629. [9] Bernstein, L. A. (1993). Financial Statement Analysis:
Theory, Application, and Interpretation, 5th ed, Home-
wood, Irwin. [20] Milojević, S., Paunović, J., Vuksanović, N. (2020). The Role of Accountants in Litigation Support and
Expert Witnessing in the Republic of Serbia, Inter-
national Academic Conference „Education and Social
Sciences Business and Economics“, Belgrade 2 March
2020, 255–260. [10] Narkunienė, J., Ulbinaitė, A. (2018). Comparative
analysis of company performance evaluation meth-
ods. Entrepreneurship and sustainability issues, 6(1),
125–138. doi.org/10.9770/jesi.2018.6.1(10). [21] Kovjanić, M. (2020). Fraudulent Financial Report-
ing as a Permanent Problem for Decision Makers,
FINIZ 2020 - People in the focus of process automa-
tion, Belgrade 04 December 2020, pp. 73–77. doi: 10.15308/fi niz-2020-73-77. [11] Ježovita, A. (2015). Variations between fi nancial
ratios for evaluating fi nancial position related to the
size of a company. Review of innovation and compet-
itiveness volume, 1(1), 115–135. [22] Кnežević, S., Barjaktarović Rakočević i D. Đurić
(2011). Primena i ograničenja racio analize fi nan-
sijskih izveštaja u poslovnom odlučivanju, Me-
nadžment: Časopis za teoriju i praksu menadžmenta,
16(61), 25–31. [12] Short, D. G., Libby. R., Libby, P. A. (2007). Financial
accounting: A global Perspective, McGraw-Hill/Irwin,
New York. [13] Gandhare, B. S., Akarte M. M. (2020). Benchmark-
ing maintenance performance in select agro-based
industry, Journal of Quality in Maintenance Engineer-
ing, ahead-of-print (ahead-of-print). LITERATURA DOI 10.1108/
JQME-02-2019-0018. [23] Vujić, M., Travica, J., Vujić, T. (2021). Evaluation of
the fi nancial performance of a hotel company, Sci-
entifi c Conference Accounting and autid in theory and
practice, Banja Luka 28 May 2021, pp. 219–230. [14] Tan, P. M. S., Chye, K. H., Chin, L. L. (1997). Stability
of Financial Ratios: A Study of Listed Companies in
Singapore, Asian Review of Accounting, 5(1), 19–39. https://doi.org/10.1108/eb060680. [24] Schellhorn, C., Sharma, R. (2013). Using the
Rasch model to rank fi rms by managerial abili-
ty, Managerial Finance, 39(3), 306–319. https://doi. org/10.1108/03074351311302818. [15] Kovjanić, M., Vukadinović, P. (2020). Evaluation
of fi nancial performance of small family business
for the purpose of effi cient fi nancial decision-mak-
ing of owners, International Academic Journal, 1(1),
13–23. [25] McLaney, E., Atrill, P. (2010). Accounting: An Intro-
duction, 5th edition, Pearson Education Limited, Har-
low. [26] Knežević, S., Mitrović, A., Vujić, M., Grgur, A. (2019). Analiza fi nansijskih izveštaja, samostalno izdanje au-
tora, Beograd. [16] Mitrović, A., Knežević, S. (2018). Uloga i značaj
računovodstvenih informacionih sistema u konteks- 10 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 [27] Koch, T. W., MacDonald, S. S. (2014). Bank Manage-
ment, 8th edition, Cengage Learning, Boston. [40] Finansijski izveštaji, Agencija za privredne registre,
pristupljeno preko https://www.apr.gov.rs/%d0%
bf%d0%be%d1%87%d0%b5%d1%82%d0%
bd%d0%b0.3.html, datum pristupa 14. 07. 2021. godine. [40] Finansijski izveštaji, Agencija za privredne registre,
pristupljeno preko https://www.apr.gov.rs/%d0%
bf%d0%be%d1%87%d0%b5%d1%82%d0%
bd%d0%b0.3.html, datum pristupa 14. 07. 2021. godine. [28] Vujić, M., Stojanović Alcaraz, J., Milojević, S., Tra-
vica, J. (2021). Financial Ratio Analysis of the Gas-
tronomy Company “Ugostiteljstvo – Simić M”: Case
Study, Specifi cs of human resources management
in family businesses, The Book of Abstracts & Con-
ference Program X International Conference of Social
and Technological Development, Trebinje, 03-06 June
2021, Republic of Srpska, B&H, 54. [41] Kostić, M., Mizdraković, V., Mitić, V. (2020). Non-dis-
closure of fi nancial statements of public companies
as an economic off ense in the Republic of Serbia,
NBP - Nauka, bezbednost, policija, 25(3), 47–58. [42] Obradović, V., Milašinović, M., Bogićević, J. (2021). Obelodanjivanja o segmentima u fi nansijskim iz-
veštajima akcionarskih društava u Republici Srbiji
i Republici Hrvatskoj, Ekonomski horizonti, 23(1),
doi:10.5937/ekonhor2101055O. [29] Gibson, C. H. (2012). Financial Reporting and Anal-
ysis: Using Financial Accounting Information. 13th edi-
tion, South-Western Cengage Learning, Mason. [30] Mitrović, A., Knežević, S. (2020). Računovodstvo,
Fakultet za hotelijerstvo i turizam u Vrnjačkoj Banji
Univerzitet u Kragujevcu, Vrnjačka Banja. LITERATURA [43] Mitrović, A., Knežević, S., Milašinović, M. (2021). Profi tability analysis of hotel companies in the Re-
public of Serbia, Hotel and Toursim Management,
9(1), 121–134. doi: 10.5937/menhottur2101121M. [31] Walsh, C. (2006). Key management ratios: The clear-
est guide to the critical number that drive your busi-
ness, Pearson Education Ltd, Harlow. [44] Kostić, M., Ratković, M., Forlani, M. (2019). Eco-ho-
tels as an example of environmental responsibility
and innovation in savings in the hotel industry, Ho-
tel and Tourism Management, 7(2), 47–56. https://doi.org/10.5937/menhottur1902047K. [32] Subramanyam, K. R. (2014). Financial Statement
Analysis, 11th edition, McGraw-Hill Education, New
York. [33] Кnežević, P. S., Dmitrović, V., Jovanović, M., Obra-
dović, T. (2011). Upravljanje potraživanjima od ku-
paca u funkciji podrške uspešnosti poslovanja, Me-
nadžment: Časopis za teoriju i praksu menadžmenta,
16(58), 49–57. [45] Akinyi Odeny, J., Maingi, S., Kurauka, J. (2020). The
role of procurement procedures in environmental
management: A case study of classifi ed hotels in
Mombasa County, Kenya, Hotel and Tourism Man-
agement, 8(1), 11–23. https://doi.org/10.5937/menhottur2001011A. [34] Žarkić Joskimović, N., Barjaktarović Rakočević,
S., Obradović, T. (2016). Upravljačko računovodstvo
– analiza, planiranje, odlučivanje, kontrola, Fakultet
organizacionih nauka, Beograd. [46] Zdravković, S., Peković, J. (2020). The analysis of
factors infl uencing tourists’ choice of green hotels,
Hotel and Tourism Management, 8(1), 69–78. https://
doi.org/10.5937/menhottur2001069Z. [35] Matan, F., Libman, A. (2007). Crash course in ac-
counting and fi nancial statement analysis, John Wiley
& Sons, Inc., Hobokenm, New Jersey. ___________________
Primljeno/Received on: 05.08.2021. Revidirano/ Revised on: 07.02.2022. Prihvaćeno/Accepted on: 08.02.2022. [36] Shim, J. K. (2008). Analysis and Uses of Financial
Statement, Global Professional Publishing, London. [37] Mitrović, A., Knežević, G. (2016). The selected ratio
activities in the context of the accounting process-
ing of the supplies from the production in progress,
The third International Scientifi c Conference FINIZ
2016 „Risks in Contemporary Business“, Belgrade 02
December 2016, рp. 63–67. ______________________________
© 2021 Authors. Published by Union of Textile Engi-
neers and Technicians of Serbia. This article is an open
access article distributed under the terms and condi-
tions of the Creative Commons Attribution 4.0 Inter-
national license (CC BY) (https://creativecommons. org/licenses/by/4.0/) ______________________________
© 2021 Authors. Published by Union of Textile Engi-
neers and Technicians of Serbia. This article is an open
access article distributed under the terms and condi-
tions of the Creative Commons Attribution 4.0 Inter-
national license (CC BY) (https://creativecommons. org/licenses/by/4.0/) [38] Weetman, P. (2011). ___________________
Primljeno/Received on: 05.08.2021.
Revidirano/ Revised on: 07.02.2022.
Prihvaćeno/Accepted on: 08.02.2022. ______________________________
© 2021 Authors. Published by Union of Textile Engi-
neers and Technicians of Serbia. This article is an open
access article distributed under the terms and condi-
tions of the Creative Commons Attribution 4.0 Inter-
national license (CC BY) (https://creativecommons.
org/licenses/by/4.0/) Primljeno/Received on: 05.08.2021. LITERATURA Financial Accounting: An Intro-
duction, 5th edition, Pearson Education, Harlow. [39] Sektor C, Beogradska berza, pristupljeno preko
https://www.belex.rs/trzista_i_hartije/sektori/C, da-
tum pristupa 14. 07. 2021. godine. 11
|
https://openalex.org/W2806838981
|
https://content.sciendo.com/downloadpdf/journals/pjct/20/2/article-p24.pdf
|
English
| null |
New bio-polyol based on white mustard seed oil for rigid PUR-PIR foams
|
Polish Journal of Chemical Technology
| 2,018
|
cc-by
| 5,759
|
INTRODUCTION polyols based on citric acid (2-hydroxy-1,2,3-propanetri-
carboxylic acid) and various glycols to obtained a rigid
polyurethane-polyisocyanurate foams. These composites
were characterized by good thermal insulation properties,
high aging resistance and low fl ammability. Polyurethanes are the important group of thermosets
because of their wide application in everyday life. The use
of these materials is directly related to their properties:
physical (density), mechanical (brittleness, compressive
strength), thermal (thermal conductivity) and fl amma-
bility. Environmental aspects (e.g reuse of waste from
this materials) and economic aspects (e.g. price of the
fi nished product) are also signifi cant. Often, the desire to
obtain specifi c properties of polyurethanes is related to
the incorporation into the technological formulation of
additives1–4. Many chemical companies strives to receive
green product solutions and keep sustainable develop-
ment strategies. Raw materials of plant origin are recently
more and more used for the production of polyurethane
materials, e.g. bio-polyols based on vegetable oils1, 5–7. They are usually produced by pressing oil plants. The
unrefi ned vegetable oil obtained is additionally subjected
to a fi ltration process to remove solid seed residues8. In
recent years there has been a huge interest in these raw
materials because of their green nature and an alterna-
tive to shrinking oil and gas reserves1. The aim of the study was to obtain a new bio-polyol
based on crude mustard oil and 2,2’-mercaptodiethanol
and to investigate its physicochemical properties. The
effect of this bio-polyol on the properties of rigid poly-
urethane-polyisocyanurate foams was also investigated. New bio-polyol based on white mustard seed oil for rigid PUR-PIR foams Kazimierz Wielki University, J.K. Chodkiewicza 30, 85-064, Bydgoszcz, Poland
*Corresponding author: e-mail: m.borowicz@ukw.edu.pl Kazimierz Wielki University, J.K. Chodkiewicza 30, 85-064, Bydgoszcz, Poland
*Corresponding author: e-mail: m.borowicz@ukw.edu.pl A new bio-polyol based on white mustard oil (Synapis alba) and 2,2’-mercaptodiethanol (2,2’-MDE) was obtained. The synthesis was carried out by two-step method. In the fi rst stage, the double bond of the unsaturated fatty acid
residues was oxidized, and in the second step the epoxy rings were opened with 2,2’-MDE. The properties of the
obtained bio-polyol for application as raw material in polyurethane-polyisocyanurate foams (PUR-PIR) - hydroxyl
number, acid number, density, viscosity, pH, water content, FTIR, 1H NMR and 13C NMR were investigated. Based on the obtained results, foaming formulations containing 0 to 0.6 R of the new bio-polyol were prepared. Signifi cant impact of bio-polyol on apparent density, compressive strength, brittleness, fl ammability, water absorption
and thermal conductivity of polyurethane composites were noted. Modifi ed foam had better functional properties
than reference foam e.g. lower brittleness, better thermal insulation properties and better fi re resistance. Keywords: polyurethane foam, white mustard seed oil, 2,2’-mercaptodiethanol, bio-polyol. 24
Pol. J. Chem. Tech., Vo 2, 2018
Polish Journal of Chemical Technology, 20, 2, 24—31, 10.2478/pjct-2018-0019 Properties of bio-polyol based on mustard oil (Sigma-Aldrich, USA). Anhydrous calcium chloride
(POCh, Poland) was used to neutralize the catalyst and
remove residual water. (Sigma-Aldrich, USA). Anhydrous calcium chloride
(POCh, Poland) was used to neutralize the catalyst and
remove residual water. New bio-polyol was subjected to physicochemical,
analytical and spectroscopic studies to determine its
suitability for the synthesis of rigid PUR-PIR foams. The properties are determined in accordance with the
standards applicable to polyol raw materials: Polyether polyol- Rokopol RF 551 – sorbitol oxypro-
pylene product with HN = 420 mgKOH/g (PCC Rokita,
Poland), was used for the synthesis of PUR-PIR foams. Purocyn B, a technical polyisocyanate (purchased in Pu-
rinova, Poland) was used as a isocyanate raw material. Main ingredient was 4,4’-diphenylmethane diisocyanate. NCO content was 31%. – Hydroxyl number (HN), was determined in accor-
dance with the factory standard of Purinova Chemical
Plant No. WT/06/07/PURINOVA. – Acid value (AV) was determined in accordance with
PN-ISO 660 standard. The catalyst system for the preparation of PUR-PIR
rigid foams was anhydrous potassium acetate (Chempur,
Gliwice) used as a 33% solution in diethylene glycol
(catalyst 12) and DABCO – 1,4-diazabicyclo [2.2.2]
octane (Hülls, Germany) used as a 33% solution in
diethylene glycol. The foam structure stabilizer was Sili-
cone L-6900 – poly-siloxane poly-oxy-alkylene surfactant
(Witco, Sweden). The foaming agent was Solkane HFC
365/227 (Solvay, Belgium) – a mixture of 1,1,1,3,3-pen-
tafl uorobutane and 1,1,1,2,3,3,3-heptafl uoropropane in
a weight ratio of 87:13. The fl ame retardant was Antiblaze
TCMP - tri[2-chloro-1-methylethyl phosphate] (Albright
and Wilson, UK). – Iodine value (IV) was determined in accordance
with PN-ISO 3961:2013-10 standard. – Epoxy value (EV) was determined in accordance
with PN-ISO 3001:1999 standard. – Viscosity of bio-polyol determined by Fungilab digital
rheometer at 20°C (293 K). Measurements were made
using a standard DIN-87 spindle co-operating with the
ULA-DIN-87 bushing. Keeping the constant tempera-
ture is ensured by a thermostat connected to the water
jacket of the sleeve. j
– Density was measured at 25oC (298 K) in an adiabatic
pycnometer in accordance with PN-92/C-04504. – Water content was determined by Carl-Fisher me-
thod using Titraqual branded reagent in accordance with
PN-81/C-04959. Material and methods For the synthesis of a new bio-polyol, the two-stage
method used unrefi ned white mustard seed (Sinapis
alba) oil, as a light yellow liquid with a density of 0.880
g/cm3 and a viscosity of 160 mPa · s, produced by the
company jedzpij.pl (Poland). The iodine value was 0.426
mol I2/100g of oil and the unsaturated fatty acid content
was 95.88 wt% of total fatty acid in the oil. The fatty
acid profi le of the oil used is shown in Table 1. A number of studies on the synthesis of bio-polyols
based on oils: soybean9–11, castor12–15 and rapeseed16–19 can
be found in the literature. Studies on the use of polyols
based on soya oil for rigid foam were conducted by Tu et
al.20. The obtained materials were characterized by a low
coeffi cient of thermal conductivity. Veronese et al.21 used
a mixture of petrochemical polyols and soybean-based
polyol to obtain the resultant transesterifi cation of the
hydroxylated oil with tri-ethanolamine to produce rigid
polyurethane foams. The resulting bio-composites exhi-
bited better mechanical properties than their equivalents
obtained from conventional raw materials. Prociak et
al.22–25 developed composites with bio-polyol based on
rapeseed oil, which was characterized by higher content
of closed cells and lower thermal conductivity. Horak and
Benes26 have used fi sh oil waste to chemically recycle
fl exible polyurethane foams. As a result of the process,
the authors obtained liquid recycled polyols that were
used to obtain new semi-rigid foams. The properties of
these materials were, however, worse than these of pure
raw materials. Liszkowska et al.27–28 with the team used Table 1. Fatty acid profi le of white mustard oil Table 1. Fatty acid profi le of white mustard oil In the fi rst stage of the synthesis, an oxidizing system
was used: 99.5% acetic acid (Chempur, Poland) and 30%
solution of hydrogen peroxide (Chempur, Poland). 96%
sulfuric acid (VI) (POCh, Poland). Anhydrous magnesium
sulphate (VI) (Chempur, Poland) was used to dry the
purifi ed epoxidized white mustard oil. p
p
98% of 2,2’-mercaptodiethanol in presence of the acid
catalyst (as above) was used to open the oxirane rings 25 Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 Synthesis of new bio-polyol 500 g of crude mustard oil (MO) with an iodine value
of 0.426 mol I2/100g, 128.55 g of 99.5% acetic acid (AC)
and 4.35g of 96% sulfuric acid (VI) (SA) were trans-
ferred into a 1000 cm3 reactor equipped with a refl ux
condenser, thermometer, addition funnel and mechanical
stirrer. The mixture was heated to 40°C at constant stirrer
speed (700 rpm) and 362.10g of 30% hydrogen peroxide
(HP) was added via dropping funnel. The molar ratio of
the reactants was 1:1:1.5:0.02 for MO:AC:HP:SA. After
adding all hydrogen peroxide solution, the mixture was
heated to 60oC. Hydrogen peroxide in the presence
of acetic acid produced peracetic acid in situ, which
oxidized the double bonds contained in the oil to the
epoxy groups. The reaction was carried out for 3 hours. During synthesis, the epoxy number of the mixture was
determined all the time. Then the reaction system was
cooled and allowed to stand for 24 hours to separate
the oil phase from the aqueous phase. The oil phase was
washed with distilled water to remove residual acetic
acid and catalyst, and the remaining water was removed
with anhydrous magnesium sulphate (VI). The obtained
intermediate (epoxidized oil) was subjected to analytical
tests and was used to further synthesis. – The pH was measured by Hanna Instruments mi-
croprocessor laboratory pH-meter (ORP/ISO/oC) with
RS 22 C connector. – Elemental analysis with the Elementar Vario EL
III CHNSO. – Elemental analysis with the Elementar Vario EL
III CHNSO. – Spectroscopic analysis of the chemical structure of
crude mustard oil, epoxidized oil and bio-polyol was
also performed: – Spectroscopic analysis of the chemical structure of
crude mustard oil, epoxidized oil and bio-polyol was
also performed: – In infrared spectroscopy was performed using the
Brücker Vector Spectrophotometer with the KBr tech-
nique in the range of 400 to 4000 cm–1. – In infrared spectroscopy was performed using the
Brücker Vector Spectrophotometer with the KBr tech-
nique in the range of 400 to 4000 cm–1. q
g
– 1H NMR and 13C NMR were performed using the
Brücker Ascend III NMR spectrometer at 400 MHz in
deuterated chloroform. q
g
– 1H NMR and 13C NMR were performed using the
Brücker Ascend III NMR spectrometer at 400 MHz in
deuterated chloroform. Preparation of PUR-PIR foams The preparation of rigid PUR-PIR foams with white
mustard oil based bio-polyol required experimental
studies to determine the optimal composition of addi-
tives (catalysts, surfactants, fl ame retardants and blow-
ing agents). The basis for determining the amount of
polyols raw materials was the hydroxyl number. Polyol
formulation was developed to keep a constant isocyanate
index. The addition of isocyanate was selected taking
into account the equivalent (R) ratio of the NCO to
OH groups in the reaction mixture, which for PUR-PIR
rigid foams was set to 3:1. It is essential for the reaction
of the NCO groups with OH to produce the urethane
bond (Scheme 1) and the trimerization of the three NCO
groups to the isocyanurate ring (Scheme 2). In the next step, 500 g of epoxidized white mustard oil
(EMO), 234.24 g of 98% 2,2’-mercaptodiethanol (MDE)
and 1.96 g of 96% sulfuric acid (VI) were transferred into
a 1000 cm3 reactor. The molar ratio of the reagents was
1:1:0.01 for EMO:MDE:SA. The mixture was heated to
120°C under continuous stirring (700 rpm). The synthesis
was performed for 4 hours to open all epoxy rings (EV
= 0). After synthesis, the obtained polyol was neutralized
and dried with solid anhydrous calcium chloride. The content of additives, i.e. catalyst of reaction be-
tween OH and NCO (1 wt%), catalyst of trimerization
reaction (2.5 wt%), physical blowing agent (10 wt%),
fl ame retardant (17 wt%) and surfactant (1.7 wt% ). The properties of the new bio-polyol (PG3) based on
white mustard oil were studied. The reaction product
was then used for the synthesis of rigid polyurethane-
-polyisocyanurate foams. The foams were prepared on a laboratory scale from
two-component system by using one-stage method. Component A was obtained by precisely mixing the Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 26 (stability of linear dimensions, change of geometrical
volume) and PN-ISO 4590:1994 (mass loss). Scheme 1. Reaction of urethane bond formation. )
(
)
– The thermal conductivity of the foams was determi-
ned by Lasercomp FOX 200. )
(
)
– The thermal conductivity of the foams was determi-
ned by Lasercomp FOX 200. p
– Absorbability and water absorption were carried out
in accordance with DIN 53433. – Absorbability and water absorption were carried out
in accordance with DIN 53433. Scheme 1. Reaction of urethane bond formation. Preparation of PUR-PIR foams – Retention (Bütler’s vertical test) was determined in
accordance with ASTM D3014-73. Scheme 2. The trimerization of isocyanate to isocyanurate ring. – Test of horizontal burning was determined in accor-
dance with PN-78 C-05012. – Oxygen index (OI) was determined by Concept Equ-
ipment apparatus in accordance with ASTM D2863-1970. RESULTS AND DISCUSSION Scheme 2. The trimerization of isocyanate to isocyanurate ring. Synthesis and characterisation of polyols appropriate amounts of polyols, catalysts, surfactant,
blowing agent and fl ame retardant. Component B was
Purocyn B. Components A and B were mixed for 10 s
by using a mechanical stirrer (1800 rpm) at a suitable
mass ratio. Then mixture was poured into a rectangular
opened mould with an internal dimension of 25 x 25 x 30
cm, where was free rise of foam. Milk-white epoxidized oil (EMO) with an iodine va-
lue of 0.039 mol I2/100g of oil and an epoxy number of
0.384 mol/100g of oil was obtained by reaction of white
mustard oil (MO) with peracetic acid produced in situ. A simplifi ed reaction on the example of an oleic acid
triglyceride, was shown in Scheme 3. A series of P3 foams (P3.0-P3.6) was added with an
increase in the equivalent of bio-polyol from 0 to 0.6
(0.1R increments) at the expense of the petrochemical
polyol (Table 2). The synthesis of PUR-PIR foams was
repeated twice. The resulting foam was cured for 4
hours at 120°C. Scheme 3. Epoxidation of unsaturated bonds in fatty acids Assessing the properties of rigid PUR-PIR foams The selected performance characteristics of the PUR-
-PIR foams have been determined according to the
accepted standards for rigid polyurethane foam: – Direction of the foaming process was analysed by
measuring of typical foaming times: start, growth and
gelation by electronic stopwatch29–30. Scheme 3. Epoxidation of unsaturated bonds in fatty acids – Apparent density foam is marked in accordance
with ISO 845-1988. The effi ciency of oxidation the double bonds was de-
termined as the ratio of the epoxy number of EMO to
the initial iodine number of MO. It was equal 89.67% 31. – Compressive strength was achieved on Instron’s
universal 5544 machine in accordance with PN 93/C-
89071 (ISO 844). q
The epoxidized mustard oil was reacted to open the
epoxy rings with the 2,2’-mercaptodiethanol according
to Scheme 4. – The brittleness of the obtained foams was determined
in accordance with ASTM C-421-61. – Aging tests of foam were determined after 48h
thermostat at 120oC in accordance with ISO 1923:1981 The physicochemical properties of obtained light yellow
bio-polyol (PG3) were shown in Table 3. Table 2. Formulation of rigid PUR-PIR foams with bio-polyol Table 2. Formulation of rigid PUR-PIR foams with bio-polyol Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 27 Scheme 4. Reaction of epoxy ring opening by 2,2’-mercapto-
diethanol Scheme 4. Reaction of epoxy ring opening by 2,2’-mercapto-
diethanol group (–OH) stretching, (2) 2950 and 2925 cm–1 of C-H
bond stretching in -CH2- and -CH3, (3) 1740 cm–1 of
carbonyl group (C=O) stretching, (4) 1465 and 1380 cm–1
of C-H bond deformation in -CH2- and -CH3, (5) 1240, The effi ciency of the epoxy ring opening process was
equal 100% because the epoxy value of the bio-polyol
was 0 mol/100g of oil. The results of the elemental analysis of the obtained
bio-polyol (PG3) are presented in Table 4. Table 3. Properties of bio-polyol based on white mustard oil
Table 4. Results of elemental analysis of oil and bio-polyol Table 3. Properties of bio-polyol based on white mustard oil 1160 and 1089 cm–1 of torsional vibration of the ester
group and (6) 725 cm–1 of oscillation of -CH2- group. There was a signifi cant increase in oxygen and sulphur
content, at the expense of carbon and hydrogen. The
increase in oxygen was due to the oxidation of double
bonds and the opening of the rings with 2,2’-mercap-
todiethanol. Assessing the properties of rigid PUR-PIR foams As a result, two -OH groups (one at the
fatty acid carbon, the other at the end of the glycol
chain) were obtained per a C=C bond. The increase in
sulphur content is attributable to the attachment of the
2,2’-mercaptodiethanol molecule. Analysis of 1H NMR spectrum (Figure 2) showed
characteristic chemical shift for: (1) protons of oleic
groups of fatty acids -CH=CH-; (2) methane protons of
glyceryl -CH2-CH-CH2-; (3) methylene protons of glyceryl
-CH2-CH-CH2-; (4) protons of hydroxyl groups at the
end of the chain -OH; (5) protons of α-CH2 groups to
the hydroxyl group -CH2-OH; (6) protons of the β-CH2
group to the hydroxyl group -CH2-CH2-OH; (7) protons
of α-CH2 groups to the sulphide group -CH2-S-CH2; (8)
protons of the α-CH2 group to the carbonyl group -CH2-
CO-; (9) protons of hydroxyl groups inside the chain
-OH; (10) protons of α-CH2 groups with respect to olefi n
group -CH2-CH=CH-; (11) protons of the β-CH2 group
to the carbonyl group -CH2-CH2-CO-; (12) protons of
CH2 groups in the fatty acid chain; (13) protons of the
end groups -CH3. Analysis of 1H NMR spectrum (Figure 2) showed
characteristic chemical shift for: (1) protons of oleic
groups of fatty acids -CH=CH-; (2) methane protons of
glyceryl -CH2-CH-CH2-; (3) methylene protons of glyceryl
-CH2-CH-CH2-; (4) protons of hydroxyl groups at the
end of the chain -OH; (5) protons of α-CH2 groups to
the hydroxyl group -CH2-OH; (6) protons of the β-CH2
group to the hydroxyl group -CH2-CH2-OH; (7) protons
of α-CH2 groups to the sulphide group -CH2-S-CH2; (8)
protons of the α-CH2 group to the carbonyl group -CH2-
CO-; (9) protons of hydroxyl groups inside the chain
-OH; (10) protons of α-CH2 groups with respect to olefi n
group -CH2-CH=CH-; (11) protons of the β-CH2 group
to the carbonyl group -CH2-CH2-CO-; (12) protons of
CH2 groups in the fatty acid chain; (13) protons of the
end groups -CH3. The presence of sulphur in the bio-polyol molecule
also has other important implications. This element
belongs to the so-called group fl ame retardant. This
suggests that it can play a dual role in the polyurethane
preparation process as a polyol component and as an
internal fl ame retardant. The chemical structure of synthesized bio-polyol was
confi rmed by spectroscopic analyses: FTIR, 1H NMR
and 13C NMR. The FTIR method (Figure 1) showed the
presence of band intensity at: (1) 3450 cm–1 of hydroxyl Figure 1. Assessing the properties of rigid PUR-PIR foams FTIR spectrum of bio-polyol based on mustard oil Figure 1. FTIR spectrum of bio-polyol based on mustard oil Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 28 the fatty acid chain; (12) carbons of penultimate groups
-CH2-CH3; (13) carbons of the terminal groups -CH3. FTIR spectroscopy, 1H NMR and 13C NMR confi r-
med the assumed chemical structure of the synthesized
bio-polyol. Analysis of the 13C NMR spectrum (Figure 3) showed
characteristic chemical shift for: (1) carbons of car-
bonyl groups >C=O; (2) carbons of olefi nic fatty acids
-CH=CH-; (3) carbons of α-CH2 groups to the alkoxyl
group -CH2-O-CH2-; (4) carbons of α-CH groups linked
to a hydroxyl group within the chain >CH-OH; (5)
Figure 2. 1H NMR spectrum of bio-polyol based on mustard oil Figure 2
1H NMR spectrum of bio polyol based on mustard oil Figure 2. 1H NMR spectrum of bio-polyol based on mustard oil Analysis of the 13C NMR spectrum (Figure 3) showed
characteristic chemical shift for: (1) carbons of car-
bonyl groups >C=O; (2) carbons of olefi nic fatty acids
-CH=CH-; (3) carbons of α-CH2 groups to the alkoxyl
group -CH2-O-CH2-; (4) carbons of α-CH groups linked
to a hydroxyl group within the chain >CH-OH; (5)
methane carbons of glyceryl -CH2-CH-CH2-; (6) meth-
ylene carbons of glyceryl -CH2-CH-CH2-; (7) carbons of
α-CH2 groups to the hydroxyl group at the end of the
chain -CH2-OH; (8) carbons of the α-CH2 group to the
sulphide group -CH2-S-CH2-; (9) carbons of β-CH2 groups
to the hydroxyl group at the end of the chain -CH2-
CH2-OH; (10) carbons of α-CH2 groups to the carbonyl
group -CH2-OOC-CH2-; (11) carbons of CH2 groups in the fatty acid chain; (12) carbons of penultimate groups
-CH2-CH3; (13) carbons of the terminal groups -CH3. the fatty acid chain; (12) carbons of penultimate groups
-CH2-CH3; (13) carbons of the terminal groups -CH3. the fatty acid chain; (12) carbons of penultimate groups
-CH2-CH3; (13) carbons of the terminal groups -CH3. FTIR spectroscopy, 1H NMR and 13C NMR confi r-
med the assumed chemical structure of the synthesized
bio-polyol. 2
3 (
)
g
p
3
FTIR spectroscopy, 1H NMR and 13C NMR confi r-
med the assumed chemical structure of the synthesized
bio-polyol. FTIR spectroscopy, 1H NMR and 13C NMR confi r-
med the assumed chemical structure of the synthesized
bio-polyol. Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 In both cases absorbability and water
absorption was due to the introduction of hydrophobic
groups derived from vegetable oil into the polyurethane
macromolecule. An important parameter for PUR-PIR rigid foams is
the apparent density. It indirectly affects compression
strength or brittleness. The increase in the share of the
new polyol raw material resulted in a decrease in appa-
rent density from 50.19 kg/m3 for the reference foam to
33.07 kg/m3 for the foam with 0.6 R. The incorporation
to polyurethane, a component containing long linear
chains results in a decrease in packing of the polymer
macromolecules. It follows that fatty acid residues are
fl exible segments with low crosslinking. Also water con-
tent in the bio-polyol had an impact on apparent density. Free water molecule could react with NCO group and
produce a chemical blowing agent – CO2. More blowing
agent in the polyurethane formulation caused apparent
density decrease. This phenomenon also signifi cantly
affected on the compressive strength of rigid foams. With the increase in the number of elastic segments and
the apparent density decrease, this parameter decreased
from 377.43 kPa (PG3.0) to 293.31 kPa (PG3.6). In spite
of a marked decrease in compressive strength, the value
of this parameter was at a satisfactory level. Another
consequence of the decrease in degree of packing was
slight decrease in stiffness. This leads to a reduction in
the brittleness of the obtained PUR-PIR rigid foams. In
case of application of bio-polyol based on white mustard
oil, the parameter was reduced from 40.17% for P3.0
foam to 3.53% for P3.6 foam (Figure 4). Figure 5. Dependence between absorbability, water absorption
and bio-polyol content Figure 5. Dependence between absorbability, water absorption
and bio-polyol content An important feature of porous polyurethane materials
is their fl ammability. Rigid polyurethane-polyisocyanurate
foams are characterized by lower fl ammability than polyu-
rethane foams due to the presence of their isocyanurate
ring. However, due to stringent fi re safety requirements,
they must have a fl ame retardant (Antiblaze TMCP
in foam series P3). Flammability tests of obtained
polyurethane materials (vertical test, horizontal test,
oxygen index) showed that the increase of the content
of bio-polyol based on white mustard oil infl uenced on
the increase of fi re resistance of these materials. Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 29 Studies of accelerated aging i.e. change of linear di-
mensions (Δl) and change of geometric volume (ΔV)
showed that bio-polyol modifi ed foam up to 0.4 R had
the same value like a reference foam, 1.3% and 6%,
respectively. The increase in bio-polyol content resulted
in an increase of Δl to 5.6% and ΔV to 10.8%. This
was due to the migration of the blowing agent closed
in the foam cells. The increased amount of long elastic
segments encouraged easier escape from gas fumes such
as 1,1,1,3,3-pentafl uorobutane and 1,1,1,2,3,3,3-heptafl uo-
ropropane (Solkane HFC 365/227). Mass loss studies
have shown that the addition of white mustard based
bio-polyol was conducive to lowering this parameter
from 3.09% for reference foam to 1.37% for foam with
0.6 R. This meant that the bio-polyol has a higher aging
resistance than petrochemical polyol. measured during foaming, showed a tendency to elongate
(Table 5). However, up to 0.4 R technology times were
similar to the reference foam. With the use of more
bio-polyol, a noticeable increase in start, growth and
gelling time. This suggests that the new white mustard-
-based bio-polyols have less reactivity than petrochemical
polyols. As long as its contribution to the formulation
is less than 0.5 R, it does not signifi cantly affect the
reactivity of the polyol premix (component A). measured during foaming, showed a tendency to elongate
(Table 5). However, up to 0.4 R technology times were
similar to the reference foam. With the use of more
bio-polyol, a noticeable increase in start, growth and
gelling time. This suggests that the new white mustard-
-based bio-polyols have less reactivity than petrochemical
polyols. As long as its contribution to the formulation
is less than 0.5 R, it does not signifi cantly affect the
reactivity of the polyol premix (component A). Table 5. Processing times of PUR-PIR foams with bio-polyol Table 5. Processing times of PUR-PIR foams with bio-polyol Absorbability and water absorption are important pa-
rameters in porous materials. The fi rst one refers to the
amount of water that is in the material immediately after
being removed from the dipping. The second one refers
to the water that has got into its interior. In both cases,
a signifi cant decrease in the value of this parameter was
noted (Figure 5). Properties of rigid polyurethane-polyisocyanurate foams The second part of the experiment consisted of the
synthesis of polyurethane-polyisocyanurate foams using
a new white mustard oil based bio-polyol (PG3) in an
amount of 0 to 0.6 molar equivalents. The formulation
of rigid PUR-PIR foams are shown in Table 2. The effect of polyol on a number of properties of new
polyurethane foams was investigated. Processing times, Figure 3. 13C NMR spectrum of bio-polyol based on mustard oil Figure 3
13C NMR spectrum of bio polyol based on mustard oil Figure 3. 13C NMR spectrum of bio-polyol based on mustard oil Figure 3. 13C NMR spectrum of bio-polyol based on mustard oil CONCLUSION New bio-polyol based on white mustard oil was synthe-
sized in a two-step method: epoxidation of double bonds
and opened it with 2,2’-mercaptodiethanol (Pat. Appl. PL422888). After testing, the obtained product showed
suitability for the synthesis of rigid polyurethane-polyiso-
cyanurate foams. It was characterized by i.e. a hydroxyl
number of 371.70 mg KOH/g, an acid value of 4.71
mg KOH/g or a water content of 0.8%. The presented
parameters were similar to those of commercial petro-
chemical polyols used in rigid foams. The bio-polyol was
used as a raw material for PUR-PIR foams, in amounts
of 0 to 0.6 equivalents in admixture with a polyol based
on product of oxypropylation of sorbitol. Properties of
the obtained materials were tested. Studies were shown
that bio-polyol based foams had lower apparent density,
compressive strength and brittleness. The presence of
polyols containing sulphur signifi cantly affected the fl ame
retardancy of these materials. 11. Garrison, T., Murawski, A. & Quirino, R.L. (2016). Bio-
-based polymers with potential for biodegradability. Polymers,
8 (7), 262. DOI: 10.3390/polym8070262. 12. Ibrahim, S., Ahmad, A. & Mohamed, N.S. (2015). Characterization of Novel Castor Oil-Based Polyurethane
Polymer Electrolytes. Polymers, 7 (4), 747–759. DOI: 10.3390/
polym7040747. 13. Abdolhosscini, F. & Besharati Givi, M.K. (2016). Characterization of a Biodegradable Polyurethane Elastomer
Derived from Castor Oil. Am. J. Polym. Sci, 6 (1), 18–27. DOI:
10.5923/j.ajps.20160601.03. 14. Noreen, A., Zia, K.M., Zuber, M., Tabasum, S. & Za-
hoor, A.F. (2016). Bio-based polyurethane: An effi cient and
environment friendly coating systems: A review. P. Org. Coat.,
91, 25–32. DOI: 10.1016/j.porgcoat.2015.11.018. 15. Fu, C., Hu, X., Yang, Z. Shen, L. & Zheng, Z. (2015). Preparation and properties of waterborne bio-based polyure-
thane/siloxane cross-linked fi lms by an in situ sol–gel process. P. Org. Coat., 84, 18–27. DOI: 10.1016/j.porgcoat.2015.02.008. 16. Kong, X., Liu, G. & Curtis J. (2011). Characterization
of canola oil based polyurethane wood adhesives. Int. J. Adh. Adh., 559–564. DOI: 10.1016/j.ijadhadh.2011.05.004. The use of bio-polyol for the synthesis of PUR com-
posites perfectly fi ts into the principle of sustainable
development. It allows partial replacement of petroche-
mical polyols, and the ability to lower fl ammability by
these compounds is an interesting issue that can lead
to reduced use of expensive and toxic fl ame retardants. 17. Prociak, A., Kurańska, M., Cabulis, U. & Kirpluks, M. (2017). Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 The
combustion residue increased from 92.77% for P3.0 to
99.15% for P3.6, the oxygen index from 24.1% to 26.0%
(Figure 6) and the burning rate decreased accordingly
from 0.167 mm/s to 0,033 mm/s. Figure 4. Dependence between brittleness, apparent density
and bio-polyol content Obtained results indicated that the increase in the
proportion of bio-polyol based on white mustard oil had
signifi cantly infl uence of the retention of rigid PUR-PIR
foams. The reason for this was the presence of sulphur
atoms in the structure (about 10%, Table 3). This con-
fi rmed that this element is included in the group of fi re Figure 4. Dependence between brittleness, apparent density
and bio-polyol content Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 30 Figure 6. Dependence between retention, oxygen index and
bio-polyol content 3. Król, P. (2009). Polyurethanes — A review of 60 years of
their syntheses and applications. Polimery, 54 (7–8), 489–500. [in Polish]. 4. Czupryński, B. (2004). Issues in chemistry and technology
of polyurethanes. Bydgoszcz, Poland: Wydaw. Akad. Bydg. [in
Polish]. ]
5. Paciorek-Sadowska, J., Czupryński, B. & Liszkowska, J. (2011). Application of waste products from agricultural-food
industry for production of rigid polyurethane-polyisocyanura-
te foams. J. Porous Mater, 18, 631–638. DOI: DOI 10.1007/
s10934-010-9419-8. 6. Kurańska, M., Prociak, A., Kirpluks, M. & Cabulis,
U. (2013). Porous polyurethane composites based on bio-
-components. J. Com. Sci. Tech., 75, 70–76. DOI: 10.1016/j. compscitech.2012.11.014. Figure 6. Dependence between retention, oxygen index and
bio-polyol content 7. Piszczyk, Ł., Strankowski, M., Danowska, M., Hejna,
A. & Haponiuk, J. (2014). Rigid polyurethane foams from
a polyglycerol-based polyol. Eur. Polym. J., 57, 143–150. DOI:
10.1016/j.eurpolymj.2014.05.012. retardants. As a result new polyol raw material was also
an internal fl ame retardant. Thermal conductivity was reduced by using bio-polyol
from 0.028 W/(m · K) for the reference foam to 0.024
W/(m · K) for modifi ed foam. This meant that rigid poly-
urethane-polyisocyanurate foams based on white mustard
oil and 2,2’-mercaptodiethanol were better insulates heat
than foam containing petrochemical counterparts in the
same formulation. 8. Bartuzi, K. (2012). Vegetable oils, characteristics and
production technology. J. Nutri Life, 9. Retrieved October 16,
2017 from http://www.NutriLife.pl/index.php?art=52 [in Polish]. 9. Miao, S., Sun, L., Wang, P., Liu, R., Su, Z. & Zhang, S. (2012). Soybean oil-based polyurethane networks as candidate
biomaterials: Synthesis and biocompatibility. Eur. J. Lipid Sci. Technol, 114, 1165–1174. DOI: 10.1002/ejlt.201200050. 10. Miao, S., Zhang, S., Su, Z. CONCLUSION Rapeseed oil as main component in synthesis of bio-
-polyurethane-polyisocyanurate porous materials modifi ed with
carbon fi bers. Polymer Testing, 59, 478–486. DOI: 10.1016/j. polymertesting.2017.03.006. Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 & Wang, P. (2013). Syn-
thesis of bio-based polyurethanes from epoxidized soybean
oil and isopropanolamine. J. App. Polym. Sci, 10, 1929–1936. DOI: 10.1002/app.37564. 2. Lubczak, J., Chmiel-Szukiewicz, E., Duliban, J., Głowacz-
-Czerwonka, D., Lubczak, R., Łukasiewicz, B., Zarzyka, I.,
Łodyga, A., Tyński, P., Kozioł, M., Majerczyk, Z. & Minda-
-Data, D. (2014). Polyurethane foams with 1,3,5-triazine ring of
improved thermal stability. Przem. Chem., 93 (10), 1690–1697.
DOI: 10.12916/przemchem.2014.1690. [in Polish]. 1. Prociak, A., Rokicki, G. & Ryszkowska, J. (2014). Po-
lyurethane materials. Warszawa, Poland: Wydaw. Naukowe
PWN. [in Polish].
2. Lubczak, J., Chmiel-Szukiewicz, E., Duliban, J., Głowacz-
-Czerwonka, D., Lubczak, R., Łukasiewicz, B., Zarzyka, I.,
Łodyga, A., Tyński, P., Kozioł, M., Majerczyk, Z. & Minda-
-Data, D. (2014). Polyurethane foams with 1,3,5-triazine ring of
improved thermal stability. Przem. Chem., 93 (10), 1690–1697.
DOI: 10.12916/przemchem.2014.1690. [in Polish]. LITERATURE CITED 18. Bueno-Ferrer, C., Hablot, E., del Carmen Garrigos, M.,
Bocchini, S., Averous, L. & Jimenez, A. (2012). Relationship
between morphology, properties and degradation parameters
of novative biobased thermoplastic polyurethanes obtained
from dimer fatty acids. Polym. Deg. Stab., 97, 1964–1969. DOI:
10.1016/j.polymdegradstab.2012.03.002. 1. Prociak, A., Rokicki, G. & Ryszkowska, J. (2014). Po-
lyurethane materials. Warszawa, Poland: Wydaw. Naukowe
PWN. [in Polish]. 2. Lubczak, J., Chmiel-Szukiewicz, E., Duliban, J., Głowacz-
-Czerwonka, D., Lubczak, R., Łukasiewicz, B., Zarzyka, I.,
Łodyga, A., Tyński, P., Kozioł, M., Majerczyk, Z. & Minda-
-Data, D. (2014). Polyurethane foams with 1,3,5-triazine ring of
improved thermal stability. Przem. Chem., 93 (10), 1690–1697. DOI: 10.12916/przemchem.2014.1690. [in Polish]. 19. Malewska, E., Bąk, S., Kurańska, M. & Prociak, A. (2016). The effect of various rapeseed oil-based polyols on
selected properties of fl exible polyurethane foams. Polimery,
61, 799–806. DOI: 10.14314/polimery.2016.799. p
y
20. Tu, Y., Kiatsimkul, P., Suppes, G. & Hsieh, F. (2007). Physical properties of water-blown rigid polyurethane foams Pol. J. Chem. Tech., Vol. 20, No. 2, 2018
31 from vegetable oil-based polyols. J. Appl. Polym. Sci., 105,
453–459. DOI: 10.1002/app.26060. from vegetable oil-based polyols. J. Appl. Polym. Sci., 105,
453–459. DOI: 10.1002/app.26060. 21. Veronese, V.B., Menger, R.K., de C. Forte, M.M. &
Petzhold, C.L. (2011). Rigid polyurethane foam based on ve-
getable oil. J. Appl. Polym. Sci., 120, 530–537. DOI: 10.1002/
app.33185. pp
22. Prociak, A. (2008). Heat-insulating properties of rigid
polyurethane foams synthesized with use of vegetable oils –
based polyols. Polimery, 53, 195–200. [in Polish]. 23. Prociak, A. (2008). New generation polyurethane thermal
insulation materials. Kraków, Poland: Wydaw. Politech.Krak. [in Polish] 24. Rojek, P. & Prociak, A. (2012). Effect of different ra-
peseed-oil-based polyols on mechanical properties of fl exible
polyurethane foams. J. Appl. Polym. Sci., 125, 2936–2945. DOI:
10.1002/app.36500. 25. Kurańska, M. & Prociak, A. (2014). Environmentally
friendly polyurethane-polyisocyanurate foams for applications
in the construction industry. Czasopismo techniczne Budow-
nictwo, 5-B, 149–152. 26. Horak, P. & Benes, H. (2015). Polyurethane foams
based entirely on recycled polyols derived from natural oils. Polimery, 60 (9), 579–585. DOI: 10.14314/polimery.2015.579. 27. Liszkowska, J., Czupryński, B., Paciorek-Sadowska, J. &
Michałowski, S. (2016). Thermal and fl ammable properties of
rigid PUR-PIR foams obtained by using new compound based
on 2-hydroxypropane-1,2,3-tricarboxylic acid. J. Cell. Plast., 52
(3), 321–341. DOI: 10.1177/0021955X15570983. 28. Liszkowska, J., Czupryński, B. & Paciorek-Sadowska, J. (2016). Thermal properties of polyurethane-polyisocyanurate
(PUR-PIR) foams modifi ed with tris (5-hydroxypenthyl) citra-
te. J. Adv. LITERATURE CITED Chem. Eng, 6, 2. DOI: 10.4172/2090-4568.1000148. g
29. Paciorek-Sadowska, J., Borowicz, M., Czupryński, B. &
Liszkowska, J. (2017). Composites of rigid polyurethane-poly-
isocyanurate foams with oak bark. Polimery, 62 (9), 666–672. DOI: 10.14314/polimery.2017.666. [in Polish]. 30. Paciorek-Sadowska, J., Borowicz, M., Czupryński, B.,
Liszkowska, J. & Tomaszewska, E. (2018). Application of
halloysite as fi ller in the production of rigid PUR-PIR foams. Polimery, 63 (3), 185–190. DOI: 14314/polimery.2018.3.3. [in
Polish]. 31. Smagowicz, A. (2011). Obtaining of epoxidized rapeseed
oil. Pubished doctoral dissertation, Zachodniopomorski Uni-
wersytet Technologiczny, Szczecin, Poland. [in Polish].
|
https://openalex.org/W4283359440
|
https://escholarship.org/content/qt8bk6n2wq/qt8bk6n2wq.pdf?t=re8lpu
|
English
| null |
Nutrient and Trace Element Contributions from Drained Islands in the Sacramento–San Joaquin Delta, California
|
San Francisco estuary and watershed science
| 2,022
|
cc-by
| 17,703
|
Authors Copyright Information
Copyright 2022 by the author(s).This work is made available under the terms of a Creative
Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ UC Davis
San Francisco Estuary and Watershed Science
Title
Nutrient and Trace Element Contributions from Drained Islands in the Sacramento–San
Joaquin Delta, California
Permalink
https://escholarship.org/uc/item/8bk6n2wq
Journal
San Francisco Estuary and Watershed Science, 20(2)
Authors
Richardson, Christina M.
Fackrell, Joseph K.
Kraus, Tamara E. C.
et al.
Publication Date
2022
DOI
10.15447/sfews.2022v20iss2art5
Supplemental Material
https://escholarship.org/uc/item/8bk6n2wq#supplemental
Copyright Information
Copyright 2022 by the author(s).This work is made available under the terms of a Creativ
Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/
Peer reviewed UC Davis
San Francisco Estuary and Watershed Science
Title
Nutrient and Trace Element Contributions from Drained Islands in the Sacramento–San
Joaquin Delta, California
Permalink
https://escholarship.org/uc/item/8bk6n2wq
Journal
San Francisco Estuary and Watershed Science, 20(2)
Authors
Richardson, Christina M. Fackrell, Joseph K. Kraus, Tamara E. C. et al. Publication Date
2022
DOI
10.15447/sfews.2022v20iss2art5
Supplemental Material
https://escholarship.org/uc/item/8bk6n2wq#supplemental
Copyright Information
Copyright 2022 by the author(s).This work is made available under the terms of a Creativ
Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/
Peer reviewed Powered by the California Digital Library
University of California eScholarship.org eScholarship.org Sponsored by the Delta Science Program and the UC Davis Muir Institute RESEARCH Nutrient and Trace Element Contributions from Drained
Islands in the Sacramento–San Joaquin Delta, California Christina M. Richardson* 1, Joseph K. Fackrell1,2, Tamara E. C. Kraus2, Megan Young3, Adina Paytan4 Christina M. Richardson* 1, Joseph K. Fackrell1,2, Tamara E. C. Kraus2, Megan Young3, Adina Paytan4 ABSTRACT concentrations in island drainage on three Delta
islands and surrounding rivers from June 2017
to September 2018. These data were used to
calculate island-level fluxes and then upscaled
to estimate Delta-wide contributions from island
drains. Based on these results, we present (1) new
estimates of gross and net nutrient and trace
element fluxes from Delta island drains, and
(2) concomitant N stable isotope data to improve
our understanding of island N cycling. Over
60% of nearly all island drainage gross nutrient
and trace element loads occurred in winter and
spring. Upscaled island drainage net annual total
nitrogen (TN), total dissolved nitrogen (TDN), and
NH4
+ loads comprised an estimated 9%, 7%, and
4%, respectively, of annual inputs to this system
in 2018, before the SRWTP upgrade. Under a
post-upgrade scenario, we estimated net annual
island drainage TDN contributions to increase to
11% and NH4
+ contributions to 45% of total Delta
inputs as the SRWTP NH4
+ load diminished to
near zero. Our results suggest that island drainage
is a measurable N source that has likely become
increasingly important now that the SRWTP
upgrade is complete. With over 200 potential
active outfalls, these inputs may affect aquatic
biogeochemical cycling in many regions of the
Delta, especially in areas with long residence
times. Inventorying nutrient and trace element sources
in the Sacramento-San Joaquin Delta (the Delta) is
critical to understanding how changes—including
alterations to point source inputs such as
upgrades to the Sacramento Regional Wastewater
Treatment Plant (SRWTP) and landscape-scale
changes related to wetland restoration—may
alter the Delta’s water quality. While island
drains are a ubiquitous feature of the Delta,
limited data exist to evaluate island drainage
mass fluxes in this system. INTRODUCTION Over the last 4 decades, the San Francisco Bay and
Sacramento-San Joaquin Delta (the Delta) have
experienced drastic ecological changes. From
the 1970s to the 1990s, primary production and
phytoplankton biomass decreased by 40% and
60%, respectively (Jassby 2008). More recent work
shows that cholorphyll a concentrations have
declined by over 70% since 1975 (Cloern 2019). At the same time, less desirable phytoplankton
species that produce cyanotoxins, like Microcystis,
are increasing in abundance (Lehman et al. 2013). The introduction of a number of invasive species,
including two clams, Potamocorbula amurensis
and Corbicula fluminea, are commonly thought
of as important catalysts of structural ecological
change in this system, and many studies suggest
that their introductions have led to at least
some of the observed declines in phytoplankton
biomass as a result of high grazing rates (Jassby
2008; Jassby et al. 2002). Winder and Jassby (2011)
showed zooplankton community shifts over a
37-year period and associated this shift with the
clam invasion. Other invasive species, including
several aquatic macrophytes (e.g., Eichhornia
crassipes, water hyacinth, and Ludwigia hexapetala,
water primrose), persist in the Delta today and are
affecting both habitat and water quality (Dahm et
al. 2016; Ta et al. 2017). Dominant N sources to the Delta at present
include upstream rivers and wastewater
treatment plants. The Sacramento River and the
San Joaquin River generally represent about 84%
and 13% of water inflow to the Delta, respectively
(Jassby and Cloern 2000). Together, they deliver
over 17 million kg of total N (TN), as particulate
and dissolved inorganic and organic N annually
(Jassby and Cloern 2000; Saleh and Domagalski
2015). Diffuse (non-point source) agricultural
sources upstream of the Delta account for
the majority of TN in these rivers (Saleh and
Domagalski 2015). A major anthropogenic point
source of TN to the Delta is the Sacramento
Regional Wastewater Treatment Facility (SRWTP),
which underwent upgrades from advanced
secondary treatment to tertiary treatment with
biological nutrient removal that were close to Higher trophic-level species have experienced
similar widespread declines. Decreases over the
past 2 decades in pelagic fish abundance, often
referred to as Pelagic Organism Decline, have
prompted a number of food web studies (Sommer
et al. 2007). However, master controls on biomass
and production trends remain elusive, likely
owing to the hydrologic and biogeochemical
complexity of the estuary. KEY WORDS worldwide (Howarth et al. 2011; Paerl et al. 1998;
Paerl et al. 2006; Seitzinger and Sanders 1997). Drainage water quality, agricultural drainage,
return flow, diversions, Delta island groundwater,
nitrogen, phosphorous, metals A number of studies have attempted to assess the
effects of nutrient forms and ratios on primary
productivity in the Delta environment (see
reviews by Senn and Novick [2014]; Dahm et al. [2016]; Ward and Paerl [2016]). Most research on
nutrients in the Delta has focused on nitrogen (N)
biogeochemistry because of both its ubiquitous
presence in human-affected watersheds and
because of the widely debated importance of N
speciation for primary production in the Delta
(Cloern 2021; Kraus et al. 2017; Ward and Paerl
2016). Cloern (2019) showed that ammonium
(NH4
+) and nitrate plus nitrite (NO3
– + NO2
–)
concentrations in the Delta have changed
significantly since the mid-1970s, with mean
annual concentrations increasing over 50%. While
previous work had suggested that nutrients were
at saturation levels for phytoplankton in the Delta
(Jassby et al. 2002), more recent work has found
that N forms and concentrations as well as N
ratios to other nutrients may play an important
role in phytoplankton ecology and uptake kinetics
(Dugdale et al. 2015; Glibert et al. 2016). These
complex relationships between nutrients and Delta
ecology highlight the importance of adequately
characterizing and accounting for all internal and
external nutrient sources in this system. ABSTRACT To better constrain
inputs from island drains, we measured monthly
discharge along with nutrient and trace element SFEWS Volume 20 | Issue 2 | Article 5
https://doi.org/10.15447/sfews.2022v20iss2art5
*
Corresponding author: cmrichar@ucsc.edu
1 University of California, Santa Cruz
Earth and Planetary Sciences
Santa Cruz, CA 95064 USA
2 US Geological Survey
California Water Science Center
Sacramento, CA 95819 USA
3 US Geological Survey
Water Mission Area Laboratory and
Analytical Services Division
Menlo Park, CA 94025 USA
4 University of California, Santa Cruz
Institute of Marine Sciences
Santa Cruz, CA 95064 USA SFEWS Volume 20 | Issue 2 | Article 5
https://doi.org/10.15447/sfews.2022v20iss2art5
*
Corresponding author: cmrichar@ucsc.edu
1 University of California, Santa Cruz
Earth and Planetary Sciences
Santa Cruz, CA 95064 USA
2 US Geological Survey
California Water Science Center
Sacramento, CA 95819 USA
3 US Geological Survey
Water Mission Area Laboratory and
Analytical Services Division
Menlo Park, CA 94025 USA
4 University of California, Santa Cruz
Institute of Marine Sciences
Santa Cruz, CA 95064 USA SFEWS Volume 20 | Issue 2 | Article 5
https://doi.org/10.15447/sfews.2022v20iss2art5
*
Corresponding author: cmrichar@ucsc.edu
1 University of California, Santa Cruz
Earth and Planetary Sciences
Santa Cruz, CA 95064 USA
2 US Geological Survey
California Water Science Center
Sacramento, CA 95819 USA
3 US Geological Survey
Water Mission Area Laboratory and
Analytical Services Division
Menlo Park, CA 94025 USA
4 University of California, Santa Cruz
Institute of Marine Sciences
Santa Cruz, CA 95064 USA 2 US Geological Survey
California Water Science Center
Sacramento, CA 95819 USA 3 US Geological Survey
Water Mission Area Laboratory and
Analytical Services Division
Menlo Park, CA 94025 USA 4 University of California, Santa Cruz
Institute of Marine Sciences
Santa Cruz, CA 95064 USA 1 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 INTRODUCTION This seasonality
in carbon cycling and transport raises important
questions regarding concomitant changes in
N species and other nutrient and trace element
concentrations in drainage waters. Fluctuations
in water table elevation and flow can shift the
oxic-anoxic boundary in the subsurface, with
subsequent effects on biogeochemical processes
that ultimately control island drainage water
quality. Microbial processing of carbon in
the saturated zone is largely controlled by the
influx of oxygenated waters, water residence
times, nutrient availability, and the availability
of carbon for respiration (Limpens et al. 2008). When O2 demand is greater than O2 influx, anoxic
conditions can develop given sufficient carbon
substrate. Naturally reduced zones (NRZs) exist
in these saturated areas where organic matter
is abundant and oxidant-consuming reactions
are continuous (Yabusaki et al. 2017). As a result,
NRZs can affect concentrations of dissolved
organic carbon (DOC), dissolved inorganic N
(DIN), dissolved organic N (DON), and reduced
metal species (Du Laing et al. 2009; Yabusaki et
al. 2017). Past work has shown that both trace
elements and organo-metal complexes, like
methylmercury, can be mobilized in Delta soils
(Alpers et al. 2014; Bachand et al. 2019; Stumpner
et al. 2015). As such, island drainage in the Delta
may contribute seasonally important fluxes of
both oxidized and reduced N species as well
as other macronutrients and trace elements
mobilized in wet-dry cycled NRZs. completion in April 2021. Before the upgrade,
SRWTP annually discharged around 4 million kg
of TN into the Sacramento River in the northern
portion of the Delta, and the SRWTP TN input
comprised roughly 32% of the Sacramento River
annual TN load, though the importance of the
SRWTP TN load was amplified during low flow
months when contributions from other sources
decreased (Saleh and Domagalski 2015). The
SRWTP TN input predominantly occurred as
NH4
+, and SRWTP NH4
+ inputs were thought
to account for over 90% of NH4
+ input into the
Sacramento River (Jassby 2008). Post-upgrade,
SRWTP NH4
+ concentrations in the discharged
effluent have decreased to near zero, and TDN
concentrations have declined by roughly 60%
to 70% (https://ciwqs.waterboards.ca.gov/). This
decrease has substantially lowered N inputs
from SRWTP. With this reduction, NH4
+ and
TDN contributions from other sources, such as
drainage from subsided islands in the central
Delta, may increase in relative importance as
sources of N to Delta waterways. INTRODUCTION While no one variable
has been able to fully account for the previously
discussed changes, water quality, especially
nutrient availability, remains an important
control on ecosystem function in estuaries 2 JUNE 2022 Recent companion work by Richardson et al. (2020) found that island drainage is an important
seasonal source of carbon to the Delta, and also
showed that seasonal increases in dissolved
carbon concentrations in island drainage are
best explained by water table rises that help
mobilize carbon in island soils. This seasonality
in carbon cycling and transport raises important
questions regarding concomitant changes in
N species and other nutrient and trace element
concentrations in drainage waters. Fluctuations
in water table elevation and flow can shift the
oxic-anoxic boundary in the subsurface, with
subsequent effects on biogeochemical processes
that ultimately control island drainage water
quality. Microbial processing of carbon in
the saturated zone is largely controlled by the
influx of oxygenated waters, water residence
times, nutrient availability, and the availability
of carbon for respiration (Limpens et al. 2008). completion in April 2021. Before the upgrade,
SRWTP annually discharged around 4 million kg
of TN into the Sacramento River in the northern
portion of the Delta, and the SRWTP TN input
comprised roughly 32% of the Sacramento River
annual TN load, though the importance of the
SRWTP TN load was amplified during low flow
months when contributions from other sources
decreased (Saleh and Domagalski 2015). The
SRWTP TN input predominantly occurred as
NH4
+, and SRWTP NH4
+ inputs were thought
to account for over 90% of NH4
+ input into the
Sacramento River (Jassby 2008). Post-upgrade,
SRWTP NH4
+ concentrations in the discharged
effluent have decreased to near zero, and TDN
concentrations have declined by roughly 60%
to 70% (https://ciwqs.waterboards.ca.gov/). This
decrease has substantially lowered N inputs
from SRWTP. With this reduction, NH4
+ and
TDN contributions from other sources, such as
drainage from subsided islands in the central
Delta, may increase in relative importance as
sources of N to Delta waterways. Recent companion work by Richardson et al. (2020) found that island drainage is an important
seasonal source of carbon to the Delta, and also
showed that seasonal increases in dissolved
carbon concentrations in island drainage are
best explained by water table rises that help
mobilize carbon in island soils. INTRODUCTION The Delta contains over fifty peat islands, many of
which are artificially drained and commercially
farmed. Long-term drainage of Delta islands for
farming has resulted in extensive land subsidence
from soil oxidation, with many islands now
residing more than 3 m below sea level (Deverel
and Leighton 2010). As a result, most Delta
islands must artificially maintain water tables
below the land surface via managed pumping. Water pumped off Delta islands is commonly
referred to as agricultural drainage, return
flow, and/or island drainage, and here we use
“island drainage” to refer to this flow. Previous
estimates of water discharge from island drainage
(~ 1.5 × 106 m3d–1) are comparable in magnitude
to those from major wastewater treatment plants,
such as SRWTP (~ 5.5 × 105 m3d–1) (Templin and
Cherry 1997). However, nutrient budgets for the
Delta generally only account for upstream river
and wastewater inputs, and Delta island drainage
inputs remain elusive since they are widely
under-studied. There may be over 200 active
island drainage outfalls in the Delta; however,
even our knowledge of drain locations is outdated
(Siegfried et al. 2014). To address this gap in knowledge, we measured
monthly island drainage nutrient (NH4
+, NO3–,
NO2
–, PO4
3–, DON, PON, SiO4
4–) and trace element
(total dissolved Fe, Mn, As) concentrations
along with discharge on Sherman, Staten, and
Twitchell islands in the Delta from June 2017
to September 2018 (Figure 1). Island drainage
discharge volumes were used together with
constituent concentrations to calculate island- https://doi.org/10.15447/sfews.2022v20iss2art5 3 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 (DOC, DIC), stable isotopes of nitrate (δ15N-NO3
–
and δ18O-NO3
–), and select trace elements (total
dissolved As, Mn, Fe) (Figure 1). Samples for total
suspended solids (TSS), δ15N‑NH4
+, δ15N-PON, and
δ15N-DON were collected quarterly during the
same sampling events. A multi-parameter water-
quality meter (YSI Pro Plus) was used to measure
ancillary water parameters (pH, dissolved oxygen,
conductivity, and temperature) at the time of
sample collection. We do not report data for sites
where monthly discharge was zero during the
study period (e.g., May 2018 to September 2018 at
SH-P4 and all of SH-P1). Similarly, all carbon data
are presented separately in the Richardson et al. (2020) companion paper. level gross fluxes. Site Description p
Drainage from three Delta islands was sampled
in this study: Sherman (SH), Staten (ST), and
Twitchell (TW) islands (Figure 1A through 1D). Island drainage from each of these Delta islands
is discharged via outfalls that connect island
pump stations to Delta channels. Sherman Island
has five pump stations and is dominated by
pastureland (67%) and cropland (30%) (Table A1). Staten Island has two pump stations and is
predominantly cropland ( 93%). Twitchell Island
has one pump station and is mixed land use made
up approximately of cropland (48%), pastureland
( 22%), and several managed wetlands ( 30%). These islands are surrounded by river channels
and sloughs from which water is siphoned or
pumped for irrigation, a practice commonly
referred to as diversion. These channels are also
a source of seepage waters onto the islands. To
characterize nutrient and element inputs onto
the islands, surrounding river channels were also
sampled. All nutrient and trace element concentrations
were determined at the Marine Analytical
Laboratory at the University of California at
Santa Cruz. Nutrients were measured on a
Lachat QuikChem 8000 Flow Injection Analyzer. DON was determined indirectly by conversion
to inorganic N using Kjeldahl digestions and
run on a Lachat QuikChem 8000 Flow Injection
Analyzer. Dissolved trace element concentrations
were determined on a Thermo ElementXR
High-Resolution Inductively Coupled Plasma
Mass Spectrometer and run together with
certified reference materials (NIST Standard
Reference Material 1643f). DOC concentrations
were measured as non-purgeable organic
carbon (NPOC) on a Shimadzu TOC-VCPH
TOC/TN Analyzer. Island drainage particulate
organic matter (POM) concentrations were
estimated from TSS concentrations as described
in Richardson et al. (2020), and molar ratios
of C to N (C:N)m of POM values were used to INTRODUCTION Inflow water volumes, taken
as the collective sum of seepage and diversions,
to Delta islands were calculated using a water
budget approach, and river nutrient and trace
element concentrations were used with the
calculated water inflow volumes to estimate
inflow gross fluxes. Island drainage net fluxes
were subsequently calculated by subtracting the
drainage fluxes (off-island) from the river inflow
fluxes (on-island). We then upscaled island-level
fluxes to the entire Delta to establish baseline
estimates of (1) the timing and magnitude of this
poorly characterized input to the larger Delta
environment and (2) the regional importance
of island drainage in the context of other major
freshwater inputs under pre- and post-SRWTP
upgrade conditions. Water samples for all analyses, except trace
elements, were vacuum filtered in the lab to
0.7 µm (pre-combusted Whatman GF/F) first and
then 0.2 µm (Millipore Nylon Membrane). Water
samples for trace elements were filtered on-site
to 0.45 µm using trace-clean certified capsule
filters (Geotech) and immediately acidified to pH
< 2 with triple-distilled trace clean HCl. Samples
were kept on ice until filtered and subsequently
frozen or refrigerated, as dictated by their storage
requirements. Water Analyses Water samples were collected monthly from
all drains on the three islands as well as from
seven surrounding river channels from June 2017
through September 2018 for nutrients (NH4
+,
NO3
–, NO2
–, DON, PO4
3–, SiO4
4–), carbon species 4 JUNE 2022 JUNE 2022 Figure 1
(A) Overview of the Sacramento–San Joaquin Delta showing the location of, the Sacramento River at Freeport, Sacramento Regional Wastewater
Treatment Plant, San Joaquin River at Vernalis, and the three islands sampled in this study. Island drain locations for the entire Delta are shown as blue
circles based on a digitized map from CDWR (1995). (B-D) Maps of Sherman, Staten and Twitchell Island indicating locations of island drain (blue circles)
and river (orange triangles) sites sampled on each island. All study area maps are oriented such that North is up. Site abbreviations are as follows: Sherman
Island (SH), Staten Island (ST), and Twitchell Island (TW). Figure 1
(A) Overview of the Sacramento–San Joaquin Delta showing the location of, the Sacramento River at Freeport, Sacramento Regional Wastewater
Treatment Plant, San Joaquin River at Vernalis, and the three islands sampled in this study. Island drain locations for the entire Delta are shown as blue
circles based on a digitized map from CDWR (1995). (B-D) Maps of Sherman, Staten and Twitchell Island indicating locations of island drain (blue circles)
and river (orange triangles) sites sampled on each island. All study area maps are oriented such that North is up. Site abbreviations are as follows: Sherman
Island (SH), Staten Island (ST), and Twitchell Island (TW). https://doi.org/10.15447/sfews.2022v20iss2art5 5 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 TW-P1 on Twitchell Island were cross-checked
with 1.5 years of daily flow meter data (AgriFlo
XCi ultrasonic sensor) collected across the 2017
and 2018 water years. This cross-comparison
indicated that the unit-power consumption
method is a relatively robust approximation of
discharge (slope = 0.87, R2
= 0.75) that slightly
underestimates actual discharge. As such, mass
fluxes generated using these discharge estimates
herein are considered conservative estimates
(e.g., actual fluxes are likely higher). estimate PON concentrations. For river samples,
we used the previously published relationship
between TSS and POC for the Delta’s rivers from
Murrell and Hollibaugh (2000) to calculate POC
concentrations, and then used (C:N)m ratios
to generate PON concentrations. Water Analyses This method
assumes around 5% of riverine TSS is POC, which
is in line with more recent work by Hernes et
al. (2020). Precision and accuracy were below
5% for all nutrients and below 8% for trace
elements. Annual means and standard deviation
of geochemical data are presented for the 2018
water year (October 2017 to September 2018), a
below-normal water year which we refer to herein
as “dry.” Island drainage fluxes (mass per unit of time) or
loads (mass) off-island into Delta waterways are
referred to herein as gross fluxes or gross loads. Island drainage gross fluxes were calculated
from monthly measured concentrations and
discharge data for each drainage site. For islands
with more than one drain (Sherman and Staten
islands), monthly loads were summed from
all drainage sites. All fluxes in this study are
reported as elemental mass per unit of time. PON fluxes were calculated quarterly, at the
same frequency as sample collection, and these
fluxes were assumed to represent their respective
seasonal contributions. Seasons were defined
as follows: fall (September through November),
winter (December through February), spring
(March through May), and summer (June through
August). Stable isotope samples (δ15N-NH4
+, δ15N-NO3
–,
δ18O-NO3
–, δ15N-PON, and δ15N-DON) were run
at the USGS Menlo Park Stable Isotope Facility
using the methods described in Kendall et al. (2015). All values are presented in permil notation
(‰) relative to Vienna Air (VAIR) for δ15N and
Vienna Standard Mean Oceanic Water (VSMOW)
for δ18O. Analytical precision for δ15N-NH4
+, δ15N-
NO3
–, and δ18O-NO3
– was 1.1‰, 0.3‰, and 0.7‰,
respectively. Analytical precision for δ15N-DON
and PON was 0.4‰. Statistical analysis was performed in two steps:
(1) a Kruskal-Wallis One Way Analysis of Variance
on Ranks was completed to determine if seasonal
values were statistically significantly different
and (2) a coupled pairwise multiple comparisons
procedure was then conducted using Dunn’s
Method to determine which seasonal values were
different from one another. P values less than 0.05
were considered as significant for all statistical
tests. Island drainage gross mass fluxes were upscaled
to Delta-wide contributions using seasonal mean
geochemistry data from this study and an annual
volumetric estimate of drainage discharge,
~5.3 × 108 m3, from Templin and Cherry (1997). Water Analyses First, to calculate monthly regional drainage
volumes, the annual discharge volume was
scaled to monthly time-steps using monthly flow
percentages calculated from discharge data in
this study, where discharge for each month was
calculated as a percentage of total water year
discharge (Table A2). Flow percentages were
generated for Sherman Island, a pastureland-
dominated land use, and Staten Island, a
cropland-dominated land use. This assumes that
the hydrologic regime of each island is, generally,
regionally representative of these two land-
use groups. Monthly discharge values for these Island drainage gross mass fluxes were upscaled
to Delta-wide contributions using seasonal mean
geochemistry data from this study and an annual
volumetric estimate of drainage discharge, Discharge and Mass Flux Estimates This method
gives greater weight to stations that have higher
discharge rather than averaging across stations
that may not discharge as much as nearby sites. The gross flux calculations resulted in two unique
estimates: (1) upscaled gross mass fluxes based
on Staten Island (cropland) flow percentages and
geochemistry, and (2) upscaled gross mass fluxes
based on Sherman Island (pastureland) flow
percentages and geochemistry. Twitchell Island
data were not used for upscaling because of its
mixed land use. Around 82% of the region within
the legal boundary of the Delta is cropland and
18% is pastureland, idle, or grassland (based on
spatial data available online from the California
Crop Mapping database https://data.cnra.ca.gov/
dataset/statewide-crop-mapping). These spatial
coverage percentages were used to weight the
previously discussed upscaled fluxes based on
dominant land use in the Delta, and the data
presented herein are the spatially weighted
averages of these two estimates. Upscaled net flux
estimates are discussed below. To estimate mass fluxes at greater temporal
resolution than yearly, we also assumed that
change in water storage was negligible seasonally
across the 2018 water year. We recognize that
this assumption probably does not reflect actual
seasonal changes in water storage on Delta
islands. The consequence of this assumption
is that our seasonal water budgets may be
overestimating water inflow when on-island
water storage decreases (likely in the spring/
summer) and underestimating water inflow when
on-island water storage increases (likely in the
fall/winter). However, the use of these seasonal
water inflow volumes is roughly supported by
inflow values estimated by the DCD model, which
were generally the same order of magnitude and
positively correlated to one another (R2 = 0.88,
slope = 0.96). As such, we believe our seasonal
net fluxes, made possible by the seasonal
water budgets, provide useful new insights that Discharge and Mass Flux Estimates Change in storage
was assumed to be negligible on an annual scale,
based on previous studies that show island water
tables are generally stable at this time-scale
(Deverel et al. 2015; Deverel et al. 2016). Since
our outflow values were measured directly, the
close alignment of ET and P estimates with DCD
suggests that our island-level inflow estimates are
relatively robust at the annual scale. Our island-
level outflow values did differ substantially and
non-linearly from DCD estimates though, with
seasonal comparisons of measured outflow from
this study vs. DCD estimates poorly correlated
(R2 = 0.02). two land-use types in the Delta were summed
to seasonal scales and then used together with
seasonal mean concentration data for each island
in subsequent gross and net flux calculations. The seasonal mean concentration data were not
averaged equally across these islands, which have
multiple drainage outlets; instead, we divided
the island-scaled seasonal gross loads by their
seasonal outflow volumes to obtain a spatially
integrated mean concentration. This method
gives greater weight to stations that have higher
discharge rather than averaging across stations
that may not discharge as much as nearby sites. The gross flux calculations resulted in two unique
estimates: (1) upscaled gross mass fluxes based
on Staten Island (cropland) flow percentages and
geochemistry, and (2) upscaled gross mass fluxes
based on Sherman Island (pastureland) flow
percentages and geochemistry. Twitchell Island
data were not used for upscaling because of its
mixed land use. Around 82% of the region within
the legal boundary of the Delta is cropland and
18% is pastureland, idle, or grassland (based on
spatial data available online from the California
Crop Mapping database https://data.cnra.ca.gov/
dataset/statewide-crop-mapping). These spatial
coverage percentages were used to weight the
previously discussed upscaled fluxes based on
dominant land use in the Delta, and the data
presented herein are the spatially weighted
averages of these two estimates. Upscaled net flux
estimates are discussed below. two land-use types in the Delta were summed
to seasonal scales and then used together with
seasonal mean concentration data for each island
in subsequent gross and net flux calculations. The seasonal mean concentration data were not
averaged equally across these islands, which have
multiple drainage outlets; instead, we divided
the island-scaled seasonal gross loads by their
seasonal outflow volumes to obtain a spatially
integrated mean concentration. Discharge and Mass Flux Estimates Water discharge from islands was measured as
discussed by Richardson et al. (2020). Briefly,
records documenting electrical usage, P (kW‑hr),
from each pump station were used together with
pump efficiency, U (kW‑hr m-3), to calculate
discharge, D (m -3), using the unit-power
consumption method where D = P/U (Ogilbee
1966; Ogilbee and Mitten 1970; Diamond and
Williamson 1983). Discharge estimates from 6 JUNE 2022 from Station 247 for Sherman Island, Station 242
for Staten Island, and Station 140 for Twitchell
Island via California Irrigation Management
Information System (CIMIS; https://cimis.water. ca.gov/). ET was calculated at a monthly scale
and summed to seasonal and annual scales by
correcting monthly reference ET rates using crop
coefficients for land use cover on each island for
the dry 2018 water year (http://www.itrc.org/etdata/
index.html). Our seasonal ET and P estimates at
the island level were in close agreement with
values estimated by the Delta Channel Depletion
(DCD) model (R2 = 0.98 for ET and R2 = 0.92 for P;
2020 email between L Liang and CM Richardson,
unreferenced, see “Notes”). Change in storage
was assumed to be negligible on an annual scale,
based on previous studies that show island water
tables are generally stable at this time-scale
(Deverel et al. 2015; Deverel et al. 2016). Since
our outflow values were measured directly, the
close alignment of ET and P estimates with DCD
suggests that our island-level inflow estimates are
relatively robust at the annual scale. Our island-
level outflow values did differ substantially and
non-linearly from DCD estimates though, with
seasonal comparisons of measured outflow from
this study vs. DCD estimates poorly correlated
(R2 = 0.02). from Station 247 for Sherman Island, Station 242
for Staten Island, and Station 140 for Twitchell
Island via California Irrigation Management
Information System (CIMIS; https://cimis.water. ca.gov/). ET was calculated at a monthly scale
and summed to seasonal and annual scales by
correcting monthly reference ET rates using crop
coefficients for land use cover on each island for
the dry 2018 water year (http://www.itrc.org/etdata/
index.html). Our seasonal ET and P estimates at
the island level were in close agreement with
values estimated by the Delta Channel Depletion
(DCD) model (R2 = 0.98 for ET and R2 = 0.92 for P;
2020 email between L Liang and CM Richardson,
unreferenced, see “Notes”). Inflow Water Flux Estimates To calculate net fluxes for Delta islands, we used
a water budget approach to first estimate annual
and seasonal water inflow to each island as
follows: I = O + ET – P where I is total inflow (or import), including
groundwater infiltration and diversions that
bring river water onto the island (m3), O is
outflow (or export) from island drainage
pumps (m3), ET is evapotranspiration (m3), and
P is precipitation (m3). Water budget data are
provided in Appendix A, Tables A3 (annual) and
A4 (seasonal). P was based on measured data where I is total inflow (or import), including
groundwater infiltration and diversions that
bring river water onto the island (m3), O is
outflow (or export) from island drainage
pumps (m3), ET is evapotranspiration (m3), and
P is precipitation (m3). Water budget data are
provided in Appendix A, Tables A3 (annual) and
A4 (seasonal). P was based on measured data https://doi.org/10.15447/sfews.2022v20iss2art5 7 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 for water year (WY) 2018 (USGS 2019). The
Sacramento River at Freeport site is located
just upstream of the SRWTP discharge point. Concentration and flow data for SRWTP fluxes
for WY 2018 were downloaded via the California
Integrated Water Quality System and used in
pre-upgrade SRWTP flux calculations (https://
www.waterboards.ca.gov/ciwqs/). Additionally,
SRWTP only had data released for TP for the first
3 months of WY 2018, while all other N species
had data availability for the full water year; our
TP estimates for WY 2018 are thus based on these
3 months. Predicted post-upgrade dissolved N
(TDN = 528 µM, NH4
+ = 11.4 µM, NO3
– = 480 µM)
and P concentrations (TP = 73 µM, unchanged)
in SRWTP effluent were used together with WY
2018 discharge volumes to estimate post-upgrade
SRWTP mass fluxes (LWA 2014). We assume
reported SRWTP TP concentrations are roughly
equivalent to PO4
3– as PO4
3– is the dominant
component of TP in SRWTP effluent. warrant discussion herein. At the same time, we
also recognize that there is uncertainty in the
estimated seasonal water inflow volumes. This
uncertainty could be better assessed with island
groundwater level data, which are generally not
currently available. To estimate Delta-wide inflow, we used the
above island-level inflow data and calculated
seasonal inflow-to-outflow ratios for pastureland-
dominated islands (Sherman Island) vs. cropland-
dominated islands (Staten Island). Net Mass Flux Estimates River nutrient and trace element concentrations
were averaged seasonally from monthly samples
for (1) only the river sites that surround each
island, to calculate island-level inflow mass flux
estimates, and (2) all river sites, to calculate
river inflow mass fluxes at the Delta-wide scale
(Table A5). The island-level and Delta-wide inflow
water volumes, used to calculate inflow mass
fluxes, were taken directly from water budget
calculations as discussed above. The inflow mass
fluxes were then subtracted from gross outflow
fluxes at the island and Delta-wide level to
calculate net mass fluxes. Monthly averaged island drainage nutrient and
trace element concentrations showed seasonal
trends for many, though not all, constituents
(Figure 3). TDN, DON, NH4
+, SiO4
4–, and total
dissolved Mn were significantly higher in the
winter and spring across all sites compared
to the summer (p < 0.05), while NO3
– and NO2
–
concentrations were more variable, but generally
higher in the winter and spring. Island drainage
TDN concentrations averaged 201 ± 104 µM
(defined as the mean ± one standard deviation) in
fall, 240 ± 109 µM in winter, 199 ± 60 µM in spring,
and 93 ± 22 µM in summer. A majority of TDN was
comprised of DON, and DON concentrations were RESULTS Island Drainage Discharge and Water Quality
Island drainage discharge was highly variable
across sites and water years, though seasonal
trends were apparent (Figure 2). Discharge
was generally greatest in the winter across all
three islands, with 49 ± 6 and 32 ± 10% of annual
discharge occurring in winter of WY 2017
and 2018, respectively (Table A2). Cumulative
discharge was 1.2 to 2.2 times greater in wet WY
2017 than dry WY 2018 across all islands. Inflow Water Flux Estimates These were
used to scale previously reported Delta-wide
island drainage water outflow (~ 5.3 × 108 m3) from
Templin and Cherry (1997) to inflow (Table A4). Our annualized Delta-wide inflow-to-outflow
ratio generally agreed with the value estimated
by the DCD model, though volumetric magnitudes
differed, with the DCD model estimating about
two times the outflow volume we use herein for
regional upscaling from Templin and Cherry
(1997). The volumetric difference between the
two methods suggests that our results can be
considered conservative estimates that likely
under-value the contribution of these waters. Pre- and Post-Upgrade Comparison of Wastewater, Island
Drainage, and River Mass Fluxes NH4
+ concentrations averaged 14 ± 11 µM
across all sites in summer months and were
notably higher at 26 ± 17 µM in fall, 61 ± 35 µM
in winter, and 60 ± 44 µM in spring, respectively
(p < 0.05). NO3
– concentrations in island drainage
averaged 10 ± 10 µM across all sites in summer
months, and were significantly higher in winter
(34 ± 58 µM) (p < 0.05), while fall and spring had
mean concentrations of 15 ± 34 µM in fall and
23 ± 35 µM in spring, respectively. Island drainage
PON concentrations did not show a consistent
seasonal pattern and averaged 210 ± 70 µM in fall,
120 ± 61 µM in winter, 156 ± 74 µM in spring, and
180 ± 58 µM in summer. PO4
3– concentrations in
drainage were variable across sites and through statistically significantly different across seasons
at 161 ± 99, 132 ± 56, 115 ± 42, and 79 ± 39 µM, in
fall, winter, spring, and summer, respectively
(p < 0.05). Relative proportions of DIN and DON
shifted seasonally as well, with DIN generally
increasing in relative proportion during winter
and spring compared to summer and fall. NH4
+
concentrations were typically higher than NO3
–
and thus comprised a larger proportion of DIN
in island drainage, except for some dates on
Staten Island where NO3
– concentrations were
elevated. NH4
+ concentrations averaged 14 ± 11 µM
across all sites in summer months and were
notably higher at 26 ± 17 µM in fall, 61 ± 35 µM
in winter, and 60 ± 44 µM in spring, respectively
(p < 0.05). NO3
– concentrations in island drainage
averaged 10 ± 10 µM across all sites in summer
months, and were significantly higher in winter
(34 ± 58 µM) (p < 0.05), while fall and spring had
mean concentrations of 15 ± 34 µM in fall and
23 ± 35 µM in spring, respectively. Island drainage
PON concentrations did not show a consistent
seasonal pattern and averaged 210 ± 70 µM in fall,
120 ± 61 µM in winter, 156 ± 74 µM in spring, and
180 ± 58 µM in summer. Pre- and Post-Upgrade Comparison of Wastewater, Island
Drainage, and River Mass Fluxes Monthly concentration (dissolved N and P)
and discharge data were aggregated from the
Sacramento River at Freeport (USGS 11447650),
San Joaquin River at Vernalis (USGS 11303500),
and SRWTP for seasonal mass flux comparisons
to our Delta-wide island drainage net fluxes 8 JUNE 2022 JUNE 2022 Figure 2
(A) Monthly precipitation and (B) discharge from Sherman, Staten, and Twitchell Islands. Precipitation data were acquired from Station 242 via
the California Irrigation Management Information System (CIMIS). Discharge data were determined using the unit-power consumption method and cross-
checked with measured flow meter estimates discussed in Methods. Figure 2
(A) Monthly precipitation and (B) discharge from Sherman, Staten, and Twitchell Islands. Precipitation data were acquired from Station 242 via
the California Irrigation Management Information System (CIMIS). Discharge data were determined using the unit-power consumption method and cross-
checked with measured flow meter estimates discussed in Methods. time as well, with concentrations only slightly
higher in summer months (2.7 ± 1.2 µM) relative to
winter (1.6 ± 0.8 µM). Concentrations of SiO4
4– in
island drainage were significantly lower in the
summer (380 ± 130 µM) relative to fall, winter, and
spring, when means ranged between 540 ± 160
µM to 600 ± 140 µM (p < 0.05). Total dissolved Mn
concentrations were significantly higher during
fall (670 ± 210 µg L–1), winter (760 ± 370 µg L–1), and
spring (1100 ± 740 µg L–1) compared to summer
(310 ± 300 µg L–1) as well (p < 0.05). Total dissolved
Fe and As concentrations showed no significant
seasonal trends, with means ranging between
820 ± 970 to 1550 ± 1450 µg L–1 for Fe and 5.5 ± 5.0 to
6.9 ± 5.0 µg L–1 for As across all seasons. statistically significantly different across seasons
at 161 ± 99, 132 ± 56, 115 ± 42, and 79 ± 39 µM, in
fall, winter, spring, and summer, respectively
(p < 0.05). Relative proportions of DIN and DON
shifted seasonally as well, with DIN generally
increasing in relative proportion during winter
and spring compared to summer and fall. NH4
+
concentrations were typically higher than NO3
–
and thus comprised a larger proportion of DIN
in island drainage, except for some dates on
Staten Island where NO3
– concentrations were
elevated. Pre- and Post-Upgrade Comparison of Wastewater, Island
Drainage, and River Mass Fluxes 10 JUNE 2022 Table 1
Mean and standard deviation of river and island drainage geochemistry collected monthly during water year (WY) 2018 between October 2017
and September 2018. WY 2017 data are not included so as not to bias the annual mean. Rivers
SH-P2
SH-P3
SH-P4a
SH-P5
ST-P1
ST-P2
TW-P1
TN
(µM)
mean
65
386
468
230
328
352
370
227
stdev
46
74
98
115
62
114
134
12
TDN
(µM)
mean
56
183
272
168
113
188
262
126
stdev
25
87
136
52
44
105
155
45
NO3
– + NO2
–
(µM)
mean
22
10
24
12
12
49
35
10
stdev
11
12
48
9
5
55
65
8
NH4
+
(µM)
mean
6
53
56
39
24
29
48
36
stdev
5
56
58
19
13
20
23
20
DON
(µM)
mean
29
119
192
113
75
107
174
80
stdev
27
35
105
48
32
67
109
39
PON
(µM)
mean
8
218
210
53
204
175
129
105
stdev
7
28
28
28
48
86
53
51
SiO4
4–
(µM)
mean
250
310
440
790
670
510
540
500
stdev
60
120
100
60
130
140
180
110
PO4
3–
(µM)
mean
1.7
1.0
1.8
2.5
2.4
2.2
1.6
3.1
stdev
0.6
0.8
1.3
1.6
0.8
2.0
1.2
1.3
As
(µg L–1)
mean
1.6
3.1
6.1
3.8
2.4
8.5
13.5
4.8
stdev
0.5
1.2
3.0
3.3
0.8
3.3
9.2
1.4
Mn
(µg L–1)
mean
30
1120
860
780
620
640
600
430
stdev
30
820
550
710
250
350
380
180
Fe
(µg L–1)
mean
60
820
310
1820
1030
1020
1650
2020
stdev
60
830
200
2070
740
770
1230
1000
δ15N-PON
(‰)
mean
4.9
3.0
3.3
0.1
-0.1
3.2
2.4
1.6
stdev
2.1
3.4
3.2
3.0
0.5
0.9
1.5
1.4
δ15N-DON
(‰)
mean
1.8
0.1
1.8
0.9
0.3
1.7
1.6
1.8
stdev
2.1
2.1
2.3
0.4
1.0
0.7
0.5
1.7
δ15N-NH4
+
(‰)
mean
9.9
9.0
9.8
9.3
9.0
11.9
11.2
10.3
stdev
5.6
4.3
2.3
1.4
1.8
1.7
2.3
2.4
δ15N-NO3
–
(‰)
mean
7.0
4.7
3.5
5.2
4.0
12.8
15.2
3.8
stdev
1.4
3.4
2.3
7.5
1.7
6.6
6.7
3.9
a. SH-P4 water year data are incomplete as data collected during net zero discharge months were not included. SH-P4 water year data are incomplete as data collected during net zero discharge months were not included. Pre- and Post-Upgrade Comparison of Wastewater, Island
Drainage, and River Mass Fluxes δ15N values of PON, DON, NH4
+, and NO3
–
indicated clear differences in stable isotope
composition amongst N pools that were common
to all island drainage sites (Figure 4A). DON and
PON pools overlapped in concentration range and
N stable isotope composition, ranging from 120 to
150 µM and 1.2 ± 0.5‰ to 2.2 ± 1.6‰, on average,
respectively. Concentrations of NH4
+ were similar
or lower than organic N pools and generally had higher δ15N values, around 10.3 ± 1.1‰ on average,
compared to PON and DON. Concentrations of
NO3
– and δ15N-NO3
– values were generally lower
and more variable than the NH4
+ pool, with mean
δ15N-NO3
– values of 6.7 ± 3.2‰. Pre- and Post-Upgrade Comparison of Wastewater, Island
Drainage, and River Mass Fluxes PO4
3– concentrations in
drainage were variable across sites and through At an annual scale, mean island drainage
TDN, NH4
+, NO2
–, DON, PON, and SiO4
4–
concentrations for WY 2018 were always greater
than surrounding rivers at all sites, while NO3
–
and PO4
3– concentrations were more variable,
with concentrations both higher and lower than
nearby rivers (Table 1). Mean annual dissolved
Mn, Fe, and As concentrations were generally
higher in island drainage, by up to two orders of
magnitude, relative to river water (Table 1). https://doi.org/10.15447/sfews.2022v20iss2art5 9 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Box plots of monthly island drainage concentrations from all sites, starting in June 2017 through September 2018, for (A) NO3
–
d organic N (DON), (E) total dissolved N (TDN), (F) SiO4
4–, (G) PO4
3–, (H) total dissolved Fe, (I) total dissolved Mn, and (J) total d
ent the bounds of the middle quartiles, and lines represent median values. Whiskers show the bounds of the outer quartiles s, starting in June 2017 through September 2018, for (A) NO3
–
4
3–, (H) total dissolved Fe, (I) total dissolved Mn, and (J) total
dian values. Whiskers show the bounds of the outer quartiles Figure 3
Box plots of monthly island drainage concentrations from all sites, starting in June 2017 through September 2018, for (A) NO3
– (B) NO2
–, (C) NH4
+,
(D) dissolved organic N (DON), (E) total dissolved N (TDN), (F) SiO4
4–, (G) PO4
3–, (H) total dissolved Fe, (I) total dissolved Mn, and (J) total dissolved As. Boxes represent the bounds of the middle quartiles, and lines represent median values. Whiskers show the bounds of the outer quartiles (5th and 95th) of
the data. Figure 3
Box plots of monthly island drainage concentrations from all sites, starting in June 2017 through September 2018, for (A) NO3
– (B) NO2
–, (C) NH4
+,
(D) dissolved organic N (DON), (E) total dissolved N (TDN), (F) SiO4
4–, (G) PO4
3–, (H) total dissolved Fe, (I) total dissolved Mn, and (J) total dissolved As. Boxes represent the bounds of the middle quartiles, and lines represent median values. Whiskers show the bounds of the outer quartiles (5th and 95th) of
the data. Island Drainage Nutrient and Trace Element Fluxes Island-level gross TN and TDN fluxes, calculated
from monthly concentration and discharge data, https://doi.org/10.15447/sfews.2022v20iss2art5 11 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Figure 4 (A) δ15N values versus N species concentration for NH4
+
(purple), NO3
– (blue), particulate organic N (PON) (dark green), and
dissolved organic N (DON) (light green). Circular markers represent
inorganic N pools and triangular markers represent organic N pools. (B) Conceptual model of the relative relationship between δ15N values
and concentration for major dissolved and particulate N pools on
Delta islands. Model shows interconnecting processes, but arrows are ranged from 70 to 230 kg d–1 and 20 to 100 kg d–1
in the summer and 170 to 320 kg d–1 and 120 to
200 kg d–1 in the winter, respectively (Table 2,
Figure 5). Organic N accounted for ~79 to 81%
of gross annual TN fluxes across all sites (with
40% to 48% and 29% to 39% of the gross TN
flux as PON and DON, respectively), while DIN
accounted for the remaining ~19% to 21%. Island
drainage gross SiO4
4– fluxes peaked in the winter
(610 to 820 kg d–1), while gross PO4
3– fluxes were
generally greatest during the summer (1 to
7 kg d–1). Island drainage gross total dissolved Mn
and Fe fluxes ranged between 10 and 70 kg d–1 and
10 to 110 kg d–1, respectively, and were seasonally
greatest in the winter on all islands (Table 2). Island drainage gross total dissolved As fluxes
ranged between 0.1 to 0.5 kg d–1 across all sites
and seasons. While nutrient and trace element
gross fluxes generally peaked in the winter, Staten
and Twitchell islands also experienced secondary
peaks in mass fluxes during summer. After accounting for inflow fluxes, island-level net
drainage fluxes were highest in the winter and
spring for all measured species (Table 2). Islands
were generally net sinks in the summer for TDN
(0 to –130 kg d–1), NO3
– + NO2
– (–20 to –50 kg d–1),
SiO4
4– (–440 to –1310 kg d–1), and PO4
3– (-3 to
-12 kg d–1), and Sherman Island was a temporary
sink for all DIN species and DON in the summer. Annual net island level fluxes were positive for
both TDN and TN across all islands, though
NO3
– + NO2
– fluxes were net negative on both
Sherman and Twitchell islands. Island Drainage Nutrient and Trace Element Fluxes Figure 4 (A) δ15N values versus N species concentration for NH4
+
(purple), NO3
– (blue), particulate organic N (PON) (dark green), and
dissolved organic N (DON) (light green). Circular markers represent
inorganic N pools and triangular markers represent organic N pools. (B) Conceptual model of the relative relationship between δ15N values
and concentration for major dissolved and particulate N pools on
Delta islands. Model shows interconnecting processes, but arrows are
qualitative and do not refer to the direction of concentration or δ15N
change. DNRA represents dissimilatory nitrate reduction to ammonium. Double curved arrows represent conversions to or from various gaseous
(g) N forms. Upscaled to annual Delta-wide contributions,
island drainage contributed an estimated total
annual gross TN load of 2.7 × 106 kg to Delta
waterways in WY 2018 (Table 3). Similar to island-
level estimates, the annual gross TN load was
compositionally dominated by PON (43%) and
DON (34%), with DIN comprising the remaining
24% (see Figure 6 for seasonal percentages). The
annual total island drainage gross SiO4
4– load was
estimated to be about 7.6 × 106 kg, while the PO4
3-
load was about 2.8 × 104 kg (Table 3). Annual gross
total dissolved Mn and Fe loads from all islands
were similar in magnitude, 3.7 × 105 to 5.9 × 105 kg,
while total dissolved As contributions were 12 JUNE 2022 Table 2
Seasonal gross and net island drainage nutrient and trace element fluxes for each island for water year 2018 calculated by season (Fall:
September to November; Winter: December to February; Spring: March to May; Summer: June to August), and for the water year (annual). the smallest of all loads and averaged around
4.4 × 103 kg annually. Annual Delta-wide net island drainage fluxes
were positive for all constituents measured except
PO4
3– (Table 3). Delta-wide net island drainage
fluxes for TN and TDN averaged 5,030 kg d–1 and
N fluxes totaled 570 kg d–1 for NH4
+, 170 kg d–1 for
NO3
– + NO2
–, 1,520 kg d–1 for DON and 2,740 kg d–1
for PON. Seasonally, Delta-wide island drainage
fluxes were net negative in the summer for
all constituents except for total dissolved Mn,
suggesting that islands may act as temporary
sinks for many constituents (Table 4). These
September to November; Winter: December to February; Spring: March to May; Summer: June to August), and for the water year (annual). Island Drainage Nutrient and Trace Element Fluxes Season
TN
(kg d–1)
TDN
(kg d–1)
NO3
– + NO2
–
(kg d–1)
NH4
+
(kg d–1)
DON
(kg d–1)
PON
(kg d–1)
SiO4
4–
(kg d–1)
PO4
3–
(kg d–1)
As
(kg d–1)
Mn
(kg d–1)
Fe
(kg d–1)
Gross
Sherman
Fall
140
90
0
10
80
50
370
2
0.1
20
20
Winter
320
200
30
60
110
110
820
2
0.2
60
40
Spring
340
150
10
50
80
190
660
2
0.2
70
50
Summer
70
20
0
0
20
50
150
1
0.1
10
10
Annual
220
110
10
30
70
100
500
2
0.1
40
30
Staten
Fall
150
60
10
10
50
80
260
2
0.2
10
20
Winter
170
140
30
30
80
30
610
1
0.4
30
80
Spring
250
110
40
30
40
140
550
1
0.3
30
20
Summer
230
100
30
20
60
130
860
7
0.5
10
40
Annual
200
100
30
20
60
100
570
3
0.4
20
40
Twitchell
Fall
110
70
0
10
50
40
460
4
0.2
20
70
Winter
200
120
10
40
70
80
820
5
0.2
20
110
Spring
90
60
10
20
40
20
570
3
0.1
20
50
Summer
130
60
0
10
40
70
440
5
0.3
10
100
Annual
130
80
10
20
50
50
570
4
0.2
20
80
Net
Sherman
Fall
50
10
–30
0
0
40
–300
–4
0.0
20
20
Winter
260
150
10
60
90
110
430
0
0.1
60
40
Spring
190
10
–40
50
10
170
–230
–6
0.0
60
30
Summer
–140
–130
–50
–10
–70
–20
–1170
–12
–0.4
–10
–10
Annual
90
10
–30
20
10
80
–320
–6
–0.1
30
20
Staten
Fall
140
50
10
10
40
80
170
1
0.2
10
20
Winter
160
140
30
30
80
30
580
1
0.4
30
80
Spring
190
60
30
20
10
130
–20
–2
0.1
30
20
Summer
50
–60
–40
–40
20
110
–1310
–8
–0.1
10
30
Annual
140
50
10
0
40
90
–150
–2
0.2
20
40
Twitchell
Fall
70
20
–20
10
30
40
20
0
0.1
20
70
Winter
130
60
–20
30
50
70
220
1
0.1
20
100
Spring
20
0
–20
10
10
20
100
–2
0.0
20
50
Summer
60
0
–20
10
10
60
–440
–3
0.0
10
100
Annual
70
20
–20
20
30
50
–20
–1
0.0
20
80 the smallest of all loads and averaged around
4.4 × 103 kg annually. Island Drainage Nutrient and Trace Element Fluxes N fluxes totaled 570 kg d–1 for NH4
+, 170 kg d–1 for
NO3
– + NO2
–, 1,520 kg d–1 for DON and 2,740 kg d–1
for PON. Seasonally, Delta-wide island drainage
fluxes were net negative in the summer for
all constituents except for total dissolved Mn,
suggesting that islands may act as temporary
sinks for many constituents (Table 4). These
upscaled net negative fluxes were overcome by the smallest of all loads and averaged around
4.4 × 103 kg annually. N fluxes totaled 570 kg d–1 for NH4
+, 170 kg d–1 for
NO3
– + NO2
–, 1,520 kg d–1 for DON and 2,740 kg d–1
for PON. Seasonally, Delta-wide island drainage
fluxes were net negative in the summer for
all constituents except for total dissolved Mn,
suggesting that islands may act as temporary
sinks for many constituents (Table 4). These
upscaled net negative fluxes were overcome by Annual Delta-wide net island drainage fluxes
were positive for all constituents measured except
PO4
3– (Table 3). Delta-wide net island drainage
fluxes for TN and TDN averaged 5,030 kg d–1 and
2,290 kg d–1 annually, respectively. Delta-wide net https://doi.org/10.15447/sfews.2022v20iss2art5 13 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE
VOLUME 20, ISSUE 2, ARTICLE
Figure 5
Seasonal (A-C) gross and (D-F) net island drainage nitrogen (N) species loads for (A, C) Sherman, (B, E) Staten, and (C, F) Twitchell Islands. Bar color refers to N species (NO3
– + NO2
–, NH4
+, dissolved organic nitrogen (DON), particulate organic N (PON). See Table 2 for values. Figure 5
Seasonal (A-C) gross and (D-F) net island drainage nitrogen (N) species loads for (A, C) Sherman, (B, E) Staten, and (C, F) Twitchell Islands. Bar color refers to N species (NO3
– + NO2
–, NH4
+, dissolved organic nitrogen (DON), particulate organic N (PON). See Table 2 for values. Controls on Island Drainage Nutrient and Trace Element
Composition Under oxic conditions, a portion of NH4
+ can also
be converted to NO3
– via nitrification, which will
also leave behind a 15N-enriched NH4
+ pool. The
spatial and temporal variability in NO3
– and NO2
–
concentrations along with δ15N-NO3
– values across
all sites suggests that these processes change
irregularly and are not spatially or temporally
consistent. Island Drainage Nutrient and Trace Element Fluxes stable isotope signature of mineralized organic
N—which would have NH4
+ with lower δ15N-NH4
+
values relative to its organic source—is overprinted
by other biogeochemical processes common to all
sites (Nadelhoffer and Fry 1994). This unexpected elevation in δ15N-NH4
+ values
relative to organic N pools is best explained
by a combination of nitrification, uptake, and
volatilization of NH4
+, all of which would lead
to preferential loss of 14NH4
+ and/or 14NH3
that leaves remaining NH4
+ enriched in 15N
(Ostrom et al. 1998; Clark 2015). The NH4
+ pool
is likely subject to uptake and volatilization in
the unsaturated zone during summer and fall
when water tables are low and plant biomass is
high. The seasonality in NH4
+ concentrations
across all sites suggests that increases in NH4
+
concentrations coincide with known winter and
spring periods of water table rises from seasonal
shifts in island hydrology (evapotranspiration,
precipitation, etc.). As such, drainage outlets
receive mineralized NH4
+ that is mobilized and
transported from shallow soil stores during
winter and spring that was previously subjected
to uptake and volatilization during the summer
and fall. Some of the material transported during
this time could be pulsed off-island as “first flush”
events, but the sustained elevation in N content in
drainage suggests that seasonal shifts in primary
water sources are the major driver of change. Figure 6
Relative proportion of individual N species as a percentage of
the seasonal upscaled Delta-wide island drainage total nitrogen (TN) flux
for WY 2018 (see text for details). Figure 6
Relative proportion of individual N species as a percentage of
the seasonal upscaled Delta-wide island drainage total nitrogen (TN) flux
for WY 2018 (see text for details). greater net positive fluxes during other seasons
though, as indicated by the positive annual island
drainage net fluxes for all constituents except
PO4
3– (Table 4). Island Drainage Nutrient and Trace Element Fluxes 14 JUNE 2022 Table 3
Upscaled Delta-wide island drainage gross fluxes, river inflow fluxes, and net fluxes for WY 2018 before any upgrades to the Sacramento Regional
Wastewater Treatment Plant Wastewater Treatment Plant
Annual island drainage load
(kg)
Island drainage gross flux
(kg d–1)
River inflow flux onto islands
(kg d–1)
Mean annual net flux
(kg d–1)
Annual net load
(kg)
TN
2.7 x 106
7,390
2,360
5,030
1.8 x 106
TDN
1.5 x 106
4,240
1,950
2,290
8.4 x 105
NO3
– + NO2
–
3.3 x 105
910
750
170
6.2 x 104
NH4
+
3.0 x 105
830
260
570
2.1 x 105
DON
9.1 x 105
2,500
970
1,520
5.6 x 105
PON
1.1 x 106
3,150
410
2,740
1.0 x 106
SiO4
4–
7.6 x 106
20,770
19,270
1,490
5.4 x 105
PO4
3–
2.8 x 104
80
170
– 90
–3.3 x 104
As
4.4 x 103
10
10
10
2.3 x 103
Mn
3.7 x 105
1,010
80
930
3.4 x 105
Fe
5.9 x 105
1,620
180
1,440
5.3 x 105 Table 4
Upscaled Delta-wide seasonal mean island drainage gross and net fluxes based on water year 2018 geochemistry data
Fall
(kg d–1)
Winter
(kg d–1)
Spring
(kg d–1)
Summer
(kg d–1)
Gross
TN
6,240
9,620
9,590
4,100
TDN
3,800
7,030
4,500
1,630
NO3
– + NO2
–
520
1,500
1,270
380
NH4
+
360
1,500
1,190
270
DON
2,930
4,040
2,040
980
PON
2,440
2,590
5,100
2,470
SiO4
4–
14,930
31,890
22,750
13,500
PO4
3–
70
70
50
110
As
10
20
10
10
Mn
770
1,620
1,430
200
Fe
1,200
3,390
1,200
670
Net
TN
5,470
9,150
6,910
– 1,410
TDN
3,120
6,600
2140
– 2,680
NO3
– + NO2
–
250
1,330
320
– 1,220
NH4
+
280
1,460
1,000
– 470
DON
2,450
3,870
770
– 990
PON
2,350
2,560
4,780
1,270
SiO4
4–
8,470
28,540
4,560
– 35,600
PO4
3–
20
50
– 100
– 330
As
10
20
10
– 10
Mn
750
1,620
1,330
10
Fe
1,180
3,360
960
260 Upscaled Delta-wide seasonal mean island drainage gross and net fluxes based on water year 2018 geochemistry data https://doi.org/10.15447/sfews.2022v20iss2art5 15 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Figure 6
Relative proportion of individual N species as a percentage of
the seasonal upscaled Delta-wide island drainage total nitrogen (TN) flux
for WY 2018 (see text for details). Trace Elements We found high concentrations of total dissolved
Mn and Fe in island drainage during winter
and spring months when compared to summer
(Figure 3H and 3I). Mobilization of Fe and Mn in
water is commonly associated with redox state,
and the observed seasonal increases in total
dissolved Fe and Mn concentrations suggest that
drainage waters receive contributions from a
reduced water source seasonally. This seasonality
has been observed for other trace elements in
temporarily flooded fields of the Delta, where
re-wetting periods are thought to mobilize
mercury species previously formed in unsaturated
soils during dry phases (Marvin-DiPasquale et al. 2014). Regionally, reduction of Fe- and Mn-oxides
commonly leads to increases in soluble Fe and
Mn species in groundwater (Bennett et al. 2006). However, we found no relationship between total
dissolved Fe and Mn concentrations across all
sites, though site-specific trends were evident at
some locations (but weakly correlated) (Figure 7). Interestingly, total dissolved Mn and DOC
concentrations (R2 = 0.29) were weakly positively
correlated, while total dissolved Fe and dissolved
oxygen concentrations (R2 = 0.38) along with pH
(R2 = 0.37) were weakly negatively correlated when
considering all sites (Figure 7). This dynamic
relationship suggests that controls on total
dissolved Mn and organic matter inputs may be At the individual site level, N species stable
isotope values were highly variable, both spatially
and temporally (Table 1). Such variability in
individual N species stable isotope values, without
context relative to other N pools, shows that
biogeochemical controls and sources are complex
at small spatial and temporal scales. However, the
clear distinctions among the N pools and stable
isotope composition in island drainage as a whole
show that there are indeed broad, common links
in N cycling across Delta islands. Nitrogen We generated estimates of N fertilizer application
amounts to each island using areal crop cover
estimates and the N application rate associated
with each crop, as available, from Rosenstock
et al. (2013); these rates are not based on data
from Delta islands and not all land cover types
have N application rate data, so estimates are
considered preliminary. Estimates of fertilizer
inputs on Staten (~800,000 kg N) and Sherman
(~230,000 kg N) islands eclipsed TDN gross
fluxes by an order of magnitude, while Twitchell
Island fertilizer application estimates were
much smaller (~38,000 kg N). These estimates
suggest that fertilizer N may be an important
source of “new” island N annually, especially
on cropland- dominated islands such as Staten
Island. Importantly, the N stable isotope data
generally suggests that any fertilizer N that leaves
via drainage is generally highly recycled. the system were also evidenced by high δ15N-
NO3
– values in several samples on Staten Island
that overlapped or were higher than δ15N-NH4
+
values. The high NO3
– concentrations and δ15N-
NO3
– values of these samples show the influence
of a N input that is most consistent with a high-
concentration, partially-denitrified fertilizer
source (Kendall and McDonnell 2012; Clark 2015). We generated estimates of N fertilizer application
amounts to each island using areal crop cover
estimates and the N application rate associated
with each crop, as available, from Rosenstock
et al. (2013); these rates are not based on data
from Delta islands and not all land cover types
have N application rate data, so estimates are
considered preliminary. Estimates of fertilizer
inputs on Staten (~800,000 kg N) and Sherman
(~230,000 kg N) islands eclipsed TDN gross
fluxes by an order of magnitude, while Twitchell
Island fertilizer application estimates were
much smaller (~38,000 kg N). These estimates
suggest that fertilizer N may be an important
source of “new” island N annually, especially
on cropland- dominated islands such as Staten
Island. Importantly, the N stable isotope data
generally suggests that any fertilizer N that leaves
via drainage is generally highly recycled. Nitrogen The multi-species stable isotope data we
collected provides new insight into the dominant
biogeochemical processes that control N species
concentrations and stable isotope composition
in island drainage. The clear distinctions in
δ15N values of inorganic and organic N pools in
drainage from all islands suggests that N is cycled
in a relatively consistent biogeochemical manner
across Delta islands (Figure 4). Similarity between
δ15N values of PON and DON indicates that DON is
mainly derived from the breakdown of larger OM,
such as PON. Drainage POM likely originates from
soil as discussed by Richardson et al. (2020), which
showed that annual mean (C:N)m ratios of POM
were generally above 10 at these sites. The higher
δ15N-NH4
+ values in nearly all samples relative to
δ15N-PON and δ15N-DON values suggests that the Values of δ15N-NO3
– were generally low with lower
NO3
– concentration relative to the NH4
+ pool,
which is consistent with partial nitrification of
NH4
+. Some of the NH4
+ appears to be nitrified
locally in the subsurface and/or in the drainage
waters under sub-oxic to oxic conditions,
possibly from hot spots and hot moments of
NO3
– and NO2
– production (McClain et al. 2003). Additional inorganic N sources external to 16 JUNE 2022 JUNE 2022 as a result of water-mineral interactions in the
subsurface. In the case of island hydrology,
mineral accumulation from evaporation
may also contribute to higher SiO4
4– in the
subsurface. In contrast, controls on drainage
PO4
3– concentrations (Figure 3G) were not clear,
although some sites showed higher concentrations
in the summer, which may indicate that most
PO4
3– is sourced from fertilizer application during
the growing season. Phosphorous is subject to
complex sorption reactions in the subsurface
that can significantly limit mobility, which
may account for the generally low drainage
PO4
3– concentrations year-round (Schoumans
2013). Studies on fertilizer applications of
soluble phosphorous show that over 50% of the
added PO4
3– is immobilized in under 3 days (do
Nascimento et al. 2018). the system were also evidenced by high δ15N-
NO3
– values in several samples on Staten Island
that overlapped or were higher than δ15N-NH4
+
values. The high NO3
– concentrations and δ15N-
NO3
– values of these samples show the influence
of a N input that is most consistent with a high-
concentration, partially-denitrified fertilizer
source (Kendall and McDonnell 2012; Clark 2015). Silicon and Phosphorous Concentrations of SiO4
4– were seasonally
elevated in the fall and winter in island
drainage (Figure 3F) and suggestive of increased
groundwater contributions in line with expected
water table fluctuations (Richardson et al. 2020). This finding is not surprising because
SiO4
4– concentrations in groundwater are
commonly high relative to those in river water https://doi.org/10.15447/sfews.2022v20iss2art5 17 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Figure 7
Island drainage total dissolved Fe, Mn, and As concentrations versus (A, D, G) dissolved oxygen, (B, E, H) pH, and (C, F, I) DOC concentration for
all 16 months of sampling in WY 2017 and WY 2018. Inset figures show cumulative R2 value for exponential regressions when considering all sites. Light blue
circles, dark blue triangles, and purple squares represent drainage sites on Sherman, Staten, and Twitchell islands, respectively. broadly related across all islands, which is not
surprising given that organic matter has a high
be a more important driver of Fe solubility and
speciation than OM in drainage waters Since
Figure 7
Island drainage total dissolved Fe, Mn, and As concentrations versus (A, D, G) dissolved oxygen, (B, E, H) pH, and (C, F, I) DOC concentration for
all 16 months of sampling in WY 2017 and WY 2018. Inset figures show cumulative R2 value for exponential regressions when considering all sites. Light blue
circles, dark blue triangles, and purple squares represent drainage sites on Sherman, Staten, and Twitchell islands, respectively. Figure 7
Island drainage total dissolved Fe, Mn, and As concentrations versus (A, D, G) dissolved oxygen, (B, E, H) pH, and (C, F, I) DOC concentration for
all 16 months of sampling in WY 2017 and WY 2018. Inset figures show cumulative R2 value for exponential regressions when considering all sites. Light blue
circles, dark blue triangles, and purple squares represent drainage sites on Sherman, Staten, and Twitchell islands, respectively. broadly related across all islands, which is not
surprising given that organic matter has a high
retention capacity for trace elements (Aiken et
al. 2011). The negative relationship between total
dissolved Fe and dissolved oxygen as well as pH,
but lack of a relationship between total dissolved
Fe and DOC, suggests that redox processes may be a more important driver of Fe solubility and
speciation than OM in drainage waters. Island Drainage Nutrient and Trace Element
Contributions to Delta Waterways Sherman, Staten, and Twitchell islands were
each a net annual source of TN, TDN, NH4
+, DON,
PON, total dissolved Mn and Fe, and a net annual
sink for SiO4
4– and PO4
3– (Table 2). Sherman and
Twitchell islands were also sinks for NO3
– + NO2
–,
while Staten Island was a net source of NO3
– + NO2
–
(Table 2). Upscaled to Delta-wide contributions,
calculated mean annual island drainage gross TN
fluxes were 7390 kg d–1 (or 2.7 × 106 kg annually),
and net TN fluxes were 5,030 kg d–1 (or 1.8 × 106 kg
annually) (Table 3). This finding complicates
many existing N box models in the Delta, which
commonly assume island drainage N inputs are
negligible or net zero (Novick et al. 2015). The
net and gross annual island drainage TN load for
WY 2018 was about 9% and 13%, respectively, of
previously reported annual TN loads from the
Sacramento River (including SRWTP) and San
Joaquin River combined (~1.8 × 107 kg) (Saleh and
Domagalski 2015) (Table A6). To further examine the relative importance of
island drainage TN and NH4
+ inputs, we revised
three existing box models (“SFEI,” “DSM2,” and
“EPA”) described in Novick et al. (2015) and
TetraTech (2006) to include our new estimates
of (1) island drainage TN and NH4
+ fluxes off-
island, and (2) river TN and NH4
+ fluxes on-island
(Table A6). We found that gross island drainage
contributions could account for ~13% to 17% of
annual TN loads into the Delta, while TN loads
from river inflow onto islands could account for
~8% to 10% of TN flow out of Delta waters. These
existing models also suggest that the Delta is a
sink for NH4
+ and TN that enters the region from
riverine inputs. Taking island drainage inputs into
account, we estimated whole-Delta TN losses of
33% to 35%, which is slightly higher in range than
original estimates of 25% from Novick et al. (2015)
and in close agreement with original estimates
of 35% from TetraTech (2006) (Table A6). Silicon and Phosphorous Since
island drainage integrates the effects of both
solute source contributions and biogeochemical
processes that change solubility and speciation, it
is hard to assess the importance of each of these
processes without detailed porewater studies. be a more important driver of Fe solubility and
speciation than OM in drainage waters. Since
island drainage integrates the effects of both
solute source contributions and biogeochemical
processes that change solubility and speciation, it
is hard to assess the importance of each of these
processes without detailed porewater studies. 18 JUNE 2022 JUNE 2022 in source geochemistry and biogeochemical
transformations during transport to drainage
ditches (and ultimately surrounding river
channels). Total dissolved As concentrations in island
drainage were more variable than Fe and
Mn, with no clear seasonal trends across sites
(Figure 3J). Reduction of As-bearing Fe- and
Mn-oxides is the primary mechanism for As
contamination of groundwater in the Delta and
areas nearby (e.g., northern San Joaquin Basin)
(Izbicki et al. 2008; Bennett and Belitz 2010). In
fact, some of the highest concentrations of acid-
extractable As—a measure of As available for
desorption from mineral surfaces—in this region
are from Delta sediments (Izbicki et al. 2008). Similar to PO4
3–, complex sorption reactions
affect As mobilization (Herath et al. 2016), and
the variability in As concentrations observed
across sites in our study is likely a reflection
of the complex As biogeochemistry in both the
subsurface and surface waters of Delta islands. Broadly, total dissolved As concentrations
in drainage were higher under low oxygen
conditions, aside from a subset of samples
collected on Sherman Island (Figure 7G). While
reductive dissolution reasonably explains As
mobilization under low oxygen conditions, this
subset of Sherman Island samples may actually
represent As mobilization from a different
biogeochemical process. In high pH oxic waters,
As can be mobilized via alkali desorption and
sulfide oxidation (Herath et al. 2016). SAN FRANCISCO ESTUARY & WATERSHED SCIENCE SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 likely shift inter-annually from differences in
island-level and Delta-wide hydrology. Inflow
to outflow ratios at the island level were
highly variable across islands and water years
(Table A3, A4). Inflow and drainage constituent
concentrations may also change year to year, and
even slight changes could affect the magnitude
of net fluxes. Richardson et al. (2020) estimated
that wet water years see greater mass fluxes of
carbon from island drainage, and these results
may scale to N. Future work should consider
the importance of water year variability, which
can affect both concentrations and discharge,
when estimating the relative contributions of
different nutrient sources internal and external
to the Delta. Seasonality also matters because
the fate and effects of these inputs will vary
with environmental conditions (e.g., flow,
temperature, and solar radiation). For example,
flows in the Delta are higher in the winter and
spring, which may facilitate rapid transport and
dilution through the system relative to other
seasons. In the summer and fall, changes in
nutrient concentrations and forms may be of
greater consequence as they overlap with warmer
temperatures and higher solar radiation that can
promote phytoplankton growth. to 87% and were similar to past estimates of 65%
to 85% from Novick et al. (2015). These revisions
to existing box models to include island drainage
loads from this study, which is a net source
of both TN and NH4
+, suggests that the Delta
is a slightly larger sink for TN (+1.9 × 105 kg to
1.8 × 106 kg) and NH4
+ (+ 2.1 × 105 kg) than previous
estimates suggest (Table A6). In contrast to N dynamics, Delta islands were
a net sink for PO4
3– during WY 2018 (Table 3). The annual gross PO4
3– load in drainage waters
pumped off islands was 2.8 × 104 kg, which
is similar in magnitude to past estimates of
~5.1 × 104 kg (TetraTech 2006). However, island
drainage exports of PO4
3– were negated by the
larger inflow of PO4
3– onto islands, resulting in
islands being a net sink for PO4
3– to the order
of –3.2 × 104 kg annually (Table 3). Relative to
previously reported annual estimates of PO4
3-
inputs to the Delta, the gross island drainage
PO4
3– contribution comprised just 1% of existing
PO4
3– contributions to the Delta (TetraTech 2006). SAN FRANCISCO ESTUARY & WATERSHED SCIENCE Delta islands were, annually, a net source of total
dissolved Mn (3.4 × 105 kg), Fe (5.3 × 105 kg), and
As (2.3 × 103 kg) to the larger Delta environment
(Table 3). Most of these loads likely precipitate
in the Delta’s oxic river waters and are deposited
in the sediments. Similar to concerns about
methylmercury in the Delta, re-suspension of
sediments via dredging or other physicochemical
processes could remobilize elements deposited
from island drainage for downstream transport,
with ultimate fate depending on concentrations
and speciation (Shipley et al. 2011). Island Drainage Nutrient and Trace Element
Contributions to Delta Waterways Our
annual NH4
+ losses in the Delta ranged from 64% Interestingly, drainage waters on Staten Island
had mean annual dissolved As concentrations
that were almost double the other drainage sites
and, at times, exceeded recommended thresholds
of total dissolved As set by the US Environmental
Protection Agency (USEPA) (> 10 µg L–1) and World
Health Organization (> 30 µg L–1). We suspect that
these high levels of total dissolved As, which were
specific to Staten Island, are related to seasonal
flooding of fields that contribute to localized
anoxic conditions. This is also evidenced by
past studies showing large CH4 fluxes during
these times, that likely allow for release of As via
reductive dissolution (Pellerin et al. 2013). Taken
together, trace element geochemistry broadly
suggests that island drainage receives water
from a seasonally reduced water source or NRZ;
future work could explicitly sample along possible
flow paths to better account for differences https://doi.org/10.15447/sfews.2022v20iss2art5 19 Importance of Island Drainage Nutrient Contributions
Using Pre- and Post-Upgrade SRWTP Scenarios Importance of Island Drainage Nutrient Contributions
Using Pre- and Post-Upgrade SRWTP Scenarios
To better understand the relative magnitude
of island drainage nutrient contributions in
the context of the larger Delta environment
under pre- and post-upgrade scenarios, we first
compared island drainage nutrient contributions
to other major Delta inflows (Sacramento and
San Joaquin rivers) along with contributions
from SRWTP, pre-upgrade, for WY 2018. We focus
on dissolved N and P based on data availability
from the above river sites and SRWTP. Annual
net island drainage TDN, NH4
+, NO3
– + NO2
–,
and PO4
3– contributions were 7%, 4%, 2%, and
–4% of total inputs to the Delta, respectively
(Figure 8, Table A7). During WY 2018, SRWTP
NH4
+ contributions were 92% of all NH4
+ inputs,
and this mass flux percentage is nearly identical
to past estimates by Jassby (2008). Seasonally, we
estimated island drainage contributed around
16% of TDN, 9% of NH4
+, 10% of NO3
– + NO2
–, and A large fraction of the previously discussed
nutrient and trace element exports from Delta
islands occurred in the winter and spring, from
increases in both concentrations and discharge
(Figure 3, Figure A1). Seasonality in the delivery
of island drainage nutrients and trace elements
to Delta waters has important implications for
mass flux and net flux comparisons. For example,
nearly 65% and 68% of the annual upscaled gross
TN and TDN load, respectively, was delivered in
winter and spring of WY 2018 (Table 4). Island
drainage nutrient and trace element fluxes also 20 JUNE 2022 JUNE 2022 Calculated mean seasonal fluxes to the Delta from the Sacramento River (SR) at Freeport, the San Joaquin River (SJR) at Verna
nage (taken as net contributions), and Sacramento Regional Wastewater Treatment Plant (SRWTP) under (A-D) pre-upgrade an
onditions. Fluxes were calculated using flow and generally monthly concentration data from water year (WY) 2018. River sites h
data, depending on species, and, as such, we present these fluxes as baseline seasonal estimates for WY 2018, a dry year. Study Limitations and Applications This study is an important first step toward better
constraining and evaluating the importance of
island drainage nutrient contributions to the
Delta. Future work should work toward resolving
water budget uncertainties and issues relating
to scale, both in space and time. Island water
budgets, which are commonly modeled in this
system, would benefit from ground truthing
where possible. In the context of this study,
explicit accounting of water inflow volumes could
improve a source of uncertainty in both net flux
estimates and island water budgets. Specifically,
our study was limited by two key assumptions
that warrant further work and improvement: (1)
that island water budgets are at steady-state, and
(2) that the regional estimates of island drainage
from Templin and Cherry (1997) are accurate. This study is an important first step toward better
constraining and evaluating the importance of
island drainage nutrient contributions to the
Delta. Future work should work toward resolving
water budget uncertainties and issues relating
to scale, both in space and time. Island water
budgets, which are commonly modeled in this
system, would benefit from ground truthing
where possible. In the context of this study,
explicit accounting of water inflow volumes could
improve a source of uncertainty in both net flux
estimates and island water budgets. Specifically,
our study was limited by two key assumptions
that warrant further work and improvement: (1)
that island water budgets are at steady-state, and
(2) that the regional estimates of island drainage
from Templin and Cherry (1997) are accurate. The steady state water budget assumptions used
herein affect the resulting mass flux estimates. A
simple sensitivity analysis shows that changing
inflow based on expected directional shifts in
on-island water storage seasonally (decreases
in spring/summer, increases in fall/winter)
increases annual and seasonal net mass fluxes
for nearly all constituents; many analytes become
smaller sinks and/or larger sources, especially in
the summer (Table A8). Additionally, if Delta-wide
drainage outflow is closer to DCD estimates (2
times greater, 1.2 109 m3 for WY 2018), the mass
flux estimates herein are clear underestimates. Predicted effluent NO3
– + NO2
–, NH4
+, PO4
3–,
and TDN concentrations for SRWTP—once
fully upgraded to tertiary treatment with
biological nutrient removal (i.e., nitrification
and denitrification)—were used to forecast
possible changes in dominant N and P sources
to and within the Delta under a post-upgrade
SRWTP scenario. Importance of Island Drainage Nutrient Contributions
Using Pre- and Post-Upgrade SRWTP Scenarios Pre-
RWTP were calculated from three months (Oct-17 to Dec-17) of data in WY 2018 due to limited concentration data, and the decre
oss upgrade scenarios for SRWTP is a residual effect of bias in the WY 2018 mean as SRWTP PO4
3– fluxes are not expected to ch Figure 8
Calculated mean seasonal fluxes to the Delta from the Sacramento River (SR) at Freeport, the San Joaquin River (SJR) at Vernalis, Delta-wide
island drainage (taken as net contributions), and Sacramento Regional Wastewater Treatment Plant (SRWTP) under (A-D) pre-upgrade and (E-H) post-
upgrade conditions. Fluxes were calculated using flow and generally monthly concentration data from water year (WY) 2018. River sites had some months
of missing data, depending on species, and, as such, we present these fluxes as baseline seasonal estimates for WY 2018, a dry year. Pre-upgrade PO4
3–
fluxes at SRWTP were calculated from three months (Oct-17 to Dec-17) of data in WY 2018 due to limited concentration data, and the decrease in PO4
3–
fluxes across upgrade scenarios for SRWTP is a residual effect of bias in the WY 2018 mean as SRWTP PO4
3– fluxes are not expected to change significantly. Figure 8
Calculated mean seasonal fluxes to the Delta from the Sacramento River (SR) at Freeport, the San Joaquin River (SJR) at Vernalis, Delta-wide
island drainage (taken as net contributions), and Sacramento Regional Wastewater Treatment Plant (SRWTP) under (A-D) pre-upgrade and (E-H) post-
upgrade conditions. Fluxes were calculated using flow and generally monthly concentration data from water year (WY) 2018. River sites had some months
of missing data, depending on species, and, as such, we present these fluxes as baseline seasonal estimates for WY 2018, a dry year. Pre-upgrade PO4
3–
fluxes at SRWTP were calculated from three months (Oct-17 to Dec-17) of data in WY 2018 due to limited concentration data, and the decrease in PO4
3–
fluxes across upgrade scenarios for SRWTP is a residual effect of bias in the WY 2018 mean as SRWTP PO4
3– fluxes are not expected to change significantly. https://doi.org/10.15447/sfews.2022v20iss2art5 21 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 biogeochemical processing and incorporation into
the food web. 2% of PO4
3– in winter months under pre-upgrade
conditions (Table A7). Importance of Island Drainage Nutrient Contributions
Using Pre- and Post-Upgrade SRWTP Scenarios In the summer and fall, net
contributions from island drainage represented
–12% and 10% of TDN, –3% and 2% of NH4
+, –47%
and 2% of NO3
– + NO2
–, and –21% and –1% of
PO4
3– total inputs under pre-upgrade conditions
(Table A7). CONCLUSIONS For more explicit source tracking, coupled
physicochemical instrumentation (e.g., water
level, chemical sensors) of island groundwater
and drainage waters across multiple islands could
provide a better understanding of biogeochemical
transformations as they occur from initial
diversion or infiltration to final discharge. Our
use of stable isotope tracers provided new insight
into N cycling on Delta islands and a similar
application of isotopic tracers could be used
in conjunction with the suggested monitoring
regime to better understand N sources and
transformations. We estimated island-level and upscaled
Sacramento-San Joaquin Delta (Delta)-wide
drainage fluxes using monthly nutrient (PON,
DON, NO3
– + NO2–, NH4
+, PO4
3–, SiO4
4–) and
trace element (total dissolved Fe, Mn, and As)
concentrations with discharge data. Upscaled
island drainage estimates for the entire Delta
suggested that islands are annual net sources
of total nitrogen (TN), total dissolved nitrogen
(TDN), NH4
+, NO3
– + NO2
–, DON, PON, SiO4
4–, total
dissolved Mn, Fe, and As, and sinks for PO4
3–. Island drainage net annual TN and TDN exports
were 1.8 × 106 and 8.4 × 105 kg, respectively. Delta-wide island drainage gross and net TN
contributions were roughly 13% and 9% of
previously reported TN loads to the system. Our
results complicate existing nutrient budgets in
the Delta—which commonly assume that N inputs
from island drainage are negligible or net zero—
and provide new information on under-studied
trace element inputs to the Delta. Beyond nutrients, work on contaminants in the
Delta suggests farmed Delta islands may also be a
source of a number of ecologically consequential
pesticides, transported in dissolved forms and/
or sorbed on soil particulates (Kuivila and Hladik
2008; De Parsia et al. 2019; Weston et al. 2019). With over 200 possible active drainage sites in
this system, new studies could examine the
possibility of delivery of these contaminants
via island drains in the Delta. Drainage waters
may also be a potential source for harmful algae
and cyanotoxins that has not been studied;
Richardson et al. (2020) found several island
drainage sites had seasonal algal blooms. Using forecasted changes in post-upgrade
Sacramento Regional Wastewater Treatment
Plant (SRWTP) dissolved N and P loads, we also
estimated how relative contributions of N and P
from island drainage and other major freshwater
inflow sources could shift in importance in
the Delta environment. Study Limitations and Applications Post-upgrade, annual net
island drainage contributions were predicted to
comprise 11% of the TDN, 45% of the NH4
+, and
1% of the NO3
– + NO2
– delivered to and within
the Delta relative to inputs from major inflows
and SRWTP (Figure 8, Table A7). Since SRWTP
does not anticipate changes to its PO4
3– loads,
net island drainage PO4
3– percent contributions
remained relatively similar, around –4% to –5%,
under pre- and post-upgrade scenarios. Pre-
versus post-upgrade seasonal percentages for
winter net island drainage N contributions shifted
from 16% to 23% for TDN, and from 9% to 65%
for NH4
+, while NO3
– + NO2
– percent contributions
remained around 8% to 10% (Table A7). Because overall NH4
+ inputs to the Delta will
be reduced as SRWTP transitions to be a more
advanced treatment plant, following the upgrade,
a large fraction of NH4
+ that enters the Delta
may originate internally from island drainage. Importantly, a majority of island drainage N
delivery to Delta waterways will occur seasonally,
in the winter and spring, when carbon and
trace element contributions from drainage
are similarly elevated (Richardson et al. 2020)
(Figure 8, Table 4). Though NO3
– will likely
dominate external inorganic N loads to the Delta
post-upgrade, the seasonal delivery of N from
island drainage will be measurable and may be
locally relevant. The spatially diffuse locations of
drainage outfalls may mean that these seasonal
loads are delivered to regions of the Delta with
long residence times that allow for extended High-frequency monitoring of drainage outlets—
for nutrient concentrations and related ancillary
water-quality parameters such as temperature,
dissolved oxygen, and pH—could be used for
better resolution of mass flux estimates and
shifting nutrient biogeochemistry in island
drainage. Similar recent work, enabled by
deployment of high-frequency sensor networks,
showed nutrient dynamics can change at time-
scales of hours, days, and weeks in the Delta
(Downing et al. 2017; Kraus et al. 2017), and we
suspect a similar high-frequency data set for
multiple island drainage sites could help resolve
some of the variability seen in this study and 22 JUNE 2022 JUNE 2022 companion work by Richardson et al. (2020). Such monitoring would also likely generate more
refined and accurate load estimates both within
and across water years. how different land uses affect constituent source
and sink dynamics could also help inform best
management practices on Delta islands. CONCLUSIONS Under a post-upgrade
scenario, annual island drainage net TDN and
NH4
+ contributions to the Delta—relative to inputs
from the San Joaquin River, the Sacramento River,
and SRWTP—increased in relative importance
from 7% to 11% and 4% to 45% based on data from
2018 (a dry water year), respectively. Both pre-
and post-upgrade NO3
– + NO2
– and PO4
3– percent
contributions from island drainage—relative
to other considered sources—were similar,
suggesting that the recent SRWTP upgrade,
which reduces total N loads while maintaining
similar P loads to the Delta, will also shift
dominant sources of N species in different ways. Of these sources, island drainage may become Finally, this study and past work show that
Delta islands are spatially heterogenous, both
within islands and across islands. This has been
observed in studies of (1) gas fluxes, which can
be remarkably variable across identical land-
use types on multiple Delta islands (Hemes
et al. 2019), and (2) aqueous fluxes of carbon,
nutrients, and trace elements, with clear site-
to-site variability in concentrations as shown
in this study and Richardson et al. (2020). Finally, this study and past work show that
Delta islands are spatially heterogenous, both
within islands and across islands. This has been
observed in studies of (1) gas fluxes, which can
be remarkably variable across identical land-
use types on multiple Delta islands (Hemes
et al. 2019), and (2) aqueous fluxes of carbon,
nutrients, and trace elements, with clear site-
to-site variability in concentrations as shown
in this study and Richardson et al. (2020). Better resolution of controls on this spatial
heterogeneity—which affects system-wide
assessments of gaseous, aqueous, and particulate
fluxes—could provide more accurate upscaling of
lateral and vertical fluxes. Similarly, identifying https://doi.org/10.15447/sfews.2022v20iss2art5 23 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Science, Inc. (CUAHSI) at http://www.hydroshare. org/resource/90464d9f9bb34c43b9c6b13eafb5e843. Any use of trade, firm, or product names is for
descriptive purposes only and does not imply
endorsement by the US Government. the dominant source of NH4
+ to Delta waterways,
at least during dry water years, though more
work is needed to assess how NH4
+ fluxes from
other sources, like wetlands and sediments, may
factor into the shifting N budget. REFERENCES Aiken GR, H Hsu-Kim H, Ryan JN. 2011. Influence
of dissolved organic matter on the environmental
fate of metals, nanoparticles, and colloids. Environ Sci Tech. [accessed 2020 Jan 1]. https://doi.org/10.1021/es103992s Alpers CN, Fleck JA, Marvin-DiPasquale M,
Stricker CA, Stephenson M, Taylor HE. 2014. Mercury cycling in agricultural and managed
wetlands, Yolo Bypass, California: Spatial and
seasonal variations in water quality. Sci Total
Environ. [accessed 2020 Jan 1];484:276-287. https://doi.org/10.1016/j.scitotenv.2013.10.096 More broadly, this work shows that island
drainage is an existing and measurable source
of nutrients and trace elements, at least during
dry water years, and highlights the importance
of accounting for temporal variability in existing
nutrient budgets. Our understanding of dominant
nutrient sources in the Delta may be biased
without further consideration of mass fluxes as
they relate to seasonal, annual, and interannual
time-scales in a system with direct water year
dependence that is projected to become even
more variable in the coming years. Bachand PA, Bachand SM, Kraus TEC, Stern D,
Liang YL, Horwath WR. 2019. Sequestration and
transformation in chemically enhanced treatment
wetlands: DOCs, DBPPs, and nutrients. J Environ
Eng. [accessed 2020 Jan 1];145(8), 04019044. https://doi.org/10.1061/(ASCE)EE.1943-7870.0001536 Bennett GL, Belitz K. 2010. Groundwater quality in
the Northern San Joaquin Valley, California. US
Geological Survey Fact Sheet 2010-3079. [accessed
2020 Jan 1]. 4 p. https://doi.org/10.3133/fs20103079 CONCLUSIONS Seasonal island
drainage net TDN fluxes (2,140 to 6,600 kg d–1) will
also be similar in magnitude to post-upgrade TDN
fluxes from SRWTP (3,110 to 3,530 kg d–1) for most
of the year (fall through spring). The increased
role of island drainage nutrients may have
implications for ecosystem dynamics, especially
in places where drains empty into long residence
time areas (sloughs, etc.) of the Delta. ACKNOWLEDGEMENTS We thank the Nature Conservancy for their
assistance in accessing sampling locations,
and the California Department of Water
Resources for helping arrange access to
sampling locations. We thank Carol Kendall
for her early support and assistance with this
work. We thank Kaylee Glenney, Carolyn Brady,
and Rob Franks for their help in the Marine
Analytical Lab. This research was supported
by grants from the International Association
of Geochemistry, National Geographic, and the
J. Casey Moore Award. Additional support for
C. Richardson was provided by the National
Science Foundation Graduate Research Fellowship
Program (DGE‐1329626), and J. Fackrell received
support from the California Sea Grant Delta
Science Fellowship (R/SF-84). Island drainage
geochemistry and flow data is publicly available
through HydroShare from the Consortium of
Universities for the Advancement of Hydrologic Bennett GL, Belitz K, Milby Dawson BH. 2006. California GAMA Program—ground-water quality
data in the northern San Joaquin basin study
unit. Sacramento (CA): US Geological Survey. Data
Series 196. [accessed 2020 Jan 1]. 122 p. Available
from: https://pubs.usgs.gov/ds/2006/196/ [CDWR] California Department of Water Resources. 1995. Sacramento-San Joaquin Delta Atlas:
State of California. [accessed 2020 Jan 1]. 121 p. Available from: https://cawaterlibrary.net/document/
sacramento-san-joaquin-delta-atlas/ Clark I. 2015. Groundwater geochemistry and
isotopes. New York (NY): CRC Press. 402 p. Clark I. 2015. Groundwater geochemistry and
isotopes. New York (NY): CRC Press. 402 p. Cloern JE. 2019. Patterns, pace, and processes
f
t
lit
i bilit
i
l
t di d Clark I. 2015. Groundwater geochemistry and
isotopes. New York (NY): CRC Press. 402 p. Cloern JE. 2019. Patterns, pace, and processes
of water‐quality variability in a long‐studied
estuary. Limnol Oceanogr. [accessed 2020 Jan
1];64(S1):S192-S208. https://doi.org/10.1002/lno.10958 Cloern JE. 2019. Patterns, pace, and processes
of water‐quality variability in a long‐studied
estuary. Limnol Oceanogr. [accessed 2020 Jan
1];64(S1):S192-S208. https://doi.org/10.1002/lno.10958 24 JUNE 2022 Cloern JE. 2021. Use care when interpreting
correlations: the ammonium example in the San
Francisco Estuary. San Franc Estuary Watershed
Sci. [accessed 2020 Jan 1];19(4). https://doi.org/10.15447/sfews.2021v19iss4art1 Cloern JE. 2021. Use care when interpreting
correlations: the ammonium example in the San
Francisco Estuary. San Franc Estuary Watershed
Sci. [accessed 2020 Jan 1];19(4). https://doi.org/10.15447/sfews.2021v19iss4art1 Downing BD, Bergamaschi BA, Kraus TEC. 2017. Synthesis of data from high-frequency nutrient
and associated biogeochemical monitoring for
the Sacramento–San Joaquin Delta, northern
California. US Geological Survey Scientific
Investigations Report 2017–5066. [accessed 2020
Jan 1]. 28 p. https://doi.org/10.3133/sir20175066 Dahm C, Parker A, Adelson A, Christman M,
Bergamaschi B. 2016. ACKNOWLEDGEMENTS Nutrient dynamics of the
Delta: effects on primary producers. San Franc
Estuary Watershed Sci. [accessed 2020 Jan 1];14(4). https://doi.org/10.15447/sfews.2016v14iss4art4 Du Laing, G, Rinklebe J, Vandecasteele B, Meers E,
Tack FM. 2009. Trace metal behaviour in estuarine
and riverine floodplain soils and sediments: a
review. Sci Total Environ. [accessed 2020 Jan
1];407(13):3972-3985. https://doi.org/10.1016/j.scitotenv.2008.07.025 De Parsia M, Woodward EE, Orlando JL, Hladik ML. 2019. Pesticide mixtures in the Sacramento–San
Joaquin Delta, 2016–17: results from Year 2 of the
Delta Regional Monitoring Program. [accessed
2020 Jan 1]. https://doi.org/10.3133/ds1120 Dugdale R, Wilkerson F, Parker AE. 2015. The
“ammonium paradox”: a summary of more than a
decade of research into phytoplankton processes
and nitrogen relationships in the northern San
Francisco Estuary. Suisun Synthesis II Report
Section 2. Prepared for the San Francisco Bay
Nutrient Management Strategy. [accessed
2020 Jan 1]. Available from: https://bacwa.org/
wp-content/uploads/2016/11/SuisunSynthesis2_
November2016_DRAFT.pdf Dugdale R, Wilkerson F, Parker AE. 2015. The
“ammonium paradox”: a summary of more than a
decade of research into phytoplankton processes
and nitrogen relationships in the northern San
Francisco Estuary. Suisun Synthesis II Report
Section 2. Prepared for the San Francisco Bay Deverel SJ, Leighton DA. 2010. Historic, recent, and
future subsidence, Sacramento-San Joaquin Delta,
California, USA. San Franc Estuary Watershed Sci. [accessed 2020 Jan 1];8(2). https://doi.org/10.15447/sfews.2010v8iss2art1 Deverel SJ, Lucero CE, Bachand S. 2015. Evolution of
arability and land use, Sacramento–San Joaquin
Delta, California. San Franc Estuary Watershed
Sci. [accessed 2020 Jan 1];13(2). https://doi.org/10.15447/sfews.2015v13iss2art4 Glibert PM, Wilkerson FP, Dugdale RC, Raven JA,
Dupont CL, Leavitt PR, Parker AE, Burkholder JM,
Kana TM. 2016. Pluses and minuses of ammonium
and nitrate uptake and assimilation by
phytoplankton and implications for productivity
and community composition, with emphasis on
nitrogen‐enriched conditions. Limnol Oceanogr. [accessed 2020 Jan 1];61(1):165-197. https://doi.org/10.1002/lno.10203 Deverel SJ, Ingrum T, Leighton D. 2016. Present-
day oxidative subsidence of organic soils and
mitigation in the Sacramento-San Joaquin Delta,
California, USA. Hydrogeology. [accessed 2020 Jan
1];24(3):569-586. https://doi.org/10.1007/s10040-016-1391-1 Diamond J, Williamson A. 1983. A summary of
ground-water pumpage in the Central Valley of
California. 1961-1977. US Geological Survey Water-
Resources Investigations Report 83-4037. [accessed
2020 Jan 1]. 70 p. Available from:
https://pubs.er.usgs.gov/publication/wri834037 Hemes KS, Chamberlain SD, Eichelmann E,
Anthony T, Valach A, Kasak K, Szutu D,
Verfaillie J, Silver WL, Baldocchi DD. 2019. Assessing the carbon and climate benefit of
restoring degraded agricultural peat soils to
managed wetlands. Agric For Meteorol. [accessed
2020 Jan 1];268:202-214. SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Hernes PJ, Dyda RY, Bergamaschi BA. 2020. Reassessing particulate organic carbon dynamics
in the highly disturbed San Francisco Bay Estuary. Front Earth Sci. [accessed 2020 Jan 1];8:185. https://doi.org/10.3389/feart.2020.00185 Kuivila K, Hladik M. 2008. Understanding the
occurrence and transport of current-use pesticides
in the San Francisco estuary watershed. San Franc
Estuary Watershed Sci. [accessed 2020 Jan 1];6(3). https://doi.org/10.15447/sfews.2008v6iss3art2 Lehman P, Marr K, Boyer G, Acuna S, Teh SJJH. 2013. Long-term trends and causal factors
associated with Microcystis abundance and
toxicity in San Francisco Estuary and implications
for climate change impacts. Hydrobiologia. [accessed 2020 Jan 1];718(1):141-158. https://doi.org/10.1007/s10750-013-1612-8 Howarth RF, Chan F, Conley DJ, Garnier J,
Doney SC, Marino R, Billen G. 2011. Coupled
biogeochemical cycles: eutrophication and
hypoxia in temperate estuaries and coastal marine
ecosystems. Front Ecol Environ. [accessed 2020
Jan 1];9(1):18-26. https://doi.org/10.1890/100008 Izbicki JA, Stamos C, Metzger LF, Kulp T, McPherson
KR, Halford K, Bennett GL. 2008. Sources,
distribution, and management of arsenic in water
from wells, Eastern San Joaquin ground-water
subbasin, California. US Geological Survey Open-
File Report 2008-1272. [accessed 2020 Jan 1]. 8 p. https://doi.org/10.3133/ofr20081272 Limpens J, Berendse F, Blodau C, Canadell J,
Freeman C, Holden J, Roulet N, Rydin H,
Schaepman-Strub G. 2008. Peatlands and the
carbon cycle: from local processes to global
implications: a synthesis. Biogeosciences. [accessed 2020 Jan 1];5(5):1475-1491. https://doi.org/10.5194/bg-5-1475-2008 Jassby AD. 2008. Phytoplankton in the upper San
Francisco Estuary: recent biomass trends, their
causes, and their trophic significance. San Franc
Estuary Watershed Sci. [accessed 2020 Jan 1];6(1). https://doi.org/sfews.2008v6iss1art2 [LWA] Larry Walker Associates]. 2014. EchoWater
Project EIR Water Quality Technical
Memorandum. [accessed 2020 Jan 1]. Marvin-DiPasquale M, Windham-Myers L, Agee JL,
Kakouros E, Kieu LH, Fleck JA, Alpers CN,
Stricker CA. 2014. Methylmercury production in
sediment from agricultural and non-agricultural
wetlands in the Yolo Bypass, California, USA. Sci
Total Environ. [accessed 2020 Jan 1];48:288-299. https://doi.org/10.1016/j.scitotenv.2013.09.098 Jassby AD, Cloern JE. 2000. Organic matter
sources and rehabilitation of the Sacramento–
San Joaquin Delta (California, USA). Aquat
Conserv: Mar Freshw Ecosyst. [accessed 2020
Jan 1];10(5):323-352. https://doi.org/10.1002/1099-
0755(200009/10)10:5<323::AID-AQC417>3.0.CO;2-J McClain ME, Boyer EW, Dent CL, Gergel SE,
Grimm NB, Groffman PM, Hart SC, Harvey JW,
Johnston CA, Mayorga E. 2003. Biogeochemical
hot spots and hot moments at the interface of
terrestrial and aquatic ecosystems. Ecosystems. [accessed 2020 Jan 1];6:301-312. https://doi.org/10.1007/s10021-003-0161-9 Jassby AD, Cloern JE, Cole BE. 2002. Annual primary
production: patterns and mechanisms of change
in a nutrient‐rich tidal ecosystem. Limnol. Oceanogr. ACKNOWLEDGEMENTS https://doi.org/10.1016/j.agrformet.2019.01.017 do Nascimento CA, Pagliari PH, Faria LdA, Vitti GC. 2018. Phosphorus mobility and behavior in soils
treated with calcium, ammonium, and magnesium
phosphates. Soil Sci Soc Am J. [accessed 2020 Jan
1];82(3):622-631. Herath I, Vithanage M, Bundschuh J, Maity JP,
Bhattacharya P. 2016. Natural arsenic in global
groundwaters: distribution and geochemical
triggers for mobilization. Curr Pollut Rep. [accessed 2020 Jan 1];2(1):68-89. https://doi.org/10.1007/s40726-016-0028-2 https://doi.org/10.15447/sfews.2022v20iss2art5 25 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE SAN FRANCISCO ESTUARY & WATERSHED SCIENCE [accessed 2020 Jan 1];47(3):698-712. https://doi.org/10.4319/lo.2002.47.3.0698 Kendall C, McDonnell JJ. 2012. Isotope tracers in
catchment hydrology. 2017. An introduction to
high-frequency nutrient and biogeochemical
monitoring for the Sacramento–San Joaquin Delta,
northern California. [accessed 2020 Jan 1]. Elsevier
https://doi.org/10.3133/sir20175071. Murrell M, Hollibaugh J. 2000. Distribution and
composition of dissolved and particulate organic
carbon in northern San Francisco Bay during low
flow conditions. Estuar Coast Shelf Sci. [accessed
2020 Jan 1];51(1):75-90. https://doi.org/10.1006/ecss.2000.0639 Kraus TEC, Carpenter K, Bergamaschi B, Parker A,
Stumpner E, Downing BD, Travis N, Wilkerson F,
Kendall C, Mussen T. 2017. A river‐scale
Lagrangian experiment examining controls on
phytoplankton dynamics in the presence and
absence of treated wastewater effluent high in
ammonium. Limnol Oceanogr. [accessed 2020 Jan
1];62(3):1234-1253. https://doi.org/10.1002/lno.10497 Nadelhoffer KJ, Fry B. 1994. Nitrogen isotope
studies in forest ecosystems. In: Lajtha K,
Michner R (editors). Stable isotopes in ecology
and environmental science. [accessed 2020 Jan 1]. 22–45 p. 26 JUNE 2022 JUNE 2022 Novick E, Holleman R, Jabusch T, Sun J,
Trowbridge P, Senn D, Guerin M, Kendall C,
Young M, Peek M. 2015. Characterizing and
quantifying nutrient sources, sinks and
transformations in the Delta: synthesis, modeling,
and recommendations for monitoring. SFEI
Contribution No. 785. Richmond (CA): San
Francisco Estuary Institute. [accessed 2020 Jan 1]. Available from:
https://www.sfei.org/documents/delta-nutrient-sources Richardson C, Fackrell J, Kraus TEC, Young M,
Paytan A. 2020. Lateral carbon exports from
drained peatlands: an understudied carbon
pathway in the Sacramento-San Joaquin Delta,
California. J Geophys Res Biogeosci. [accessed
2020 Jan 1];125. https://doi.org/10.1029/2020JG005883 Rosenstock TS, Liptzin K, Six J, Tomich TP. 2013. Nitrogen fertilizer use in California: assessing
the data, trends and a way forward. Calif Agric. [accessed 2020 Jan 1];67(1). Saleh D, Domagalski J. 2015. SPARROW modeling
of nitrogen sources and transport in rivers and
streams of California and adjacent states, US. J Am Water Resour Assoc. [accessed 2020 Jan
1];51(6):1487-1507. https://doi.org/10.1111/1752-1688.12325 Ogilbee W. 1966. Progress report - Methods for
estimating ground-water withdrawals in Madera
County, California. US Geological Survey Open-
File Report. [accessed 2020 Jan 1]. 42 p. Available
from: https://pubs.usgs.gov/wri/1989/4107/report.pdf Ogilbee W, Mitten. H 1970. A continuing program for
estimating ground-water pumpage in California—
Methods. US Geological Survey Open-File Report
70-246. 22 p. [accessed 2020 Jan 1]. Available from:
https://pubs.er.usgs.gov/publication/ofr70246 Schoumans OF. 2013. Description of the phosphorus
sorption and desorption processes in lowland
peaty clay soils. Soil Sci. [accessed 2020 Jan
1];178(6):291-300. https://doi.org/10.1097/SS.0b013e31829ef054 Ostrom NE, Knoke KE, Hedin LO, Robertson GP,
Smucker AJ. 1998. SAN FRANCISCO ESTUARY & WATERSHED SCIENCE https://doi.org/10.3133/ds950 Stumpner EB, Kraus TEC, Fleck JA, Hansen AM,
Bachand SM, Horwath WR, DeWild JF,
Krabbenhoft DP, Bachand PA. 2015. Mercury,
monomethyl mercury, and dissolved organic
carbon concentrations in surface water entering
and exiting constructed wetlands treated with
metal-based coagulants, Twitchell Island,
California. Data Series 950. [Reston (VA)]: US
Geological Survey. 26 p. [accessed 2020 Jan 1];26. https://doi.org/10.3133/ds950 Weston DP, Moschet C, Young TM, Johanif N,
Poynton HC, Major KM, Connon RE, Hasenbein S
2019. Chemical and toxicological effects on Cache
Slough after storm-driven contaminant inputs. San Franc Estuary Watershed Sci. [accessed 2020
Jan 1];17(3). https://doi.org/10.15447/sfews.2019v17iss3art3 Winder M, Jassby A. 2011. Shifts in zooplankton
community structure: implications for food web
processes in the upper San Francisco Estuary. Estuaries Coasts. [accessed 2020 Jan 1];34(4):675-
690. https://doi.org/10.1007/s12237-010-9342-x Ta J, Anderson LW, Christman MA, Khanna, S
Kratville D, Madsen JD, Moran PJ, Viers JH. 2017. Invasive aquatic vegetation management in the
Sacramento–San Joaquin River Delta: status and
recommendations. San Franc Estuary Watershed
Sci. [accessed 2020 Jan 1];15(4). https://doi.org/10.15447/sfews.2017v15iss4art5
Templin WE, Cherry DE. 1997. Drainage-return,
surface-water withdrawal, and land-use data for
the Sacramento-San Joaquin Delta, with emphasis
on Twitchell Island, California. US Geological
Survey Open-file Report 97-350. [accessed 2020
Jan 1]. 31 p. https://doi.org/10.3133/ofr97350 Yabusaki SB, Wilkins MJ, Fang Y, Williams KH,
Arora B, Bargar J, Beller HR, Bouskill NJ,
Brodie EL, Christensen JN, et al. 2017. Water
table dynamics and biogeochemical cycling in a
shallow, variably-saturated floodplain. Environ
Sci Tech. [accessed 2020 Jan 1];51(6):3307-3317. https://doi.org/10.1021/acs.est.6b04873 https://doi.org/10.15447/sfews.2017v15iss4art5
Templin WE, Cherry DE. 1997. Drainage-return,
surface-water withdrawal, and land-use data for
the Sacramento-San Joaquin Delta, with emphasis
on Twitchell Island, California. US Geological
Survey Open-file Report 97-350. [accessed 2020
Jan 1]. 31 p. https://doi.org/10.3133/ofr97350 Liang L. 2020. Email communication between C.
Richardson and Lan Liang, California Department
of Water Resources, regarding island discharge
comparisons in the Delta in July 2020. SAN FRANCISCO ESTUARY & WATERSHED SCIENCE Temporal trends in nitrogen
isotope values of nitrate leaching from an
agricultural soil. Chem Geol. [accessed 2020 Jan
1];146(3-4):219-227. https://doi.org/10.1016/S0009-2541(98)00012-6 Seitzinger S, Sanders R. 1997. Contribution of
dissolved organic nitrogen from rivers to
estuarine eutrophication. Mar Ecol Prog Ser. [accessed 2020 Jan 1];159:1-12. Senn D, Novick E. 2014. Suisun Bay Ammonium
synthesis report. Richmond (CA): San Francisco
Estuary Institute. SFEI Contribution No. 706. p 191. [accessed 2020 Jan 1]. Available from: https://www. sfei.org/documents/suisun-bay-ammonium-synthesis Paerl HW, Pinckney JL, Fear JM, Peierls BL. 1998. Ecosystem responses to internal and watershed
organic matter loading: consequences for hypoxia
in the eutrophying Neuse River Estuary, North
Carolina, USA. Mar Ecol Prog Ser. [accessed 2020
Jan 1];166:17-25. Shipley HJ, Gao Y, Kan AT, Tomson MB 2011. Mobilization of trace metals and inorganic
compounds during resuspension of anoxic
sediments from Trepangier Bayou, Louisiana. J
Environ Qual. [accessed 2020 Jan 1];40(2):484-491. https://doi.org/10.2134/jeq2009.0124 Paerl HW, Valdes LM, Peierls BL, Adolf JE, Harding
LJ. 2006. Anthropogenic and climatic influences
on the eutrophication of large estuarine
ecosystems. Limnol Oceanogr. [accessed 2020
Jan 1];51:448-462. https://doi.org/10.4319/lo.2006.51.1_part_2.0448 Siegfried LJ, Fleenor WE, Lund JR. 2014. Physically
based modeling of Delta Island consumptive use:
Fabian Tract and Staten Island, California. San
Franc Estuary Watershed Sci. [accessed 2020 Jan
1];12(4). https://doi.org/10.15447/sfews.2014v12iss4art2 Pellerin B, Anderson F, Bergamaschi B. 2013. Assessing the role of winter flooding on baseline
greenhouse gas fluxes from corn fields in the
Sacramento-San Joaquin Bay Delta. Energy
Research and Development Division, Final Project
Report. A report prepared for the California
Energy Commission. [accessed 2020 Jan 1]. Pellerin B, Anderson F, Bergamaschi B. 2013. Assessing the role of winter flooding on baseline
greenhouse gas fluxes from corn fields in the
Sacramento-San Joaquin Bay Delta. Energy Sommer T, Armor C, Baxter R, Breuer R, Brown L,
Chotkowski M, Culberson S, Feyrer F, Gingras M,
Herbold B. 2007. The collapse of pelagic fishes
in the upper San Francisco Estuary. Fisheries. [accessed 2020 Jan 1];32(6):270-277. https://doi. org/10.1577/1548-8446(2007)32[270:TCOPFI]2.0.CO;2 https://doi.org/10.15447/sfews.2022v20iss2art5 27 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Stumpner EB, Kraus TEC, Fleck JA, Hansen AM,
Bachand SM, Horwath WR, DeWild JF,
Krabbenhoft DP, Bachand PA. 2015. Mercury,
monomethyl mercury, and dissolved organic
carbon concentrations in surface water entering
and exiting constructed wetlands treated with
metal-based coagulants, Twitchell Island,
California. Data Series 950. [Reston (VA)]: US
Geological Survey. 26 p. [accessed 2020 Jan 1];26. NOTES Liang L. 2020. Email communication between C. Richardson and Lan Liang, California Department
of Water Resources, regarding island discharge
comparisons in the Delta in July 2020. Tetra Tech, Inc. 2006. Conceptual model for
nutrients in the Central Valley and Sacramento-
San Joaquin Delta. A technical report prepared
for the US Environmental Protection Agency and
the Central Valley Drinking Water Public Policy
Workgroup. [accessed 2020 Jan 1]. Available from:
https://www.waterboards.ca.gov/centralvalley/water_
issues/drinking_water_policy/final_nutrient_report_
lowres.pdf [USGS] United States Geological Survey. 2019. National Water Information System: US Geological
Survey Web Interface. [accessed 2020 Jan 1]. Available from:
https://doi.org/10.5066/F7P55KJN Ward AK, Paerl HW. 2016. Delta nutrients forms
and ratios public workshop: “Role of nutrients in
shifts in phytoplankton abundance and species
composition in the Sacramento-San Joaquin
Delta.” Sacramento (CA): November 29-30, 2016. [accessed 2020 Jan 1]. Available from: https://
www.waterboards.ca.gov/centralvalley/water_issues/
delta_water_quality/delta_nutrient_research_plan/
science_work_groups/2017_0530_phyto_wp.pdf 28
|
W3207073491.txt
|
https://eprints.leedsbeckett.ac.uk/id/eprint/9728/1/LocalAgencyForThePublicPurposeDissectingAndEvaluatingTheEmergingDiscoursesOfMunicipalEntrepreneurshipInTheUkAM-BARNETT.pdf
|
en
|
Local agency for the public purpose? Dissecting and evaluating the emerging discourses of municipal entrepreneurship in the UK
|
Local government studies
| 2,021
|
public-domain
| 8,471
|
Citation:
Barnett, N and Griggs, S and Hall, S and Howarth, D (2021) Local agency for the public
purpose?
Dissecting and evaluating the emerging discourses of municipal entrepreneurship in the UK. Local Government Studies, 48 (5). pp. 907-928. ISSN 0300-3930 DOI:
https://doi.org/10.1080/03003930.2021.1988935
Link to Leeds Beckett Repository record:
https://eprints.leedsbeckett.ac.uk/id/eprint/9728/
Document Version:
Article (Accepted Version)
Creative Commons: Attribution-Noncommercial 4.0
This is an Accepted Manuscript of an article published by Taylor & Francis in Local Government
Studies on 31st Oct 2021, available at: https://doi.org/10.1080/03003930.2021.1988935
The aim of the Leeds Beckett Repository is to provide open access to our research, as required by
funder policies and permitted by publishers and copyright law.
The Leeds Beckett repository holds a wide range of publications, each of which has been
checked for copyright and the relevant embargo period has been applied by the Research Services
team.
We operate on a standard take-down policy. If you are the author or publisher of an output
and you would like it removed from the repository, please contact us and we will investigate on a
case-by-case basis.
Each thesis in the repository has been cleared where necessary by the author for third party
copyright. If you would like a thesis to be removed from the repository or believe there is an issue
with copyright, please contact us on openaccess@leedsbeckett.ac.uk and we will investigate on a
case-by-case basis.
1
Local Agency for the Public Purpose? Dissecting and evaluating the emerging
discourses of municipal entrepreneurship in the UK
Neil Barnett1
Steven Griggs2
Stephen Hall3
David Howarth4
1
Leeds Beckett University, UK
De Montfort University, UK
3
University of the West of England, UK
4
University of Essex, UK
2
Abstract
This article explores the contested politics and interpretations of the new practices of
municipal entrepreneurship across local government in the UK. Drawing on empirical
evidence from six cases studies of entrepreneurship in local councils, selfcharacterisations of income-generating projects in thirty authorities, and a series of
semi-structured interviews, we identify, name and characterise an emergent discourse
of municipal entrepreneurship for the public purpose. We argue that this novel strand
of discourse within the wider field of urban entrepreneurialism confers a degree of
political agency to local authorities under austerity, while redescribing and attaching
commercialism and entrepreneurship to the public good. In so doing, we challenge
overly reductionist accounts of local state agency under austerity, and articulate and
evaluate the potentials and obstacles for a progressive interventionism in this
discursive space.
Key words
Municipalism
Entrepreneurship
Commercialisation
Local government
Agency
Austerity
Discourse
Word count
7998
2
Austerity governance has transformed embedded practices of budgetary stewardship
and service delivery across local authorities in the UK (Ferry and Eckersley, 2020). 1
Reductions in grant support from central government, only partially offset by business
rates and increases in council tax, have seen council spending on local services drop
by 24 per cent in England (from 2009 to 2017), 11.5 per cent in Scotland, and 12 per
cent in Wales (Gray and Barford, 2018, p. 554). Working within highly centralised
financial and legal regimes, with few powers of local taxation, and often limited local
resource bases, especially in disadvantaged communities in ‘old’ industrial towns and
cities, local authorities have increasingly turned towards new strategies of incomegeneration and commercialisation, as they endeavour to fill the ‘funding gaps’ left by
reductions in traditional sources of revenue (Gray and Barford, 2018).
The generation of alternative revenue streams - the ‘marketisation of income’ (Taylor,
Haynes and Darking, 2021) - has embraced a multiplicity of commercial and
entrepreneurial logics and practices of municipal action (Thompson, 2020). Councils
have entered into partnerships with real estate developers, while investing some £6.6
billion in commercial property such as hotels, offices and shopping centres from
2016/17 to 2018/19 (National Audit Office, 2020, p.4). They have also made novel
trading and charging interventions in local markets, launching direct ‘for profit’
trading companies in municipal goods and services; creating public service
cooperatives and mutuals in collaboration with communities; exploiting procurement
policies as a tool to support local businesses and social enterprises; and driving
authority-wide culture change towards entrepreneurship and financial self-sufficiency
(Ferry et al., 2018).
This article evaluates the under-researched motivations and meanings attached by
local actors to such commercial and entrepreneurial forms of income-generation
(Shearmur and Poirier, 2017). Answering calls for a ‘greater sensitivity’ to local
agency under conditions of austerity (Fuller, 2018), it reveals and assesses how
1
We thank the Editor and two anonymous reviewers for their constructive
engagement with our work and for their helpful suggestions on how we might refine
and develop our arguments. Of course, responsibility for the claims remains with the
authors.
3
officers make sense of entrepreneurship in their everyday practices; the role of local
agency in relation to the demands of austerity governance; and the reconfiguration of
the local state. In so doing, we recognise and build upon existing accounts that have
identified global varieties of urban entrepreneurship (Phelps and Miao, 2020). While
we acknowledge variations in cross-national patterns and uneven geographical
‘reach’, we identify and name four dominant problematisations of entrepreneurial
practices which are alleged to best fit the case of UK local government: municipal
financialization, progressive interventionism, social innovation, and progressive selforganisation. Secondly, we discern and characterize a particular mode or variant of
the emergent discourse of ‘municipal entrepreneurialism’, which we name ‘municipal
entrepreneurialism for the public purpose’, where entrepreneurship is assocated with
the provision of a diverse array of commercial services, municipal stewardship and
public goods. Thirdly, we detect a number of potential tensions in this discourse of
municipal entrepreneurship, including its amplificiation of political risks; clashes in
organisational culture over demands for flexibility; grievances over the naming of
‘surpluses’ and traditional budgetary logics; and the transformation of the roles and
responsibilities of officers. Fourthly, while recognising the risks of drawing narrow
definitions of such phenomena, we conclude that this emerging discourse creates new
possibilities of agency for local authorities that resonate with and contribute to
problematisations of what we have named ‘progressive interventionism’.
RESEARCH STRATEGY
In developing a research strategy to address our objects of inquiry, our initial task was
to describe the core elements and features that constituted the discourse of municipal
entrepreneurship. This involved an analysis of the interpretations and statements of
representative subjects and institutional actors - chief officers, project leads, local
councillors, and frontline staff - who spoke or wrote about the practices and activities
in which they were engaged. Here the main aim of the exercise was to characterize
the beliefs and interpretations of these selected subjects and institutional actors in
different contexts, using our judgement to discern and test the underlying rules and
logics of the discourses. More fully, drawing on the resources of poststructuralist
4
policy analysis, we focussed on the signifier of ‘entrepreneurship’, tracking and
tracing its various iterations across multiple contexts, while using these descriptions to
construct the discourse. Our approach also contained an implicit logic of comparison,
as our descriptions sought to bring out similarities and differences in the multiple
articulations of ‘entrepreneurship’ across local authorities (cf. Robinson, 2016). Here
the objective was to sketch out a grammar of the different usages of ‘municipal
entrepreneurship’, so as to provide a perspicuous representation of the discursive
field, and to determine the different variants at work in this field.
Of course, this strategy presupposes a particular conception of discourse and
statements. In our approach, discourses are best defined as linguistic and nonlinguistic practices that connect ideas, things and activities together to produce
specific systems of meaning. This perspective contrasts with other methods of
discourse analysis, which focus either on the more restricted analysis of speeches or
texts, or more broadly on the role of arguments and the semiotic dimension of social
practice (e.g. Fairclough, 2013; Hajer, 2005). Discourses are thus sets of articulatory
practices that connect and modify the meaning of contingent elements to form
systems of signification, where such systems are demarcated and unified by the
creation of boundaries with other discourses. Moreover, in seeking to determine the
regularities and linkages between different statements, beliefs and actions in a
particular field of discourse, our conception focusses on the way differences are
established between contending positions and the creation of political divisions
between discourses (XXXX, TBI).
Finally, using the work of Foucault, we define statements as ‘serious speech acts’ that
are enunciated by officers and policymakers when describing their practices and
programmes in particular local contexts (Foucault, 1972). Here the notion of a ‘speech
act’ highlights the performative dimension of their utterances and written expressions
– ‘saying as doing’ – while the idea of ‘serious’ captures the way these linguistic
performances seek to accurately and licitly describe their beliefs, thoughts and
practices (Dreyfus and Rabinow, 1982, pp. 45-56). Our aim was to establish the core
statements in the discourse, and to explore their repetition, resonance, reiteration and
5
transformation in particular spaces and places, as well as their dissemination and takeup in other comparable cases.
More practically, empirical fieldwork and data generation was undertaken in three
steps. We began by discerning and naming four dominant problematisations of
entrepreneurial practices in local government in different contexts. Secondly, we
analysed the council initiatives nominated for the Association for Public Service
Excellence’s annual award for commercialisation and entrepreneurship. The
Association is a member-owned local authority body, which works with over 300
councils across the UK. As an advocate of new forms of income-generation, it
introduced the award for commercialisation and entrepreneurship in 2015, and by
2019 over 37 initiatives had been shortlisted as finalists for the award, with
nominations spread across 30 authorities. Thirdly, we undertook case studies of
entrepreneurship in six local councils, which have been widely identified as being at
the forefront of income-generation activities (see Table 1). The empirical cases were
not selected as ‘critical cases’, which would enable us to generate or test universal
explanations or predictions, but rather because they were considered to be exemplary
sites through which to explore the discursive work of ‘entrepreneurship’.
INSERT TABLE 1 HERE
For each case study, the research team assembled and analysed an archive of policy
documents, including briefings, strategic plans, project reports and evaluations. This
was followed by a series of semi-structured interviews with a total of 21 participants
drawn from project and corporate teams. Interviews lasted an hour, and the sample
included chief officers, project leads and frontline staff. Questions explored the
competing rationales underpinning initiatives, everyday practices of implementation,
and the barriers and opportunities for change. Interviews were then coded
thematically, as we focused on exemplary statements pertaining to entrepreneurship,
the articulation of demands, and the equivalences and differences between elements of
the discourse.
6
In analysing this archive, we focussed on the way actors interpreted their situations,
and the repetition of statements that constituted the specific goals, subjects, strategies,
and objects of the discourse of entrepreneurship. At each stage, we undertook repeated
readings of the texts, using ‘manual processing’ to isolate and describe the core
statements that emerged or disappeared in different contexts (Keller, 2013, p.97).
Through this to-and-fro movement between our empirical data and our research
puzzles, we engaged in a process of articulation. We did not ‘find’ the discourse of
municipal entrepreneurship ‘hiding’ in the text. Rather, we assembled and named its
logic, character, and value through our judgements and our situated knowledge of
local government, as we mediated and negotiatied our research between our
theoretical assumptions and the four problematisations of entrepreneurship that we
identified at the outset of the study. It is to these problematisations that we now turn.
PROBLEMATISING COMMERCIALISM AND ENTREPRENEURSHIP
UNDER AUSTERITY GOVERNANCE
Having outlined our research strategy, we now turn to the problematisation of the
existing interpretations of commercialism and entrepreneurship under austerity
governance in the UK. Here we identify and analyse the four main problematisations
of entrepreneurial practices within existing accounts of these ideas and practices.
Municipal Financialisation
The first problematisation emphasizes the way that practices of local
commercialisation have transformed urban infrastuctures into financial assets and
revenue streams for local government. In the process, the local state has evolved from
that of a ‘facilitator and enabler’ of the private sector to that of an ‘active executor’ of
neoliberal financialisation (Beswick and Penny, 2018). This transformation of the
local state is a consequence of the logics of austerity governance and top-down cuts to
public funding, which do not ‘enabl[e]’ local actors ‘to behave differently’, if they are
to strengthen the fiscal and political capacities of the local state (Christophers, 2019,
p. 583; see also Penny, 2018). Subordinated to central government, they are left with
7
little freedom of manoeuvre as to ‘where and how they can raise or generate funds’,
except through the financialisation of local assets (Beswick and Penny, 2018, p. 624).
This financialisation, however, arguably increases the contradictions facing the local
state, as it is caught between the demands of value-extraction, speculation, risk-taking
and those of public intervention, policy coordination, planning and regulatuion
(Beswick and Penny, 2018, p. 612; Pike et al., 2019, p. 4; Raco and De Souza, 2018).
In pessimistic renditions of this approach, local actors adopt entrepreneurial practices
as ‘much by dull compulsion as ideological zeal’, so that the actions of local actors
ultimately mask the implementation of an ‘aggressively commercial and speculative
mode of governance new to local government’ (Beswick and Penny, 2018, p. 625;
Penny, 2017, p. 1370). Such interpretations resonate with ‘austerian realist’ accounts
of local agency, where actors in local government deny their own agency, leading to
the consolidation of state power (Davies et al., 2020, p.63).
More optimistic interpretations privilege the uneven nature of processes of
financialisation, drawing attention to the agency of local actors, as well as the political
and economic contexts, which ultimately shape outcomes (O’Brien, O’ Neil and Pike,
2019). They thus characterise practices of local entrepreneurship as part of a
‘financialisation-in-motion’ (Pike et al., 2020, p.792), that is, a set of messy,
incomplete and recursive processes, which are marked by the continued interactions
of managerial, financialised and entrepreneurial logics (p. 793), where actors are
‘actively financialising and being financialised’ (p. 792). In this view, the motivations
of local actors and the potential outcomes of commercialisation and entrepreneurship
are highly contingent and contextual, so that until the outcomes of programmes prove
otherwise, ‘there is no compelling reason not to give councils the benefit of the doubt’
(Christophers, 2019, p. 582).
Progressive Interventionism
In arguing that the pro-active entrepreneurial strategies of the local state can drive
forward civic and collective provision of goods and services as an integral component
of a ‘more inclusive, holistic and integrated place-based economic strategy’, the
second problematisation goes further than merely giving councils the ‘benefit of the
8
doubt’ (Thompson et al., 2020, p. 1191; Gardner and Lowndes, 2016). Typically,
Thompson et al. (2020) mobilise a Polanyian understanding of the socially embedded
economy to foreground the agency of the local state in developing ‘accelerators’ of
economic growth in combination with ‘stabilisers’ of social welfare and protection.
Characterising such practices as ‘entrepreneurial municipalism’, they argue that the
local state goes beyond generating funds to fill the ‘holes’ in central grants, to invest
‘directly in self-sustaining projects, which harness the value of (de-commodified)
land, (cooperative) labour and (patient) capital to ground economic development in
people and place’ (p. 1188).
This approach thus suggests that interventions in the foundational economy represent
an emergent and nuanced, yet distinct, strategy available to local state actors, who are
duly assigned agency on route to the production of locally-embedded social value.
Similar conclusions are drawn by Shearmur and Poirier (2017) and Aldag et al.
(2019), who assert that the origins of municipal entrepreneurship are not necessarily
intertwined with competition between cities and local authorities to attract inward
investment. On the contrary, they argue that local government can exercise its agency
to protect public services in a ‘progressive push back’ against central cuts. Indeed,
Shearmur and Poirier (2017) suggest that municipal entrepreneurship can be driven
and sanctioned by the demands of the local population, civil society and businesses.
Social Innovation
The claims of Shearmur and Poirier (2017) act as a bridge to our third
problematisation. In this approach, entrepreneurship is tied to public value creation,
thus privileging local officers and political leaders as the key agents of change in the
response to austerity, as well as longer-term shifts in the policy environment, which
have been caused by deregulation, privatisation, asset sales, and collaboration (Bello
et al., 2018). Local state actors are perceived to exercise innovative forms of agency
within a ‘relational’ form of governance, which stands in marked contrast to a
bureaucratic, ‘traditional’ top-down government (Liddle and McElwee, 2019). Local
actors, it is claimed, are increasingly ‘freed’ within such relational modes of
governance to facilitate and leverage public value, as they are encouraged to pursue
forms of ‘collective entrepreneurship [that have] the capacity to provide greater
9
options, and allow bottom up solutions to emerge’ (Liddle and McElwee, 2019; Klein
et al., 2010). In such spaces, the local state assumes the role of an ‘animateur’, which
in part acts as a catalyst for economic development by ensuring network leadership
(Quinn and Courtney, 2016, p. 144). In this perspetive, practices of entrepreneurship
and innovation are thus framed as ‘co-operative in nature, emphasising mutuality
rather than voracious profit-making’ (Johnson and Fenwick, 2018, p. 212).
Progressive Self-organisation
The fourth problematisation tends to eschew any explicit reference to the signifier
‘entrepreneurship’. Practices of ‘smart procurement’ and interventions into the
foundational cooperative economy, which might otherwise be characterised as the
activity of the municipal ‘entrepreneur’, are understood in terms of ‘community
wealth building’ and ‘progressive new municipalism’ (O’Neil and Howard, 2018;
Russell, 2019). Rejecting urban entrepreneurialism or the regime of inter-urban
competition over inward investment (Harvey, 1989), this problematisation promotes
the democractisation of the economy, alternative forms of ownership and service
delivery (Russell, 2019).
Importantly for our analysis the innovative forces of social change are thus situated
‘outside’ the local state in the prefigurative micro-settings of self-management and the
spaces of informality created by the accelerated disaggregation of the local state under
austerity (Beveridge and Koch, 2019, pp.11-12). The local state remains a flawed and
somewhat damaged vehicle, hampered by weak powers, financial scarcity, and a lack
of capabilities to address the issues and constraints generated by the global political
economy (Blanco, Salazar and Bianchi, 2020). More pessimistically, it is replete with
bureaucratic hierarchies, asymmetrical power relations and illegitimate forms of
authority (see Russell, 2019). Proponents of progressive self-organisation thus
advocate the further hybridisation of the local state so as to exploit its organisational
resources to the advantage of community activists and to embed the local state in
‘everyday urban politics’ (Beveridge and Koch, 2019, pp.11-12).
THE EMERGING DISCOURSES OF MUNICIPAL ENTREPRENEURSHIP
10
In order to evaluate these problematisations, we now set out the results of our
empirical research of municipal entrepreneurship. Our initial empirical mapping and
analysis of the emerging discourse of municipal entrepreneurship yields six
interconnected themes, which function as the quilting points that knit together the
overlapping strands and threads. Together they form a loose system of storylines,
often in contradiction with one another, which weave together the different strands of
the emerging discourse in a dispersed regularity. The internal unities and limits of this
‘dispersed regularity’ are in turn shaped by a series of suble divisions and exclusions,
which mark the boundaries between this formation and others.
Income-Generation and Local Agency
One core strand of the discourse of municipal entrepreneurship constructed
commercialisation and income-generation as ‘necessary’ strategic responses to the
‘perfect storm’ of rising demands on council services (Statement 1) and reduced
government funding ( Statements 8). In this context, entrepreneurial narratives
portrayed local government as the ‘triumphant underdog’ winning out against the
odds (like the eponymous hero in the Rocky boxing films) in a challenge to the
‘mentality of cuts’ (Statement 7). Aspirations for greater local agency were clearly
emphasized in the naming of the initiatives, with repeated references to ‘taking
control’ (Statement 8), ‘shaping the future’ (Statement 6) and challenging the
‘mentality of cuts’ (Statement 12).
INSERT FIGURE 1 HERE
For the Public Purpose
The narrow instrumental goal of income-generation was framed as a means for
generating public value or social benefit, be it tackling social care demands
(Statement 3), addressing fuel poverty (Statement 2) or mitigating climate change
(Statement 4). Entrepreneurship was thus firmly anchored in the public domain, while
a series of equivalences were drawn in the discourse between practices of
commercialisation, income-generation, and innovation and resilience in service
delivery. Manifesting itself in multiple forms in our case studies, the leitmotif of
11
‘entrepreneurship for the public purpose’ shaped diverse initiatives across different
local authority contexts from house building through to property investment,
promoting healthy lifestyles, and smart procurement. The East Riding Leisure
Services programme, for example, working in partnership with local GPs, referred
patients identified at risk of inactive lifestyles or poor diet to leisure services for
physical exercise and support via a bespoke on-line system, rather than
recommending costly clinical care. But, importantly, the programme was couched in
an entrepreneurial narrative, which connected support for patients to become longterm participants in physical activity to the economic case for reducing demand on
over-stretched public services through the prioritisation of prevention and the
collaborative ‘buy-in’ of multiple partners. The initiative foregrounded savings of
some £800,000 per annum to the NHS, evident in reduced numbers of bariatric
surgery procedures and a £200,000 accrual to the local authority from increased takeup of leisure services.
Stewards of the Local Economy
A third theme of the discourse evoked images of the authority as the stewards of the
local economy, ‘nurturing the growth of local people and businesses’ (Statement 5).
When asked to explain their role in service delivery in Enterprising DG, the direct
service arm of Dumfries and Galloway council, one officer initially described it
narrowly as ‘basically making money for the council’, yet immediately countered that
their work also aimed to make the most of local authority assets within communities,
and building local supply chains. Such legitimising narratives ultimately resonated
with place-based appeals of the council as an ‘energiser’ authority, driving forward
local growth by supporting local companies to retain economic benefits within what
was deemed to be a peripheral locality poorly served by the market. Local
procurements of goods and services, which rose from £44 million in 2015/16 to £62.4
million in 2018/19, while developing a network of preferred supplier relationships
with over 300 local small and medium-sized companies (Dumfries and Galloway
Council, 2020), was regularly portrayed as a means of creating social value and
promoting social cohesion within local communities.
12
Allusions to place-based stewardship were constructed in opposition to markets
through the rhetoric of negative exteralities and market failures, prioritising
‘stakeholder not shareholder’ value (Statement 6). The mission of Birmingham
Municipal Housing Trust (BMHT) was thus legitimised by senior officers as a means
of countering the failure of private developers to provide an adequate quantity and
quality of new homes across the city. One outome of this initiative, for example, was
the provision of housing in disadvantaged inner-city neighbourhoods with large
BAME communities, where it was wrongly assumed that there was low levels of
demand. For BMHT officers, this market failure stemmed from: the flawed business
model adopted by the national, private house building sector, which privileged
development on large sites in areas of ‘demonstrable’ demand (typically suburbs); the
primacy of shareholder value, requiring a high return on investment (typically 25 per
cent); and the provision of standardised house types, notwithstanding local context. In
this context, BMHT positioned itself as a distinctive niche as a bespoke, local
alternative to the ‘budget and scarper’ blueprint of volume house builders (interview
with senior officer). It opposed its development model against those of its private
competitors, thus offsetting market failure by absorbing risks traditionally borne by
the private sector. The strategy involved the development of homes designed by
council architects, exclusively on council owned land (with planning consent presecured), with construction, sales and marketing contracted to the private sector, with
whom surpluses were shared. In this process, BMHT has built to date more than 3,000
homes for sale or rent, becoming the largest house builder in the West Midlands.
Importantly, this discursive strand also established divisions between local and
national/global companies and markets. In Dumfries and Galloway, such antagonisms
typically rested on demands to tackle the ‘Klondike economy’, whereby national
contractors and global companies ‘come into our authority […] take the money and
the benefits [..] and then disappear’ (interview with senior officer). ‘Othering’ large
external providers in an emergent discourse of patriotic localism, the local was
privileged over the national and global, while autonomous strategies of local
development were prioritised over external interdependency. It followed that
networking with local suppliers and contractors was framed as a means of promoting
place-based inclusive economies, ensuring that the benefits of growth were retained
13
locally, notably by using local authority procurement rules to generate demand for
goods and services from local suppliers. In short, working alongside local firms,
councils used their political powers to open up access to new markets, from which
previously, local businesses might have been excluded, because of their obligations to
bid for large contracts and compete against national contractors.
Redefining Risk
The affirmations of local agency and market intervention were made possible by the
reframing of risk and risk-taking by local authorities, as they drew equivalences
between, on the one hand, the often conflicting institutional logics of budgetary
stewardship and, on the other hand, service improvement and innovation (Ferry and
Eckersley, 2020). In the first instance, municipal entrepreneurship was reframed as
‘astute’ risk-taking with ‘a clear sense of purpose’ (Statement 11), implicity drawing
boundaries with forms of allegedly ‘uncalculated’ risk-taking, while foregrounding
the capacity of councils to manage risk and contingencies. In our case studies,
commercial risks attached to the use of public funds were compared to the ‘horrific’
risks of inaction and the failure to defend services and local communities under
austerity. In this way, the property investment strategy of Sevenoaks District Council
weighed the risks of commercial investments against the generation of a revenue
stream (a minimum yield of 5 per cent per annum on investments), the value of
interventions in local markets to maintain employment property, and the aspiration of
the authority to be become financially self-sufficient within ten years (from 2013). In
short, the meaning of stewardship was itself redescribed to incorporate budgetary
demands, the defence of public services, and the risks of inaction.
But it is also noteworthy that in this discourse the notion of ‘risk’ is primarily
constructed as political risk, and is conceptualised in terms of negative electoral
outcomes for local councillors, including, for example, fears that the council would be
seen to be competing with local business. In Birmingham, one officer noted,
‘commercialism has always been classified as a “dirty word”, for [it assumes] council
departments competing against anybody who is local […]’. Such political risks were
negated by appeals to collaboration with local small and medium firms, and niche
market provision (Statement 9), which it was claimed met specifically local needs,
14
while avoiding direct competition with established local businesses. The ventures thus
opened up opportunities to compete against national contractors in local and external
markets. For example, Oxford City Council rebranded itself as a ‘social enterprise
council’, as it explored different ways to trade and charge for its services within and,
crucially, beyond Oxford itself. It also recycled dividends produced for local benefit
via a municipal trading company - Oxford Direct Services - which generated in
2018/19, a £1.3m dividend for the Council, while employing over 600 employees and
supporting 1,251 jobs (Oxford Direct Services, 2020). In discursive terms, then,
collaborative and niche provision serves as a logic that can incorporate the defence of
local business into the basket of other aims and demands advocated by the appeals to
municipal entrepreneurship. It thus seeks to negate political risk surrounding market
interventions, while allaying the fears of elected members that competition with local
businesses might undermine local political support.
Driving Forward Cultural Change
Appeals to commercialisation and entrepreneurship were frequently couched in the
rhetoric of ‘behavioral change’, ‘embedding a commercial culture’ and ‘working for
our citizens’ (Statement 10). In our case studies, municipal entrepreneurship was
defined in opposition to standard ‘checks and balances’, which were interpreted as a
barrier to exploiting new opportunities or as a competitive disadvantage. In response
to such demands, authorities introduced new managerial models and practices of
delegation that were deemed to fit with the different business demands and modes of
intervention of municipal entrepreneurship. Councils established practices for signing
off initiatives by key individuals - chief officers and portfolio holders - without
recourse to cabinet, full council, or relevant committees.
Most importantly, however, practices of municipal entrepreneurship introduced new
financial and budgetary logics into the running of the council, triggering new conflicts
and antagonisms. In particular, these tensions concerned the reinvestment of
surpluses, when only part of the council was engaged in such entrepreneurial
practices. Indeed, the discourse of municipal entrepreneurship embeds a logic of
needs-led funding that establishes local needs and levels of service provision and then
seeks to generate funding to meet such outcomes. It redirects thinking away from
15
standard budget-setting processes that assume ‘fixed’ levels of resources and
expenditure patterns, while privileging the generation of efficiencies to meet service
outcomes. In so doing, it challenges protectionist practices of silo funding. As such,
the City & County of Swansea inverted traditional budgetary mechanisms, adopting a
method of ‘co-produced’ budgeting that brought senior officers, policy officers and
frontline service staff into dialogue in outcome-focussed service reviews. These
reviews assumed zero-based budgets and explored service delivery as part of an ‘endto-end, needs-based’ process. Yet practices of income-generation also triggered
conflicts between departments about the use of ‘additional incomes’, and the absence
of incentives to generate income, if any such resources were not re-invested in the
department that generated them. Across authorities, such conflicts about the
‘ownership’ of commercial funding triggered the construction of rival discursive
projects which sought to name additional income as ‘surpluses’, ‘savings’ or ‘profits’
- each term legitimising different uses of additional incomes.
The Innovative Puzzler
The emergent discourse also constructed novel subject-positions and voices for
officers. Practices of municipal entrerpreneurship were not typically associated with
innovative ruptures or breaks with established ways of working. Practitioners spoke of
a ‘long journey’, which involved the morphing of services by building upon past
activities or by bringing existing assets together in new combinations to respond to
local needs. In Birmingham, such narratives resonated with appeals to ‘start small and
have a plan B’ (interview with senior officer). In fact, such practices privileged the
subject-position of the ‘innovative puzzler’, where the ‘puzzle’ to be solved was
represented not as some intellectual conundrum, but the contradictions between - and
the exclusions of - customary practices, organisational styles, and policy
commitments (see Spinosa, Flores, Dreyfus, 1997, pp. 22-9). Across all case studies,
many local officers thus criticized existing practices and organisational styles of
councils, using phrases and tropes that highlighted the inconsistencies between
traditional ways of working, the council stated aims, and the service-delivery
outcomes within communities. New fora and dialogues emerged across councils to
accommodate such ‘voices’. Typically, one senior officer spoke of how it is vital that
all staff are ‘not afraid to come up with a duff idea’. Another referred to the value of
16
working in environments in which there is ‘permission to fail’ and a ‘no handcuffs
policy’, which gives frontline staff the confidence to work outside risk adverse
cultures which stifle innovation. To this end, Dumfries and Galloway council
established its own centre of excellence, which was a two-way learning space to
engage other council services, as well as to transfer ‘good practice’ lessons across the
authority and facilitate culture change. Its operational values typically sought to
enshrine ‘promoting a responsibility culture not a blame culture’ (DG First, undated,
p.15). Importantly for our analysis, such practices are intrinsically tied to perceptions
of local agency, for ‘puzzling’ opens up the perspective of making political choices,
re-politicising taken-for-granted ways of working, and imagining new visions.
RE-INTERPRETING MUNICIPAL ENTREPRENEURSHIP AND LOCAL
AGENCY UNDER AUSTERITY
In the light of our empirical evidence, and our mapping of the emergent discourse, we
now turn back to the critical evaluation of our findings. These are developed in
relation to the four rival problematisations that we identified at the start of this article.
The Dangers of Municipal Financialisation
We began by noting that pessimistic interpretations of municipal financialisation
criticize the way that practices of commercialisation and entrepreneurship accelerate
the neoliberalization of the local state, transforming the local state from a facilitator of
private entrepreneurship into an active entrepreneur in its own right. However, our
interpretation of the empirical evidence suggests an alternative possibility, in which
the local state is a site and driver of progressive interventions in local markets. Indeed,
this strand of the emergent discourse of municipal entrepreneurship challenges the
long-held assumptions of the neoliberal model of the enabling council, which
advocated councils contracting out and divesting themselves of local services (Smith,
2000). In contrast, municipal entrepreneurship – whether in the form of house
building, smart procurement, promotion of active and healthy lifestyles, property
investment or social enterprise - leads authorities to adopt interventionist stewardship
17
strategies that reconnect with the ‘big ticket’ policy issues facing local communities
(Stoker, 2011).
At the same time, the discourse of municipal entrepreneurship also potentially moves
beyond the confines of optimistic accounts of municipal financialisation. Such
accounts argue that commercialisation generates additional funding to fill the gaps in
local authority budgets due to austerity. But we argue that in advancing the
progressive capabilities of the local state, municipal entrepreneurship for the public
purpose goes further, articulating demands for income-generation with market
intervention, stewardship, and the advancement of the public good. It thus severs the
enterprise narrative from connotations of neo-liberal, market-led growth and favours
thinking about commercialisation and entrepreneurship in ways in which ‘take back’
the local state from capital (Cumbers, 2015, p. 74). That is to say, the emerging
discourse recognises the contingency of the institutional configurations of the local
state and local economies, so that under certain conditions the state can reframe
commercialisation to advance the common interest and social well-being, notably
through the tackling of public problems (Sheamur and Poirier, 2017, pp. 721-4).
The Agency of the Local State
Seen in this way, the discourse of municipal entrepreneurship resonates with the
demands of the new municipalism of ‘self-organising progressives’, as well as with
the public value orientation of ‘social innovators.’ Indeed, in all our case studies, the
rhetoric of municipal entrepreneurship often endorses appeals to practices of:
community wealth building; local inclusive growth; the use of procurement policy to
support local businesses and social enterprises; and the facilitation and leverage of
public and private capabilities in the pursuit of public value. We argue that such
demands are advanced by both self-organising progressives and social innovators (see
Johnson and Fenwick, 2018; O’Neil and Howard, 2018).
Yet, in contrast to these two problematisations, our research suggests that the
discourse of municipal entrepreneurship discloses an alternative reading of the agency
of the local state, opening up new avenues of inquiry for the transformative role of
local agency, and the formal arenas and practices of the local state. In fact, the
18
discourse of municipal entrepreneurship for the public purpose, which we discern in
our findings, calls into question the risk of ‘state phobia’ and ‘the fear of the formal’
often associated with accounts of progressive self-organisation and social innovation
(Lopdrup-Hjorth and du Gay, 2019). Indeed, although the latter two problematisations
acknowledge the progressive potentials that are afforded by the control of the local
state, they still ultimately ground the origins of innovation and entrepreneurship in
civil society movements and the informal politics that operate beyond the local state,
and the local state remains dominated by the hierarchy of the centre and the ‘push’ for
formality.
Progressive Pragmatism
By contrast, our empirical analysis shows that the drive for innovation and
entrepreneurship cannot be disentangled from the agency of local state actors within
the formal arena of local authorities. The practices of local enterprise that
characterised our our case studies were driven by actors inside the formal apparatus
of the local state, working in and against established practices of local policymaking.
The logics of municipal entrepreneurship challenged established cultures and
budgetary practices, while redefining perceived risks and subject positions for
officers. Such logics went beyond the ‘tinkering’ and the ‘active politics of the
present’. Instead, they privileged the subject-position of the officer as the ‘innovative
puzzler’, who is generally intent on challenging the rupture between the declared
strategic aims of councils and everyday practices and outcomes. Our case study
participants thereby rearticulated the demands of commercialisation and incomegeneration, as they rhetorically redescribed such demands as entrepreneurship for the
public purpose.
Our analysis thus exposes the political work of local agents and the rhetorical
reframing of ‘commercialisation’ and ‘entrepreneurship’ in and through their
articulation with municipalism. We foreground how local actors within the local state
were able to move beyond the practices of ‘tinkering’ to generate an alternative
mobilising vision for local government in response to the demands of austerity
governance. Importantly, such evidence supports calls for a more actor-focussed
19
orientation that trawls beneath the ‘surface appearances’ of financialisation (Pike et
al, 2020, p. 794).
Stewards of Place
The upshot of our interpretation is that the discourse of municipal entrepreneurship
resonates with and supports the problematisation of progressive interventionism. This
problematisation recognises the capacity of the local state to act as a ‘countermovement’ to the predominant logics of competitive, market-led growth,
implementing entrepreneurial strategies, which disrupt local markets by investing in
foundational services and infrastructures, so as to address economic inequalities
across communities (Thompson et al., 2020, pp. 1178-80). The interpretation of our
evidence suggests that markets can operate according to different logics, so that
municipal interventions in local economies can challenge logics of economic
necessity or technocracy to harness markets for the delivery of the public good (Hay
and Payne, 2015). Indeed, the analysis of our case studies adds weight to claims that
local state actors act as ‘activist-entrepreneurs’, who are able to intervene to address
market failure, sometimes through the logic of market disruption, as they seek to
‘reshape’ or redesign the operations of local markets. At the same time, they can
mobilise and generate resources in the form of pragmatic public actions that challenge
economic injustices, rather than extending the harmfully competitive and exclusionary
logics of neoliberalism (Thompson et al., 2020; Aldag et al., 2019).
Having said this, the evidence generated in our study also questions the ideological
grounding and temporal fixing of progressive interventionism, opening up a dialogue
about why such interventions ‘grip’ local officers and politicians. It will be recalled
that Thompson et al characterise progressive intervention as practices of
‘entrepreneurial municipalism’, associating such practices with the project of
democratic socialism (2020, p. 1180). This definition rests on their aim of intregrating
social justice into economic structures, identifying entrepreneurial municipalism as a
more or less progressive and left-leaning set of practices, which can be aligned with
the so-called ‘new municipalism’ and the fearless cities movement. However, it is
clear that in the local authorities that we have studied the patterns of political
leadership cannot be narrowly confined to the left of the political spectrum. The party
20
affiliations of council leaders included the Labour Party and Scottish Labour, as well
as the Conservative Party and the Scottish National Party (not to mention their
different internal party positionings).
Equally, officers within the local state also played a primary role in advancing the
discourse of municipal entrepreneurship, so that the practical interventions cannot
easily be defined as politically-led. We thus posit an alternative reading of
entrepreneurship across local authorities, which foregrounds the potential resonance
of practices of municipal entrepreneurship for the public purpose with the embedded
discourse of stewardship of place. Our evidence suggests that innovative practices and
visions of public entrepreneurship traverse party political allegiances, and that the
‘grip’ of such practices rests less on the political alignment with progressive politics,
and more on the sedimented discourse of place stewardship embedded within political
and administrative leaderships in the different cultures and systems of local
government.
CONCLUSION: MUNICIPAL ENTREPRENEURSHIP, LOCAL
DEMOCRACY AND THE PUBLIC PURPOSE
In characterising and naming an emergent discourse of municipal entrepreneurship for
the public purpose, this article has critically assessed the income-generation practices
of local government authorities. Adding to our knowledge of how local actors
interpret their practices and roles when they engage in entrepreneurship, the analysis
also challenges the continued salience of ‘hollow’ accounts of local agency under
austerity. Although it is tempting to show a clear complicity between these new
discursive practices and other tendencies, including logics of neoliberalization, the
rise of the ‘new municipalism’ or the constraints of ‘austerian realism’, our analysis
discloses a more nuanced and richer set of processes, which have complex lines of
descent.
One upshot of our findings is to caution against a too rapid desire to overgeneralise,
leading to the positing of universal forms that embody a clear essence and a fixed set
21
of features. Instead, we argue that the discourses and practices of commercialisation
and entrepreneurship can take multiple forms. This is not to say that this discourse
cannot slip into logics of financialisation or forms of social innovation, which have
been identified in ther problematisations we have evaluated. On the contrary, under
certain conditions, commercialisation and entrepreneurship may manifest itself as a
form of self-organising progression or social innovation. In equal fashion, the
discourse of municipal entrepreneurship for the public purpose is not without its
tensions and contradictions, as it is always open to contestation and re-articulation, as
well as to economic risk and market competition. Indeed, it remains open to counteraccusations of providing ideological cover for growing inequalities and government
retreat, harbouring the real risks that the narrow income-generation concerns of
commercialisation come to dominate over the public purpose. Under such
circumstances, municipal entrepreneurship carries the danger of being rearticulated as
little more than a justificatory narrative for ‘go-it-alone’ competitive localism and
reductionist strategies of community betterment and divestment.
Of course, the sustainability of such entrepreneurial practices has also been called into
question by COVID-19 and its impacts on commercialisation revenues. At the time of
writing, the Local Government Association (2020) has predicted that local authorities
in England will lose £2.8 billion in falling commercial income due to the pandemic.
Equally, the National Audit Office (2020) has raised concerns about the exposure of
local authorities to economic and market risk, particularly where they rely on rental
incomes to fund services. Yet, our study suggests that the stewardship garned by the
discourse of municipal entrepreneurship for the public purpose could offer local
government a privileged means of supporting local economies, particularly the pillars
of the foundational economy, in a post-COVID recovery.
REFERENCES
Aldag, A., Kim, Y. and Warner, M. (2019) ‘Austerity urbanism or pragmatic
municipalism?’, Environment and Planning A, 51(6): 1287-1305.
22
Bello, B., Downe, J., Andrews, R. and Martin, S. (2018) ‘Does austerity drive public
service innovation?’, Public Money & Management, 38(2): 131-138.
Beswick, J, and Penny, J. (2018) ‘Demolishing the present to sell of the future?’
International Journal of Urban and Regional Research, 42 (4): 612-632.
Beveridge, R. and Koch, P. (2019) ‘Contesting austerity, de-centring the state’,
Environment and Planning C, 39(3): 451-68.
Blanco, I., Salazar, Y. and Bianchi, I. (2020) ‘Urban governance and political change
under a radical left government’, Journal of Urban Affairs, 42(1): 18-38.
Christophers, B. (2019) ‘Putting financialisation in its financial context’, Transactions
of the Institute of British Geographers, 44(3): 571-586.
Cumbers, A. (2015) ‘Constructing a global commons in, against and beyond the
state’, Space and Polity, 19 (1): 62-75.
Davies, J., Bua, A., Cortina-Oriol, M. and Thompson, E. (2020) ‘Why is austerity
governable?’, Journal of Urban Affairs, 42(1): 56-74.
DG First (Undated), Business Plan 2015-2018, Dumfries and Galloway Council.
Dreyfus, H.L. and Rabinow, P. (1982) Michel Foucault, Brighton: Harvester.
Dumfries and Galloway Council (2020) Procurement annual report, 2018/19,
https://www.dumgal.gov.uk/media/20817/Procurement-Annual-Report-for-20181/pdf/Procurement_Annual_Report_for_Financial_Year_18-19__appendix.pdf?m=637038048383900000 accessed 30 June 2020.
Fairclough, N. (2013) ‘Critical discourse analysis and critical policy studies’, Critical
Policy Studies, 7(2): 177-97.
23
Ferry, L., Andrews, R., Skelcher, C. and Wegorowski, P. (2018) ‘New development:
Corporatization of local authorities in England in the wake of austerity, 2010-2016’,
Public Money & Management, 38(6): 477-480.
Ferry, L. and Eckersley, P. (2020) ‘Hybridizing the institutional logics of performance
management and budgetary stewardship in English and Welsh local government’
Public Policy & Administration, 35(1), 45-64.
Foucault, M. (1972) The Archaeology of Knowledge, London: Tavistock.
Fuller, C. (2018) ‘Entrpreneurial urbanism, austerity and economic governance’
Cambridge Journal of Regions, Economy and Society, 11(3), 565–585.
Gardner, A. and Lowndes, V. (2016). ‘Negotiating austerity and local tradition’. In M.
Bevir & R. A. W. Rhodes (Eds.), Rethinking governance, (pp. 125-143). Abingdon:
Routledge.
Gray, M. and Barford, A. (2018) ‘The depths of the cuts’, Cambridge Journal of
Regions, Economy and Society, 11(3): 541-563.
Hajer, M. (2005) ‘Coalitions, practices, and meaning in environmental politics’. In D.
Howarth & J. Torfing, Discourse Theory in European Politics, (pp. 297-315).
London: Palgrave Macmillan.
Harvey, B. (1989) ‘From managerialism to entrepreneurialism’, Geografiska Annaler:
B, 71(1): 3-17.
Johnson, L. and Fenwick, J. (2018) ‘Public Entrepeneurship’. In R. Kerley, J. Liddle
and P. Dunning (eds.) The Routledge Handbook of International Local Government,
(pp. 203-217). Abingdon: Routledge.
Keller, R. (2013) Doing Discourse Research. London: Sage.
24
Klein, P., Mahoney, J., McGahan, A. and Pitelis, A. (2010) ‘Toward a Theory of
Public Entrepreneurship’, European Management Review, 7: 1-15.
Liddle, J. and McElwee, G. (2019) ‘Theoretical perspectives on public
entrepreneurship’, Journal of Entrepreneurial Behaviour & Research, 25(6): 13081320.
Local Government Association (2020) LGA analysis: COVID-19 council funding gap
widens to £7.4 billion, https://www.local.gov.uk/lga-analysis-covid-19-councilfunding-gap-widens-ps74-billion accessed 17 August 2020.
Lopdrup-Hjorth, T and du Gay, P. Speaking truth to power?,
https://doi.org/10.1177/1350508419830622 accessed 30 June 2020.
National Audit Office (2020) Local Authority Investment in Commerical Property,
London: NAO.
Oxford Direct Services (2020) A doing good company,
https://www.odsgroup.co.uk/getmedia/ac3f5154-9a35-48a8-9c2a-dac3ab8fa76d/ODSAnnual-Report-2018-19.pdf, accessed 30 June 2020.
O’Brien, P., O’Neil, P. and Pike, A. (2019) ‘Funding, financing and governing urban
infrastructures’, Urban Studies, 56(7): 1291-1303.
O’Neill, M. and Howard, T. (2018) ‘Beyond extraction’, Renewal, 26(2): 46-53.
Penny, J. (2017) ‘Between coercion and consent’, Urban Geography, 38(9): 13521373.
Penny, J. (2018) ‘The “Cooperative Council” or the “Cop-Out Council”?’. In T.
Enrigth and U. Rossi (Eds.), The Urban Political Thought, (pp. 147–169).
Basingstoke: Palgrave Macmillan.
25
Phelps, N. and Miao, J. (2020) ‘Varieties of urbam entrepreneurialism’, Dialogues in
Human Geography, 10(3): 304-21.
Pike, A., O’Brien, P., Strickland, T., Thrower, G. and Tomaney, J. (2019)
Financialising City Statecraft and Infrastructure, Cheltenham: Edward Elgar.
Pike, A., O’Brien, P., Strickland, T., Thrower, G. and Tomaney, J. (2020)
‘Financialising city statecraft and infrastructure’, Environment and Planning A,
52(4): 791-796.
Quinn, M. and Courtney, R. (2016), ‘The Public Sector as an Entrepreneur?’, New
Perspectives on Research, Policy & Practice in Public Entrepreneurship Vol. 6,
Bradford: Emerald, pp. 127-146.
Raco, M and de Souza, T.M. (2018) ‘Urban development, small business
communities and the entrepreneurialisation of English local government’ Town
Planning Review 89(2), 145-165.
Robinson,W. (2016) ‘Comparative Urbanism’, International Journal of Urban and
Regional Research 40(1): 187-199.
Russell, B., (2019) ‘Beyond the Local Trap’, Antipode 51(3): 989–1010
Shearmur, R. and Poirier, V. (2017) ‘Conceptualising non-market municipal
entrepreneurship’, Urban Affairs Review, 53(4): 718-51.
Smith, B. (2000) ‘The concept of the “enabling” local authority’, Environment and
Planning C, 18(1): 79-94.
Spinosa, C. Flores, F. and Dreyfus, H. (1999) Disclosing New Worlds, Cambridge,
Mass.: MIT.
26
Stoker, G. (2011) ‘Was local governance such a good idea?’, Public
Administration, 89(1): 15-31.
Taylor, L. Haynes, P. and Darking, M. (2021) ‘English local government finance on
transition’, Public Management Review, 32(7): 1081-1106.
Thompson, M. (2020) ‘What so new about New Municipalism?’ Progress in Human
Geography, 45(2): 317-342.
Thompson, M., Nowak, V., Southern, A., Davies, J. and Furmedge, P. (2020) ‘Regrounding the city with Polyani’, Environment and Planning A, 52(6): 1171-1194.
27
Table 1: Case Study Councils
Birmingham City
Municipal Housing Trust, local
authority housing company
Dumfries and Galloway
Enterprising DG, in-house
service delivery arm
East Riding of Yorkshire
Leisure services partnership with
GPs
Oxford City
Oxford Direct Services, local
authority social enterprise
Sevenoaks District
Property investment strategy
Swansea City and
House building programme
County
28
Figure 1: Statements: Commercialisation and Entrepreneurship, APSE Nominees
2015-2019
1. ‘Commercialising our services to meet the challenge of reduced Government funding.’
(Nottingham City Council, New DLO for Commercial Gas and Electrics, 2015)
2. ‘To deliver free residential solar PV to residents in the city and deliver a community benefit
fund and income to the Council whilst offering some of the City’s residents an opportunity to
address fuel poverty issues.’ (Peterborough City Council, Empower Peterborough, 2015)
3. ‘Business reengineering project that has (…) already helped safeguard and enhance the
lives of over 5,000 residents and has delivered cashable savings of over 70% to the public
purse.’ (North Hertfordshire District Council, Hertfordshire Careline - Social
Entrepreneurship in Practice, 2016)
4. ‘Growth of 110% in external income, financial savings of £168k a year, reduction annually
in 17,000 tons of emissions and an on target, projected additional income stream of over £2.9
million over the next five years.’ (GS Plus Ltd – Royal Borough of Greenwich, Creating a
Prosperous Future!, 2016)
5. ‘The company was created to nurture the growth of people, businesses and the region.’
(Cheshire East Council, The Skills and Growth Company, 2017)
6. ‘Our USP as the only social caterer in town: ‘Stakeholders NOT Shareholders’ whilst
maximising quality in delivery, income growth and penetration.’ (Birmingham City Council,
Building on the Past - Shaping the Future, 2017)
7. ‘The Rocky Balboa-style story that has taken KWL from being the underdog to a wellrespected champion in its field.’ (Kingstown Works Ltd, Ten Years On and Trading Strong,
2017)
8. ‘Commercialisation is a key enabler in tackling the perfect storm of austerity and rising
demand on council services.’ (Warrington Borough Council, Taking Control of Our Future,
2018)
9. ‘We were able to assist 200 new clients and deliver nearly £200K in surpluses back to our
host authority in just one year.’ One West, Bath and North East Somerset Council, Delivering
Good Governance Through Commercialisation, 2019)
10. ‘To influence substantial behavioural change, positively impact upon trading imperatives
and embed a commercial culture – changing the perception of every council employee from ‘I
work for the Council’ to ‘I work for our Citizens’.’ (Birmingham City Council, The
Commercial Business Hub - Providing the Oxygen for Growth, 2019)
11. ‘Astute commercial investments with a clear sense of purpose.’ (Cheltenham Borough
Council, Calculated Risk Taking, 2019)
12. ‘Being an effective commercial business managing its budget, generating income,
reducing costs and expenditure, effectively commissioning and procuring and ensuring a
focus on outcomes and value for money rather than ‘the mentality of cuts’.’ (Thurrock
Council, The Commercial Council, 2019)
|
|
https://openalex.org/W2960196667
|
https://bmcurol.biomedcentral.com/track/pdf/10.1186/s12894-019-0495-z
|
English
| null |
Expression of components of the urothelial cholinergic system in bladder and cultivated primary urothelial cells of the pig
|
BMC urology
| 2,019
|
cc-by
| 6,285
|
Leonhäuser et al. BMC Urology (2019) 19:62
https://doi.org/10.1186/s12894-019-0495-z Leonhäuser et al. BMC Urology (2019) 19:62
https://doi.org/10.1186/s12894-019-0495-z Open Access Expression of components of the urothelial
cholinergic system in bladder and
cultivated primary urothelial cells of the pig Dorothea Leonhäuser1, Jasmin Kranz2, Regina Leidolf2, Patrick Arndt1, Ulrich Schwantes3, Joachim Geyer2 and
Joachim O. Grosse1* Abstract Background: Porcine urinary bladders are widely used for uro-pharmacological examinations due to their
resemblance to the human organ. However, characterisations of the porcine urothelium at the molecular level
are scarce up to now. As it has become clear over the last years that this tissue plays an important role in
the signaling-pathways of the bladder, we examined whether the transporter and receptor pattern (with focus
on the transmitter acetylcholine) is comparable to the human urothelium. With regard to in vitro studies, we
also investigated if there is a difference between the native tissue and cultivated primary urothelial cells in culture. Methods: Urothelium from German Landrace and Göttingen Minipig bladders was collected. One part of the German
Landrace tissue was used for cultivation, and different passages of the urothelial cells were collected. The actual mRNA
expression of different transporters and receptors was examined via quantitative real-time PCR. These included
the vesicular acetylcholine transporter (VAChT), the choline acetyl transferase (ChAT), organic cation transporters 1–3
(OCT1–3), organic anion transporting polypeptide 1A2 (OATP1A2), P-glycoprotein (ABCB1), the carnitine acetyl-
transferase (CarAT), as well as the muscarinic receptors 1–5 (M1–5). Results: There is a strong qualitative resemblance between the human and the porcine urothelium with regard to the
investigated cholinergic receptors, enzymes and transporters. CarAT, OCT1–3, OATP1A2 and ABCB1 could be detected
in the urothelium of both pig races. Moreover, all 5 M-receptors were prominent with an emphasis on M2 and M3. VAChT and ChAT could not be detected at all. Cultures of the derived urothelial cells showed decreased expression of
all targets apart from ABCB1 and CarAT. Conclusions: Based on the expression pattern of receptors, transporters and enzymes of the cholinergic system, the
porcine urinary bladder can be regarded as a good model for pharmacological studies. However, cultivation of primary
urothelial cells resulted in a significant drop in mRNA expression of the targets. Therefore, it can be concluded that the
intact porcine urothelium, or the whole pig bladder, may be appropriate models for studies with anticholinergic drugs,
whereas cultivated urothelial cells have some limitation due to significant changes in the expression levels of relevant
targets. Abstract Keywords: Urothelium, Urinary bladder, Acetylcholine, Transporter, Muscarinic receptor, German landrace pig,
Göttingen Minipig Urothelium, Urinary bladder, Acetylcholine, Transporter, Muscarinic receptor, German landrace pig,
Minipig * Correspondence: joachim.grosse1963@gmail.com
1Department of Urology, RWTH Aachen University Hospital, Pauwelsstraße
30, 52074 Aachen, Germany
Full list of author information is available at the end of the article Harvest and storage of urothelial tissue and cell culture
of urothelial cells (UC) Harvest and storage of urothelial tissue and cell culture
of urothelial cells (UC) All experiments on animals were performed in com-
pliance with German legislation governing animal stud-
ies and the Guide for the Care and Use of Laboratory
Animals (National Institutes of Health (NIH), Publica-
tion No 85–23, revised 2011). The pig urinary bladders
were obtained from other working groups in our animal
facility in order to reduce the number of animal experi-
ments (3-R principle). These animal experiments were
approved by the Governmental Animal Care and Use
Committee (LANUV Recklinghausen). Göttingen Mini-
pigs (GM) were about 2 years old whereas the German
Landrace (GL) pigs were 6 months old. Besides the
anaesthetics, no other drugs or treatments were applied
to the pigs which could affect the bladder tissue. The
pigs were euthanized by the original working group
using 0.16 g/kg barbiturate i.v. (Narcoren®, Merial, Hall-
bergmoos, Germany), and the intact bladders were
transported to the cell culture facility in a 37 °C pre-
warmed Modified Eagle’s Medium (MEM, Life Tech-
nologies, Braunschweig, Germany). Some molecular examinations of detrusor and urothe-
lial tissue have been performed on human material
concerning transporters and receptors of anticholinergic
drugs, such as trospium chloride, which are used as the
clinical standard in the treatment of overactive bladder
(OAB) [6–9]. Although it is clear that anticholinergic
drugs block muscarinic (M-) acetylcholine (ACh) recep-
tors, it is not completely understood how these drugs
interact with the cellular machinery of the urothelium
and the detrusor. Goepel et al. [10], as well as Sellers et
al. [11], showed that muscarinic receptors M2 and M3
are prominent in porcine as well as human detrusor
tissue, and in the human urothelium all five known M-
receptor subtypes are present [12–14]. g
g
y)
Via a Y-shaped incision, bladders from GM and GL were
opened, and the urothelial tissue was carefully dissected
and minced with scissors. Whole urothelial tissue of GM
and one segment of GL tissue was frozen in liquid nitro-
gen and stored at −80 °C. The other segment of GL tissue
was used for cell culture as described previously [20]. Briefly, the urothelium was incubated in MEM containing
400 μg/ml collagenase (Liberase®, Roche Applied Sciences,
Penzberg, Germany) for 1 h at 37 °C. © 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. Leonhäuser et al. BMC Urology (2019) 19:62 Page 2 of 8 Leonhäuser et al. BMC Urology (2019) 19:62 Page 2 of 8 Page 2 of 8 Methods Over the last years, the use of urinary pig bladders,
and the pig itself as a large animal model, have be-
come extremely popular for urological examinations. Especially pharmacological studies depend on the test-
ing of drugs using porcine material [1–3]. This is
justified on the basis that the porcine bladder closely
resembles the human organ both anatomically and
physiologically [4, 5]. Nevertheless, not much research
on the molecular basics has as yet been conducted
which justifies unlimited use and comparison of the
porcine material [5]. Harvest and storage of urothelial tissue and cell culture
of urothelial cells (UC) The cell suspension
was then filtered, washed with MEM containing 10% fetal
calf serum (FCS, Thermo Scientific) and transferred into
collagen-coated (Biochrom AG, Berlin, Germany) cell
culture flasks (Nunclon™, Thermo Scientific). Incubation
of the urothelial cells (UC) for the first 24 h was per-
formed in MEM containing 20% FCS, 1% gentamicin
(PAA, GE Healthcare, Frankfurt am Main, Germany) and
1% amphotericin B (PAA). After 24 h, the medium was
changed to Keratinocyte-SFM (Life Technologies) and
subsequently changed twice a week. Examination of the
cells in culture was performed with a Leica DMI 4000B
(Leica Microsystems GmbH, Wetzlar, Germany) with
integrated software Diskus (4.80.5909, Hilgers, Tech-
nisches Büro, Königswinter, Germany). The cells were
split at confluence and 1 × 106 cells were transferred into
a new 75 cm2 flask. The remaining cells were frozen in
liquid nitrogen and stored at −80 °C. Besides the M-receptors, other possible drug targets
exist in the human bladder. Lips et al. [15] found proof
for different transporters and enzymes responsible for
synthesis and storage of ACh in the murine and human
urothelium, including the carnitine acetyl-transferase
(CarAT), whereas the classical ACh synthesising enzyme
of neuronal cells,
choline
acetyltransferase (ChAT),
seems to be absent in the urothelium. Furthermore, they
demonstrated expression of the organic cation trans-
porters OCT1–3 in the human urothelium. Interestingly,
all of them are active in transporting TrCL [16]. More-
over, the solute carrier organic anion transporting poly-
peptide 1A2 (OATP1A2), involved in the cellular uptake
of TrCL, as well as P-glycoprotein (syn. ATP-binding
cassette transporter ABCB1, encoded by the multi-drug
resistance gene MDR1), involved in the efflux of TrCL,
are expressed in the normal urothelium [17–19]. The
vesicular ACh transporter VAChT, which transports
ACh in synaptic vesicles of neuronal cells, appears to be
absent in the urothelium [13]. With regard to the pig as a model for pharmaco-
logical studies on anticholinergic drugs in the bladder,
our aim was to determine whether these relevant
transporters, enzymes and receptors are present in
the porcine urothelium with
the same expression
pattern and to the same extent as in the human. Fur-
thermore, their molecular stability during cell culture
was to be examined. Results Cell culture of urothelial cells and proof of cell type
During the cultivation period, UCs from GL could be
passaged up to three times. This resulted in five
samples per GL pig; the untreated bladder biopsy and
four immediately consecutive passages of UCs. From
these GL pig bladders and three additional GM blad-
ders, untreated urothelial tissue samples were frozen to
compare the native tissues of the different races. Immu-
nohistochemical staining of the cells against panCK
confirmed an urothelial phenotype (Fig. 1). The pres-
ence
of
muscarinic
receptors
M2
and
M3,
most
relevant for storing and voiding of urine in the bladder,
could also be visualized in the native urothelium of the
German Landrace pig (Fig. 2a+b). Antigen retrieval of deparaffinised sections was per-
formed using citrate-buffer (Zytomed Systems GmbH,
Berlin, Germany) in a steamer for 30 min. Primary
monoclonal and polyclonal antibodies (Table 1) were
incubated for 1 h, and secondary antibody and chromo-
gen development (DAKO Real EnVision HRP rabbit/
mouse with DAB) were applied according to the manu-
facturer’s protocol. Counterstaining was performed using
haemalaun (Merck). Staining was observed using a Leica
DM6000B and integrated software Diskus (4.80.5909,
Hilgers, Technisches Büro, Königswinter, Germany). Immunohistochemical staining of tissue and cells Validation of the cell type was performed via immu-
nohistochemistry. Therefore, confluent UC were de-
tached with trypsin-EDTA (Life Technologies), washed Page 3 of 8 Leonhäuser et al. BMC Urology (2019) 19:62 was performed using OriginPro (2017G, Origin Lab
Corporation, Northampton, USA). The Shapiro-Wilks
test was used to test for normal distribution, and One-
Way ANOVA with the Tukey-Post Hoc test was used to
determine statistical differences. Values of p ≤0.05 were
considered to be significant. with phosphate buffered saline (PBS) (Life Technologies)
and fixed in 4% (w/v) phosphate buffered formaldehyde
(Merck). After further centrifugation, supernatant for-
maldehyde was removed and the cells were mixed with
3% (w/v) agarose (Biozym Scientific GmbH, Hessisch Old-
endorf, Germany). The agarose cell-hybrids were cooled
for 3 min. in a fridge at 4 °C. Native bladder tissue served
as the control and was also used for staining of muscar-
inic receptors M2 and M3 and therefore fixed in phos-
phate buffered formaldehyde. The cell-hybrids and the
native tissue were dehydrated, embedded in paraffin
and cut into 3 μm sections. Quantitative real-time polymerase chain reaction In general, quantitative real-time PCR expression analysis
of native porcine urothelium was very similar concerning
the presence of the examined transporters and receptors
as in humans [13, 15]. The enzyme CarAT and the trans-
porter ABCB1 were most commonly expressed. The or-
ganic cation transporters OCT1–3, as well as OATP1A2,
could be detected, but to a lesser extent (Fig. 3a). The
mRNA expression for all muscarinic receptors was found
in the porcine urothelium, especially M2 and M3 (Fig. 3b). VaChT and ChAT, as in human urothelium, were not
detectable. There were no significant differences in the
expression pattern between the two pig breeds. Quantitative real time polymerase chain reaction
TriReagent (Sigma Aldrich) was used to extract RNA from
cultured cells and tissues according to the manufacturer’s
protocol. The isolated RNA was reverse-transcribed using
the SuperScript III system (Life technologies GmbH,
Darmstadt, Germany). Quantitative real-time polymerase
chain reaction (qRT-PCR) was performed using the Taq-
Man GEX Master Mix (Life Technologies). Four of the ob-
tained assays (VAChT, OCT3, CHRM4, and CHRM5) had
to be custom-made as there were no such probes available
for the pig. All other TaqMan assays were ordered
according to the reference numbers shown in Table 2. Glyercinaldehyde-3-phosphate-dehydrogenase
(GAPDH)
was used as the housekeeping gene. All assays were tested
using
a
pig
tissue
cDNA
panel
(BioCat
GmbH,
Heidelberg, Germany) prior to the initial examinations. Subsequently, it was analysed whether the enzyme/
transporter/receptor expression changed during culti-
vation of the porcine urothelium. In general, most of the
analysed targets were down-regulated following repeated
passaging of the cells. This down-regulation was most
noticeable for OCT1 and OCT2 as well as M1-M3,
whereas the expression level of the M4 and M5 recep-
tors was almost maintained up to passage 3 (P3) (Fig. 4). Statistical analysis Cell cultures from three different animals were exa-
mined using technical triplicates. Statistical evaluation Table 1 Primary antibodies for immunohistochemistry
Antibody
Clone
number
Reactivity
Dilution Incubation
time
Company
PanCK
AE1/AE3
Monoclonal mouse, anti-
human
1:300
1 h
Dako GmbH, Hamburg,
Germany
Anti-Muscarinic Acetylcholine Receptor 2
31-1D1
Monoclonal mouse, anti-
human
1:100
1 h
Abcam, Cambridge, United
Kingdom
Anti-Muscarinic Acetylcholine Receptor M3
antibody
–
Polyclonal rabbit, anti-pig
1:100
1 h
Abcam, Cambridge, United
Kingdom Table 1 Primary antibodies for immunohistochemistry Leonhäuser et al. BMC Urology (2019) 19:62 Page 4 of 8 Leonhäuser et al. BMC Urology Table 2 TaqMan gene expression assays used for real-time PCR expression analysis
Assay
Order No. VAChT-Pig
Custom Gene ex assay, AIWR3FN, referred to the cDNA sequence with GenBank Accession No. XM_013983446
ChAT-Pig
Taqman Gene ex assay MTO, sm, Ss03391504_m1
CarAT-Pig
Taqman Gene ex assay MTO, sm, Ss03389781_m1
ABCB1-Pig
Taqman Gene ex assay MTO, sm, Ss03373435_m1
OCT1-Pig
Taqman Gene ex assay MTO, sm, Ss03391173_m1
OCT2-Pig
Taqman Gene ex assay MTO, sm, Ss03390935
OCT3-Pig
Custom Gene ex assay, JD89KCA, referred to the cDNA sequence with GenBank Accession No. XM_003121106
OATP1A2-Pig
Taqman Gene ex assay MTO, sm, Ss03375623_u1
CHRM1-Pig
Taqman Gene ex assay MTO, sm, Ss03393581_u1
CHRM2-Pig
Taqman Gene ex assay MTO, sm, Ss03383697_u1
CHRM3-Pig
Taqman Gene ex assay MTO, sm, Ss03387661_u1
CHRM4-Pig
Custom plus Tqmn RNA Assays, AJPADK0, referred to the cDNA sequence with GenBank Accession No. XM_003122828
CHRM5-Pig
Custom plus Tqmn RNA Assays, AJN1FES, referred to the cDNA sequence with GenBank Accession No. XM_021099908
GAPDH-Pig
FG, Off the shelf GX Set, Ss03374854_g1 Table 2 TaqMan gene expression assays used for real-time PCR expression analysis Table 2 TaqMan gene expression assays used for real-time PCR expression analysis The downregulation of muscarinic receptors M2 and
M3 could also be visualized by immunohistochemical
staining (Fig. 2c+d). The downregulation of muscarinic receptors M2 and
M3 could also be visualized by immunohistochemical
staining (Fig. 2c+d). process have been overlooked. This might be due to the
fact, that this special tissue was only assumed to be a
barrier with no physiological impact on the bladder func-
tion [21, 22]. As it has become increasingly clear that the
urothelium plays an essential role, it is even more import-
ant to re-examine the animal models already used for trans-
lational examinations applicable to the human bladder. Discussion Previously, systematic examinations on the urothelium as
an active component of the bladder storage and micturition Fig. 1 Isolation of urothelial cells from German Landrace pig urothelium. a Urothelial layer which was dissected from the underlying lamina
propria (HE staining). b Urothelial cells in culture at confluence. c Immunostaining of UCs with panCK embedded in agarose. d Positive control of
porcine urothelium, immunostained with panCK Fig. 1 Isolation of urothelial cells from German Landrace pig urothelium. a Urothelial layer which was dissected from the underlying lamina
propria (HE staining). b Urothelial cells in culture at confluence. c Immunostaining of UCs with panCK embedded in agarose. d Positive control of
porcine urothelium, immunostained with panCK Leonhäuser et al. BMC Urology (2019) 19:62 Page 5 of 8 Leonhäuser et al. BMC Urology Fig. 2 Immunohistochemical staining of muscarinic receptors in native bladder tissue and derived urothelial cells. a+c Muscarinic receptor M2
and b+d M3 could be visualized in a+b native bladder tissue of the German Landrace pig but c+d not in the derived urothelial cells at passage
1. Scale bar = 50 μm Fig. 2 Immunohistochemical staining of muscarinic receptors in native bladder tissue and derived urothelial cells. a+c Muscarinic receptor M2
and b+d M3 could be visualized in a+b native bladder tissue of the German Landrace pig but c+d not in the derived urothelial cells at passage
1. Scale bar = 50 μm Fig. 2 Immunohistochemical staining of muscarinic receptors in native bladder tissue and derived urothelial cells. a+c Muscarinic receptor M2
and b+d M3 could be visualized in a+b native bladder tissue of the German Landrace pig but c+d not in the derived urothelial cells at passage
1. Scale bar = 50 μm So far, we have only established the M2 and M3
immunohistostaining for the targets also analysed via
qRT-PCR. As it is not easy to find antibodies that are
applicable for pig tissue, it is often necessary to use anti-
bodies for human or other species and hope for cross-
reactions. Therefore, we also tried a GAPDH antibody
which turned out to react very unspecific. As the use of
immune staining is more for visual effects, we post-
poned the establishment of other antibodies. cholinergic system, a comparable expression pattern was
detected in the pig in the present study, as has pre-
viously been described for humans [13, 15, 18, 19]. Discussion * Significant down-regulation compared to tissue with p < 0.05 limits the usability of these cells, for example, for trans-
port or receptor binding experiments at higher passaging
frequencies. Bexten et al. were able to show that, on the
one hand, TrCL is a substrate of the aforementioned solute
carriers OCT1 and OATP1A2 (uptake), but on the other
hand also for the efflux carrier ABCB1 [19]. The in vitro
downregulation of the uptake transporters in combination
with the still relatively high amount of the ABCB1 efflux
carrier would lead to a non-physiological shift compared to
the actual in vivo situation. This cultivation-related down-
regulation could also be observed for the muscarinic re-
ceptors, especially M2 and M3, which play a crucial role in
the storing and voiding mechanisms of the bladder. M3 is
known to mediate the contractile response and thus is
addressed by the main muscarinic receptor antagonist
TrCL [12]. This has to be considered for uptake studies
with anticholinergic drugs like TrCL. However, down-
regulation of M1-M5 has also been demonstrated for hu-
man urothelial cells in culture by Tyagi et al. [14]. tissue so far. Additionally, in the present study, expres-
sion of the classical ACh synthesising enzyme ChAT
could not be detected in the porcine urothelium as has
previously been reported for the human [13]. However,
the porcine urothelium showed high mRNA expression
levels for CarAT, which is an alternative source of ACh
synthesis in the urothelium [13]. In the pig, all three
OCTs (OCT1–3) were detected in the urothelium in the
order OCT3 > OCT2 > OCT1, and all of them were also
present in the human urothelium [13]. The solute carrier
OATP1A2, uptake carrier for endogenous substances
and pharmaceuticals in the human urothelium, has been
identified as a transporter for TrCL by Bexten et al. [19],
and could also be detected in the pig urothelium by our
group. ABCB1 plays an important role in the efflux of
many drugs, also including the anticholinergic drug
TrCL [23]. ABCB1 is highly expressed in normal human
urothelium [18, 19] and the present study also con-
firmed its expression in the porcine urothelium. Finally,
all 5 M-receptors have been reported in the human
urothelium [13, 14], and could be detected in the
present study with a similar expression pattern in the
pig, with the highest expression levels for M2 and M3. Discussion Although VAChT is an important vesicular transporter
for ACh, it could not be detected in the porcine uro-
thelium. This too, is in accordance with the findings in
the human urothelium, where the presence of VAChT
could not be proved [15]. However, as a limitation it has
to be mentioned that the gene expression assay for
VAChT was derived from a predicted cDNA sequence
(see Table 1) and could not be confirmed in any porcine Using qRT-PCR expression analysis of selected en-
zymes, transporters and receptors of the urothelial Fig. 3 Relative gene expression in the urothelium of German Landrace pigs and Göttingen minipigs (n = 3). VAChT and ChAT expression could
not be detected. All enzymes/transporters (a) as well as receptors (b) relevant for anticholinergic therapy showed comparable expression pattern
for GL and GM. Highest expression was detected for CarAT, ABCB1, OCT3, OATP1A2, M2 and M3. GAPDH expression was used for normalization pigs and Göttingen minipigs (n
3) VAChT and ChAT expression could Fig. 3 Relative gene expression in the urothelium of German Landrace pigs and Göttingen minipigs (n = 3). VAChT and ChAT expression could
not be detected. All enzymes/transporters (a) as well as receptors (b) relevant for anticholinergic therapy showed comparable expression pattern
for GL and GM. Highest expression was detected for CarAT, ABCB1, OCT3, OATP1A2, M2 and M3. GAPDH expression was used for normalization Leonhäuser et al. BMC Urology (2019) 19:62 Page 6 of 8 Leonhäuser et al. BMC Urology Fig. 4 Relative gene expression in the urothelium from German Landrace pigs (“Tissue”) as well as derived UCs (“P0-P3”) (n = 3). All enzymes/
transporters (a) as well as receptors (b) relevant for anticholinergic therapy were down-regulated during repeated passaging, with the exception
of M4. In particular, expression levels of OCT2 and M1 dramatically dropped to nearly undetectable levels. GAPDH expression was used for
normalization. * Significant down-regulation compared to tissue with p < 0.05 i
(“Ti
”)
ll
d i
d UC (“P0 P3”) (
3) All
/ Fig. 4 Relative gene expression in the urothelium from German Landrace pigs (“Tissue”) as well as derived UCs (“P0-P3”) (n = 3). All enzymes/
transporters (a) as well as receptors (b) relevant for anticholinergic therapy were down-regulated during repeated passaging, with the exception
of M4. In particular, expression levels of OCT2 and M1 dramatically dropped to nearly undetectable levels. GAPDH expression was used for
normalization. Conclusions This study was able to show that the porcine urothelium
of the GL and GM pig is very similar to the human
urothelium concerning the investigated ACh-dependent
targets. We therefore conclude that both pig races are
appropriate as a pharmacological animal model for in vivo
and ex vivo investigations. Nevertheless, the use of UCs
from healthy animals is limited due to the down-
regulation of the afore-mentioned targets. Therefore, the
development and use of an animal model with OAB or IC
could provide more insight into the working mechanism
of these diseases. Funding
h
d This study was funded by the Dr. R. Pfleger GmbH, Germany. The Dr. R. Pfleger GmbH had no control over the study conduct, or the collection and
analysis of the data. Abbreviations
b d ABCB1: ATP-binding cassette B1; ACh: Acetylcholine; CarAT: Carnitine acetyl-
transferase; ChAT: Choline acetyl transferase; GAPDH: Glyercinaldehyde-3-
phosphate-dehydrogenase; GL: German Landrace pig; GM: Göttingen
Minipig; IC: Interstitial cystitis; M1–5: Muscarinic receptors 1–5; MEM: Modified
Eagle’s Medium; OAB: Overactive bladder; OATP1A2: Organic anion
transporting polypeptide 1A2; OCT1–3: Organic cation transporters 1–3;
PBS: Phosphate buffered saline; TrCL: Trospium chloride; UC: Urothelial cell;
VAChT: Vesicular acetylcholine transporter 2. Yamanishi T, Chapple CR, Yasuda K, Chess-Williams R. The role of M-2-
muscarinic receptors in mediating contraction of the pig urinary bladder in
vitro. Br J Pharmacol. 2000;131(7):1482–8. 3. Parsons BA, Drake MJ, Gammie A, Fry CH, Vahabi B. The validation of a
functional, isolated pig bladder model for physiological experimentation. Front Pharmacol. 2012;3:52. 4. Huppertz ND, Tolba RH, Grosse JO. Micturition in Gottingen minipigs: first
reference invivo data for urological research and review of literature. Lab
Anim. 2015;49(4):336–44. Discussion Interestingly, ABCB1 showed slight upregulation dur-
ing the first round of cultivation in comparison to the
tissue samples. This upregulation of ABCB1 might be
triggered by cell culture supplements such as antibiotics,
as the main role of this transporter is the efflux of po-
tential hazardous substances out of the cell [23, 24]. Two different pig races were analysed i.e. GL and GM,
and both showed comparable expression patterns to
each other and the human. Taking this into account, it
could be expected that pharmacological studies in pigs,
for example with anticholinergic drugs, would sub-
stantially reflect the situation in man. UCs in culture are a promising model for phar-
macological in vitro studies with anticholinergic drugs. However, such studies would be disadvantaged by down-
regulation of relevant transporters and receptors for
ACh
and
/
or
anticholinergic
drugs. Nevertheless,
Mukerji et al. [25], as well as Gupta et al. [26], could
show that urothelial cells of patients with interstitial
cystitis (IC) retained their phenotype under cell culture
conditions. Furthermore, the animals in this study were
mature but not old [27, 28] and thus, do not represent
the aged population which suffer from bladder dysfunc-
tions. Therefore, a corresponding animal model with IC Cultivation of urothelial cells has previously been estab-
lished in order to avoid repeated in vivo experiments in
pigs, and to enable in vitro studies with urothelial derived
cells. For this reason, the expression of the relevant en-
zymes, transporters, and receptors was also analysed
under cell culture conditions during repeated passaging. Unfortunately, most of the analysed targets revealed
continuous downregulation over time, which in general Page 7 of 8 Leonhäuser et al. BMC Urology (2019) 19:62 Page 7 of 8 Page 7 of 8 Leonhäuser et al. BMC Urology (2019) 19:62 or OAB could provide more insight in the mechanisms
of these diseases at a molecular level. References 1. Templeman L, Chapple CR, Chess-Williams R. Urothelium derived inhibitory
factor and cross-talk among receptors in the trigone of the bladder of the
pig. J Urol. 2002;167(2):742–5. 2. Yamanishi T, Chapple CR, Yasuda K, Chess-Williams R. The role of M-2-
muscarinic receptors in mediating contraction of the pig urinary bladder in
vitro. Br J Pharmacol. 2000;131(7):1482–8. 3. Parsons BA, Drake MJ, Gammie A, Fry CH, Vahabi B. The validation of a
functional, isolated pig bladder model for physiological experimentation. Front Pharmacol. 2012;3:52. 4. Huppertz ND, Tolba RH, Grosse JO. Micturition in Gottingen minipigs: first
reference invivo data for urological research and review of literature. Lab
Anim. 2015;49(4):336–44. 5. Bode G, Clausing P, Gervais F, Loegsted J, Luft J, Nogues V, et al. The utility
of the minipig as an animal model in regulatory toxicology. J Pharmacol
Toxicol Methods. 2010;62(3):196–220. 6. Abrams P, Andersson KE. Muscarinic receptor antagonists for overactive
bladder. BJU Int. 2007;100(5):987–1006. 7. Andersson KE, Yoshida M. Antimuscarinics and the overactive detrusor -
which is the main mechanism of action? Eur Urol. 2003;43(1):1–5. 8. Abrams P, Andersson KE, Buccafusco JJ, Chapple C, de Groat WC, Fryer
AD, et al. Muscarinic receptors: their distribution and function in body 1. Templeman L, Chapple CR, Chess-Williams R. Urothelium derived inhibitory
factor and cross-talk among receptors in the trigone of the bladder of the
pig. J Urol. 2002;167(2):742–5. 1. Templeman L, Chapple CR, Chess-Williams R. Urothelium derived inhibitory
factor and cross-talk among receptors in the trigone of the bladder of the
pig. J Urol. 2002;167(2):742–5. Authors’ contributions All authors have made substantial contributions to conception and design,
acquisition of data, or analysis and interpretation of data: DL designed the
study, performed the experiments and analysed the data, JK performed the
experiments and analysed the data, RL and PA performed the experiments JG
and JOG designed the study and analysed the data. US, as an employee of the
sponsor, was involved in the determination of the study design, but had no
control over the study conduct, or the collection and analysis of data. All
authors have been involved in drafting the manuscript or revising it critically for
important intellectual content; All authors have given final approval of the
version to be published and take public responsibility for appropriate portions
of the content. All authors have agreed to be accountable for all aspects of the
work in ensuring that questions related to the accuracy or integrity of any part
of the work are appropriately investigated and resolved. The authors have read
and approve of the final version. It remains that physiological and pharmacological stu-
dies on the urinary bladder are mainly conducted in ro-
dent models [29–32]. These animals are cheap, easy to
handle, and a large number of individuals can be investi-
gated. However, data generated in rodents may not be
fully comparable to the situation in humans, as the ana-
tomy, physiology and the day-night-rhythm of these
animals is somewhat different. Therefore, pigs were ana-
lysed in the present study as these show a more com-
parable bladder physiology to humans [4]. Initially the study was intended only with GL pigs as
these are cheaper to obtain and therefore used more
commonly in animal studies. Additionally, it is common
practice to perform whole bladder experiments and drug
studies using pig bladders from an abattoir [1–3, 33]. However, the GM has proven itself to be a comparable
animal model for urological in vivo long-term studies,
not only on the physiological [4] but also on the molecu-
lar level, as was also demonstrated in the present study. Based on the data presented here, both pig races are
appropriate as a pharmacological animal model concer-
ning the investigated targets. Competing interests DL, JK, RL, PA, JG, and JOG declare that they have no competing interests. US is an employee of Dr. R. Pfleger GmbH, Bamberg. Availability of data and materials The datasets used and/or analysed during the current study are available
from the corresponding author on reasonable request. Author details
1
f 1Department of Urology, RWTH Aachen University Hospital, Pauwelsstraße
30, 52074 Aachen, Germany. 2Institute of Pharmacology and Toxicology,
Biomedical Research Center BFS, Justus Liebig University Giessen, Giessen,
Germany. 3Dr. R. Pfleger GmbH, Bamberg, Germany. Biomedical Research Center BFS, Justus Liebig University Giessen, Giessen,
Germany. 3Dr. R. Pfleger GmbH, Bamberg, Germany. Received: 15 February 2018 Accepted: 4 July 2019 Ethics approval and consent to participate pp
p
p
All projects for which our study group received subordinate urinary bladder
material were either notified or approved by our licensing authority, the
LANUV (Landesamt für Natur-, Umwelt und Verbraucherschutz in
Recklinghausen). The secondary use of tissues or organs from dead animals
does not require approval by the authorities according to animal welfare
regulations in Germany. Publisher’s Note 10. Goepel M, Gronewald A, Krege S, Michel MC. Muscarinic receptor subtypes
in porcine detrusor: comparison with humans and regulation by bladder
augmentation. Urol Res. 1998;26(2):149–54. 10. Goepel M, Gronewald A, Krege S, Michel MC. Muscarinic receptor subtypes
in porcine detrusor: comparison with humans and regulation by bladder
augmentation. Urol Res. 1998;26(2):149–54. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. 11. Sellers DJ, Yamanishi T, Chapple CR, Couldwell C, Yasuda K, Chess-Williams
R. M-3 muscarinic receptors but not M-2 mediate contraction of the porcine
detrusor muscle in vitro. J Auton Pharmacol. 2000;20(3):171–6. 12. Mansfield KJ, Liu L, Mitchelson FJ, Moore KH, Millard RJ, Burcher E. Muscarinic receptor subtypes in human bladder detrusor and mucosa,
studied by radioligand binding and quantitative competitive RT-PCR:
changes in ageing. Br J Pharmacol. 2005;144(8):1089–99. 12. Mansfield KJ, Liu L, Mitchelson FJ, Moore KH, Millard RJ, Burcher E. Muscarinic receptor subtypes in human bladder detrusor and mucosa,
studied by radioligand binding and quantitative competitive RT-PCR:
changes in ageing. Br J Pharmacol. 2005;144(8):1089–99. 13. Bschleipfer T, Schukowski K, Weidner W, Grando SA, Schwantes U, Kummer
W, et al. Expression and distribution of cholinergic receptors in the human
urothelium. Life Sci. 2007;80(24–25):2303–7. 13. Bschleipfer T, Schukowski K, Weidner W, Grando SA, Schwantes U, Kummer
W, et al. Expression and distribution of cholinergic receptors in the human
urothelium. Life Sci. 2007;80(24–25):2303–7. 14. Tyagi S, Tyagi P, Van-le S, Yoshimura N, Chancellor MB, de Miguel F. Qualitative and quantitative expression profile of muscarinic receptors in
human urothelium and detrusor. J Urol. 2006;176(4). 14. Tyagi S, Tyagi P, Van-le S, Yoshimura N, Chancellor MB, de Miguel F. Qualitative and quantitative expression profile of muscarinic receptors in
human urothelium and detrusor. J Urol. 2006;176(4). 15. Lips KS, Wunsch J, Zarghooni S, Bschleipfer T, Schukowski K, Weidner W, et
al. Acetylcholine and molecular components of its synthesis and release
machinery in the urothelium. Eur Urol. 2007;51(4):1042–53. 15. Lips KS, Wunsch J, Zarghooni S, Bschleipfer T, Schukowski K, Weidner W, et
al. Acetylcholine and molecular components of its synthesis and release
machinery in the urothelium. Eur Urol. 2007;51(4):1042–53. 16. Wenge B, Geyer J, Bönisch H. The anticholinergic drugs oxybutynin and
trospium chloride are substrates of the human organic cation transporters. In: Naunyn-Schmiedeberg’s archives of pharmacology, vol. 383. Berlin
Heidelberg: Springer; 2010. p. 203–8. 17. Franke RM, Scherkenbach LA, Sparreboom A. Acknowledgements The authors thank the research group of the Department of Urology,
especially Katja Stollenwerk and Mykhailo Reidman for technical support, and
the Institute of Laboratory Animal Sciences at RWTH Aachen University
Hospital especially Taddhäus Stopinski for excellent animal care. Furthermore,
we thank Stephanie Schmidt of the Institute of Pharmacology and
Toxicology, Giessen for her scientific input. This work was also supported by
the Cell Culture Facility, a Core Facility of the Interdisciplinary Center for
Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH
Aachen University. 5. Bode G, Clausing P, Gervais F, Loegsted J, Luft J, Nogues V, et al. The utility
of the minipig as an animal model in regulatory toxicology. J Pharmacol
Toxicol Methods. 2010;62(3):196–220. Page 8 of 8 Page 8 of 8 Page 8 of 8 Leonhäuser et al. BMC Urology (2019) 19:62 (2019) 19:62 Leonhäuser et al. BMC Urology (2019) 19:62 33. Williams NA, Lee KM, Allender CJ, Bowen JL, Gumbleton M, Harrah T, et al. Investigating detrusor muscle concentrations of oxybutynin after Intravesical
delivery in an ex vivo porcine model. J Pharm Sci. 2015;104(7):2233–40. 33. Williams NA, Lee KM, Allender CJ, Bowen JL, Gumbleton M, Harrah T, et al. Investigating detrusor muscle concentrations of oxybutynin after Intravesical
delivery in an ex vivo porcine model. J Pharm Sci. 2015;104(7):2233–40. systems, and the implications for treating overactive bladder. Br J
Pharmacol. 2006;148(5):565–78. systems, and the implications for treating overactive bladder. Br J
Pharmacol. 2006;148(5):565–78. systems, and the implications for treating overactive bladder. Br J
Pharmacol. 2006;148(5):565–78. 9. Finney SM, Andersson KE, Gillespie JI, Stewart LH. Antimuscarinic drugs in
detrusor overactivity and the overactive bladder syndrome: motor or
sensory actions? BJU Int. 2006;98(3):503–7. 9. Finney SM, Andersson KE, Gillespie JI, Stewart LH. Antimuscarinic drugs in
detrusor overactivity and the overactive bladder syndrome: motor or
sensory actions? BJU Int. 2006;98(3):503–7. Publisher’s Note Pharmacogenetics of the organic
anion transporting polypeptide 1A2. Pharmacogenomics. 2009;10(3):339–44. 18. Clifford SC, Neal DE, Lunec J. High level expression of the multidrug resistance
(MDRI) gene in the normal bladder urothelium: a potential involvement in
protection against carcinogens? Carcinogenesis. 1996;17(3):601–4. 19. Bexten M, Oswald S, Grube M, Jia J, Graf T, Zimmermann U, et al. Expression of
drug transporters and drug metabolizing enzymes in the bladder Urothelium in
man and affinity of the bladder spasmolytic Trospium chloride to transporters
likely involved in its pharmacokinetics. Mol Pharm. 2015;12(1):171–8. 20. Leonhäuser D, Vogt M, Tolba RH, Grosse JO. Potential in two types of
collagen scaffolds for urological tissue engineering applications - are there
differrences in growth behaviour of juvenile and adult vesical cells? J
Biomater Appl. 2016;30(7):961–73. 21. Birder LA. More than just a barrier: urothelium as a drug target for urinary
bladder pain. Am J Physiol Renal Physiol. 2005;289(3):F489–F95. 22. Apodaca G. The uroepithelium: not just a passive barrier. Traffic. 2004;5(3):117–28. 23. Geyer J, Gavrilova O, Petzinger E. The role of P-glycoprotein in limiting brain
penetration of the peripherally acting anticholinergic overactive bladder
drug Trospium chloride. Drug Metab Dispos. 2009;37(7):1371–4. 24. Oswald S, Grube M, Siegmund W, Kroemer HK. Transporter-mediated
uptake into cellular compartments. Xenobiotica. 2007;37(10–11):1171–95. 24. Oswald S, Grube M, Siegmund W, Kroemer HK. Transporter-mediated
uptake into cellular compartments. Xenobiotica. 2007;37(10–11):1171–95. 25. Mukerji G, Yiangou Y, Grogono J, Underwood J, Agarwal SK, Khullar V, et al. Localization of M-2 and M-3 muscarinic receptors in human bladder
disorders and their clinical correlations. J Urol. 2006;176(1):367–73. 25. Mukerji G, Yiangou Y, Grogono J, Underwood J, Agarwal SK, Khullar V, et al. Localization of M-2 and M-3 muscarinic receptors in human bladder
disorders and their clinical correlations. J Urol. 2006;176(1):367–73. 26. Gupta GN, Lu SG, Gold MS, Chail TC. Bladder urothelial cells from patients
with interstitial cystitis have an increased sensitivity to carbachol. Neurourol
Urodyn. 2009;28(8):1022–7. 26. Gupta GN, Lu SG, Gold MS, Chail TC. Bladder urothelial cells from patients
with interstitial cystitis have an increased sensitivity to carbachol. Neurourol
Urodyn. 2009;28(8):1022–7. 27. Vahabi B, Sellers DJ, Bijos DA, Drake MJ. Phasic contractions in urinary
bladder from juvenile versus adult pigs. Plos One. 2013;8(3). 27. Vahabi B, Sellers DJ, Bijos DA, Drake MJ. Phasic contractions in urinary
bladder from juvenile versus adult pigs. Plos One. 2013;8(3). 28. Bollen P, Ellegaard L. The Gottingen minipig in pharmacology and
toxicology. Pharmacol Toxicol. 1997;80:3–4. 28. Publisher’s Note Bollen P, Ellegaard L. The Gottingen minipig in pharmacology and
toxicology. Pharmacol Toxicol. 1997;80:3–4. 29. Zarghooni S, Wunsch J, Bodenbenner M, Brueggmann D, Grando SA,
Schwantes U, et al. Expression of muscarinic and nicotinic acetylcholine
receptors in the mouse urothelium. Life Sci. 2007;80(24–25). 29. Zarghooni S, Wunsch J, Bodenbenner M, Brueggmann D, Grando SA,
Schwantes U, et al. Expression of muscarinic and nicotinic acetylcholine
receptors in the mouse urothelium. Life Sci. 2007;80(24–25). 30. Hanna-Mitchell AT, Beckel JM, Barbadora S, Kanai AJ, de Groat WC, Birder
LA. Non-neuronal acetylcholine and urinary bladder urothelium. Life Sci. 2007;80(24–25):2298–302. 31. Matsumoto Y, Miyazato M, Furuta A, Torimoto K, Hirao Y, Chancellor MB, et
al. Differential roles of M2 and M3 muscarinic receptor subtypes in
modulation of bladder afferent activity in rats. Urology. 2010;75(4):862–7. 32. Nandigama R, Ibanez-Tallon I, Lips KS, Schwantes U, Kummer W, Bschleipfer
T. Expression of nicotinic acetylcholine receptor subunit mRNA in mouse
bladder afferent neurons. Neuroscience. 2013;229:27–35.
|
https://openalex.org/W2803709792
|
https://europepmc.org/articles/pmc5981288?pdf=render
|
English
| null |
Application of a Protocol Based on Trap-Neuter-Return (TNR) to Manage Unowned Urban Cats on an Australian University Campus
|
Animals
| 2,018
|
cc-by
| 17,260
|
Received: 16 April 2018; Accepted: 11 May 2018; Published: 17 May 2018 Simple Summary: In Australia, management of the unowned urban cat population is a continuing
challenge. This is because the numbers of cats culled in trap-and-kill programs are inadequate to
balance the breeding rate of the remaining cats, and also because of immigration of sexually active cats
from surrounding areas in a “vacuum” effect into areas where culling has been applied. In contrast,
programs based on management of cat reproduction, such as trap-neuter-return (TNR), supplemented
by rehoming of socializable cats and kittens, have been shown to produce significant reductions in
free-living cat populations. However, evidence is lacking that these approaches are effective in an
Australian context. In this paper, we document a nine-year TNR program on an Australian university
campus, supplemented by rehoming, that reduced a free-living cat population from 69 to 15 cats,
while also rehoming 19 campus-born kittens and managing a further 34 immigrant cats that either
joined the resident colony (n = 16), were rehomed (n = 15), or died/disappeared (n = 3). Subsequent
institutional support for the program was strong because of a reduction in complaints from campus
staff and students, the minimal institutional costs, and the improved health status of the remaining
cats, all of which are desexed, microchipped, registered and fed on a daily basis. Abstract: In August 2008, the University of New South Wales (UNSW) in Sydney, Australia,
commenced a trap-neuter-return (TNR) program to manage the population of approximately
69 free-roaming unowned urban cats on its Kensington campus. The goals of the program
included an ongoing audit of cats on campus, stabilization of cat numbers through TNR, and a
subsequent reduction in cat numbers over time while maintaining the health of remaining campus cats. Continuation of the TNR program over nine years resulted in a current population, as of September
2017, of 15 cats, all desexed (78% reduction). Regular monitoring of the cats through a daily feeding
program identified a further 34 cats that immigrated on to campus since initiation of the program;
these comprised 28 adult cats (16 unsocialized, 12 socialized) and six solitary kittens. In addition,
19 kittens were born on campus, 14 of which were born to immigrant pregnant females. Unsocialized
adult immigrants were absorbed into the resident campus population. Where possible, socialized
adult immigrants, solitary kittens, and campus-born kittens were removed from campus through
rehoming. animals animals animals animals animals Article Helen Swarbrick 1,2,3,* ID and Jacquie Rand 1,4
1
Australian Pet Welfare Foundation, Kenmore, QLD 4069, Australia; jacquie@petwelfare.org.au
2
Campus Cats NSW, Kensington, NSW 2033, Australia; unswcampuscats@yahoo.com
3
School of Optometry and Vision Science, The University of New South Wales, Sydney, NSW 2052, Australia
4
School of Veterinary Science, The University of Queensland, Gatton, QLD 4343, Australia; j.rand@uq.edu.au
*
Correspondence: h.swarbrick@unsw.edu.au; Tel.: +61-427-992-048 Helen Swarbrick 1,2,3,* ID and Jacquie Rand 1,4
1
Australian Pet Welfare Foundation, Kenmore, QLD 4069, Australia; jacquie@petwelfare.org.au
2
Campus Cats NSW, Kensington, NSW 2033, Australia; unswcampuscats@yahoo.com
3
School of Optometry and Vision Science, The University of New South Wales, Sydney, NSW 2052, Australia
4
School of Veterinary Science, The University of Queensland, Gatton, QLD 4343, Australia; j.rand@uq.edu.au
*
Correspondence: h.swarbrick@unsw.edu.au; Tel.: +61-427-992-048 1. Introduction Under Australian legislation, cat populations are often categorized as domestic (owned) or
non-domestic (feral), with unowned urban cats occupying a grey area between these two categories. Unowned urban cats may be considered as domestic or non-domestic depending on individual state
legislation. If classed as non-domestic they are subject to legislation relating to feral pests, and if
domestic, they are subject to animal welfare legislation including laws relating to animal abandonment. Unowned urban cats are found in every urban and peri-urban area in Australia. They live close to
or around human structures such as houses, factories, schools and shopping strips [1], and survive
primarily through casual or regular feeding by the local community, by scavenging human rubbish
and by catching prey, mainly introduced mice and rats. Such cats often congregate in colonies and thus
are sometimes termed “colony cats”. Other terms often used to refer to unowned urban cats include
“free-living”, “free-roaming”, “stray”, “street” or “community” cats. Although they are of the same species (Felis catus), unowned urban cats are usually considered to
be distinct from truly feral cats, which do not depend on humans for shelter or food, and are usually
found in rural or forested areas some distance from human habitation [2,3]. The primary food source
for feral cats is through predation, and thus they may impact significantly on Australian native wildlife. Management of feral cats is a significant challenge [2] and is beyond the scope of this paper. Unowned urban cats largely comprise semi-owned or unowned domestic cats that may receive
some care from persons who do not perceive themselves as owning the cat. In an Australian internet
survey, 9% of respondents fed a cat daily they did not perceive that they owned [4], and 22% of
households provided occasional care to an unowned cat [5]. Some apparently unowned urban cats
are lost or wandering owned cats. The distinctions between unowned and owned domestic cats can
become blurred and cats may move across categories in certain circumstances [4,6,7]. For example,
an owned domestic cat that is lost or abandoned may join a colony of unowned urban cats. Similarly,
unowned urban cats may be adopted to become an owned domestic cat. To add to the complexity, some
semi-owned cats are desexed and microchipped and some owned domestic cats are not [4]. Unowned
and semi-owned domestic cats constitute a large proportion of animal shelter admissions [8,9]. Received: 16 April 2018; Accepted: 11 May 2018; Published: 17 May 2018 Overall, reasons for reductions in the cat population (original residents, immigrants,
campus-born kittens; n = 122) included rehoming or return to owner (30%), death/euthanasia
(30%) and disappearance (29%). This successful animal management program received some initial
funding from the university to support desexing, but was subsequently funded through donations,
and continues with the university’s approval and support. Keywords: unowned urban cat; cat management; trap-neuter-return; TNR www.mdpi.com/journal/animals www.mdpi.com/journal/animals Animals 2018, 8, 77; doi:10.3390/ani8050077 2 of 23 Animals 2018, 8, 77 1. Introduction The approach to management of unowned urban cats varies considerably around Australia
depending on state laws, council by-laws and practices, and local community attitudes. Perceived
problems with unowned urban cat populations include uncontrolled breeding and population growth,
nuisance behaviors such as fighting, spraying and roaming, the potential for cats to harbor diseases
such as toxoplasmosis, and the perceived threat to native wildlife [3,10–15]. On the other hand, many
individuals and community groups, either openly or covertly, support and feed colonies of unowned
cats, often combining this with rescue and adoption of young cats and kittens, and management
of reproduction through desexing [1,3,6,16]. These people are motivated by compassionate reasons,
including a desire to stop cats breeding and to make their life better and safer, positive perceptions of
themselves and from others for feeding a stray cat, and also the role of free-roaming cats in rodent
control [1,4,6]. The principal official approach taken to manage unwanted urban cat populations is to trap and
kill (cull) those colonies that give rise to persistent complaints from the public. This approach to
cat population management has not been demonstrated to be effective in reducing cat numbers in
the medium to long term [17,18], mainly because the number of cats that can reasonably be culled
is an insufficient proportion of the population to exceed the replacement rate through breeding and
immigration [10,19,20]. In particular, culling creates a “vacuum” effect that encourages immigration of
other cats from surrounding areas [2,12–14]. This management strategy also places major stresses on
shelters and municipal pounds (local government animal facilities) and veterinary clinics that must
cope with euthanasia of otherwise healthy cats and kittens, not only in terms of resources, but also
in emotional and ethical costs for staff involved, including post-traumatic stress which can lead to
depression, substance abuse, high blood pressure and, tragically, suicide [21–24]. There is currently
significant interest in Australia, and elsewhere, in reducing this burden on pounds and shelters, such as 3 of 23 Animals 2018, 8, 77 demonstrated by the activities of the G2Z organization (www.g2z.org.au), and the Australian Pet
Welfare Foundation (www.petwelfare.org.au). demonstrated by the activities of the G2Z organization (www.g2z.org.au), and the Australian Pet
Welfare Foundation (www.petwelfare.org.au). An alternative management strategy, which is being used increasingly in other countries such as
the USA and in Europe, is trap-neuter-return (TNR) [7,12–14,16–18]. 1. Introduction This strategy involves trapping
unowned cats, desexing them, and returning them to the location where they were trapped to resume
their life with their colony. This management strategy frequently also involves removing young kittens
and socialized adult cats for rehoming, contributing to a rapid reduction in colony size, and this is
considered best practice [1]. In TNR programs, associated animal management at the time of desexing
typically includes vaccination and parasite treatment, treatment for minor health issues, and in some
cases microchipping, or ear-tipping or tattooing for later visual identification. Rarely, a cat cannot be returned to the location where it was trapped because of safety,
environmental or practical reasons—in this situation, an alternative location must be found for
release [25]. Cats considered too unhealthy to be returned to their home location are humanely
euthanized, typically fewer than 1% of cats [26]. A number of published studies, including from the USA, UK and Italy, have demonstrated that
TNR in a targeted area can be successful in reducing a cat population over time, particularly when
augmented with rehoming of kittens and socialized adults. For example, Levy and co-workers [7]
reported a reduction in free-roaming cat numbers on a Florida university campus from 155 to 23 over
a 11-year period under a TNR program. Natoli and colleagues described a reduction in free-roaming
cat numbers in Rome after a period of TNR management [16]. In a recent paper, Spehar and Wolf [17]
described a TNR program conducted in Massachusetts, which reduced free-roaming cat numbers from
approximately 300 to zero over a 17-year period. In a separate paper, Spehar and Wolf [18] also detailed
a reduction in cat numbers in an area of Chicago managed over a 10-year period by a neighborhood
TNR program. Earlier studies by Neville and Remfry [12], Neville [13] and Zaunbrecher and Smith [14]
also detailed reduction or stabilization of small cat colonies using TNR. Recently, a survey of TNR
practices in Australia reported that this management approach resulted in a median reduction in colony
numbers from 12 to 7 cats over a median period of two years [1]. Although this study was uncontrolled
and relied on self-reports of people engaged in TNR, it does suggest that this management approach
may be successful in an Australian context for reducing urban stray cat numbers. Campus Background The University of New South Wales (UNSW) campus at Kensington is an urban university campus
of approximately 38 hectares in the eastern suburbs of Sydney, which was established in 1949 on the
site of previous racing stables and racecourse. Its immediate environs include a major racecourse
and stables to the north (Royal Randwick racecourse), a suburban shopping strip to the south
(Kingsford shops), a large hospital complex to the east (Prince of Wales hospital), and surrounding
suburban residential areas (Kensington, Randwick and Kingsford). The campus currently has a human
population of approximately 53,000 students and 6000 academic and support staff. The campus has probably always carried a resident unowned cat population from its days as
a horse racing property, when cats may well have been encouraged to control vermin. Since its
establishment as a university, the cats on campus have been sporadically fed by students, staff and
community volunteers, and occasional attempts were made to manage the increasing population
through trapping and desexing. However, there was minimal involvement of university management
in these activities, and no financial support. Consequently, attempts to limit and manage the university
cat population had little success, and the population was largely uncontrolled. In 2000, an informal organization called the Campus Cat Coalition (CCC) was formed to apply
TNR principles for management of the cat population, with regular cat feeding by university staff. There was some consultation with university management but no formal support. Indeed, strong
opposition to the presence of any cats on the campus was voiced in some parts of the university,
mainly in relation to potential impacts on campus wildlife and the threat of disease to campus users. Because of a lack of university support or funding, the CCC program began to falter. In January 2008, some kittens were found dead in the basement of a university building,
concomitant with a flea infestation in that building which adversely affected the health of some
university staff. A decision was made by university management that the presence of cats on the
campus constituted a work health and safety (WHS) issue, and a cat trapping and eradication program
was instigated by the university. A pest management company was contracted to remove the cats. In February 2008, four campus
cats were trapped over a five-day period. 1. Introduction In studies involving larger populations rather than well-characterized colonies, it is challenging
to enumerate the cat population accurately to document effectiveness of management approaches over
time, although methods are available to monitor change in cat density in a subset of the population [27]. Changes in rates of shelter impoundment are therefore often used as a surrogate for reductions in the
overall unowned cat population. Thus, when TNR was applied rigorously in a Florida zip code, shelter
impoundment of unowned cats decreased by 66% over a 2-year period, representing a decrease in
admissions from 13 to 4 cats per 1000 residents, compared to a 12% reduction in non-target areas [26]. On the other hand, there have been reports of TNR programs where cat numbers have not reduced
or have even increased [28–30]. In these reports, the reasons for failure to reduce overall cat numbers
included not desexing a significant proportion of the female cats [28,29], immigration of cats into the
colony from surrounding areas and failure to manage these adults and resulting kittens by desexing or
adoption [29], and dumping by humans of unwanted animals [30]. These effects, which counteract the
impact of TNR in managing populations, have also been noted as a factor reducing the effectiveness of
population control even in reports of successful TNR programs [14,16,17]. In any non-lethal cat management program, it must therefore be recognized that the TNR process
acts to reduce or limit the growth rate of the colony through reproduction, but may not in itself
reduce cat numbers. Any population reduction occurs primarily through adoption of socialized
cats and kittens, natural death or euthanasia of sick animals, and disappearance or emigration of
cats [6,7,12–14,16–18,31]. TNR works together with these factors to reduce reproduction and thus to
minimize replacement of animals lost from the colony. Nevertheless, other factors such as immigration
of cats from surrounding areas can counteract this effect. Thus, the impact of TNR interventions on 4 of 23 Animals 2018, 8, 77 unowned urban cat populations can be complex, and ongoing management of colonies becomes an
important component in optimizing reductions in the cat population [6,7,12,28]. The purpose of this paper is to report outcomes of a carefully-managed and rigorously-applied
TNR program conducted over nine years on an Australian university campus. 1. Introduction This study provides
strong evidence for the effectiveness of targeted TNR, supplemented by rehoming and management of
immigrant cats, to humanely reduce the numbers of unowned urban cats in an Australian context. 2.1. Data Collection and Management From the beginning of the TNR program in August 2008, an ongoing careful audit of campus
cats was conducted on the basis of daily observations of cats seen at feeding sites and other campus
locations, by members of the CCC and other interested members of university staff and students. Cats were given names for easier identification, and photographs and descriptions were recorded and
cross-referenced amongst observers. Later in the program, outdoor battery-operated motion-activated
infrared trail cameras were used to document and identify campus cats. A database of cats observed on
campus was established, including names, approximate ages, descriptions of markings and behavioral
characteristics, sex and desexed status, vaccination status and microchip numbers. This database was
updated on a regular and ongoing basis. Input from members of the CCC who had cared for the
campus cats prior to initiation of the TNR program was also incorporated. Over the course of the
nine-year program, the CCC team was able to estimate the campus cat population on an ongoing basis
with a high level of confidence. For this study, details of the cats on campus commencing in August 2008 were retrieved from
database records retained by the CCC, email correspondence, annual reports to the university
management, and reports to Annual General Meetings of Campus Cats NSW, the registered animal
welfare charity overseeing the campus cat management program. Extensive cross-referencing was
employed to maximize data accuracy and to resolve inconsistencies in cat statistics where possible. Data were summarized at three-monthly and annual intervals commencing in September 2008 until
September 2017. Data were stratified in terms of sex of cats and desexed status, where possible. Records had not
been kept on whether campus cats were entire or desexed before the start of the program. Much of
this information was determined from anecdotal evidence, based on the memories of those who had
been caring for the cats previously. In other cases, the status of campus cats was determined over time
based on observations of physical characteristics and behavior (spraying, fighting, testicles for male
cats; pregnancies for female cats), or when cats were trapped for desexing. Data for ages of cats were not readily available because of the absence of records prior to the start
of the TNR program. Campus Background Discussions with university management resulted in
approval, in August 2008, for a one-year trial of TNR with the following defined goals and targets: •
Immediate and ongoing audit of cat numbers on campus •
Stabilize current cat numbers through a TNR program, prioritizing desexing of females,
then males; 95% desexing target by the end of 2009 •
Stabilize current cat numbers through a TNR program, prioritizing desexing of females,
then males; 95% desexing target by the end of 2009 •
Reduce cat numbers over time through rehoming, humane euthanasia of sick animals, and natural
attrition; target 20% reduction by the end of 2009, eventual target 50% reduction in five years 5 of 23 Animals 2018, 8, 77 •
Maintain the health of the campus cat population by regular feeding, routine vaccination and
parasite management, and veterinary care where necessary •
Maintain the health of the campus cat population by regular feeding, routine vaccination and
parasite management, and veterinary care where necessary •
Maintain the health of the campus cat population by regular feeding, routine vaccination and
parasite management, and veterinary care where necessary •
Educate and inform the university community about the cat management program •
Educate and inform the university community about the cat management program
•
Facilitate ongoing oversight of the campus cat program through regular meetings with university •
Educate and inform the university community about the cat management program
•
Facilitate ongoing oversight of the campus cat program through regular meetings with university
management, and effective rapid response to emerging problems •
Facilitate ongoing oversight of the campus cat program through regular meetings with
t
d ff
ti
id
t
i
bl g
g
g
p
p
g
g
g
g
y
management, and effective rapid response to emerging problems management, and effective rapid response to emerging problems •
Comprehensive review of program outcomes and progress towards goals at the end o Comprehensive review of program outcomes and progress towards goals at the end of 2009. Although some funding (approximately $9000 AUD) was provided by the university to support
initial desexing and microchipping, the program was intended subsequently to become financially
sustainable through donations and fundraising by the CCC. The TNR program achieved its initial goals set with the university by the end of December 2009,
and has continued with the approval of university management over subsequent years. Campus Background This paper
reports on the outcomes of the program over the nine-year period from its establishment at the end of
August 2008 when a formal agreement was signed with university management, up until the most
recent audit of cats at the end of September 2017. Campus Background One cat was euthanized, one kitten was rehomed from
the local council pound (local government animal control facility), and two cats were released from
the council pound to CCC members for rehoming. In April 2008, over a ten-day period a further
12 cats were trapped and all were euthanized. Because it was subsequently established that some of
the euthanized cats were already microchipped and desexed, and also as a result of opposition from
some university staff and others to this culling operation, trapping ceased late in April 2008. In June 2008, the CCC made a formal approach to the peak university WHS committee, proposing
an alternative, more effective and humane approach to management of the campus cat population
based on rigorous application of TNR principles. 2.2. Desexing, Feeding, Monitoring and Health Management Cats undergoing TNR were desexed at two local private veterinary clinics. At the time of desexing,
all cats were vaccinated, treated for parasites, and received minor health care if needed. Identification
was by microchipping and registration on a state database through the local government agency,
as mandated by state law. Cats were registered as owned by Campus Cats NSW, and one of the
office-bearers of the organization was named as primary carer. Daily feeding of all cats on campus was achieved through the establishment of a cat feeding roster
that involved volunteer students and staff of the university and some local community volunteers
associated with animal welfare agencies. Cats were routinely fed once a day at around dusk at a
number of locations around the campus, discretely hidden from passersby in gardens or behind hedges,
and behind or under buildings or external staircases. Cats fed at these sites ranged from approximately
one to eight cats per site. Both wet canned food and dry kibble were provided, and some feeders
occasionally included raw meat or raw chicken wings as a supplement. Fresh water was also always
available at all feeding sites and replenished daily. Some feeding stations incorporated small feeding
shelters to protect food from the elements and scavenging birds. As well as helping to maintain the health of the cats, the regular feeding routine provided an
opportunity for volunteers to check cats for signs of ill health, identify new (immigrant) cats that
appeared on campus, and alert CCC management if any cats were not seen for a few days so that
searches for missing cats could be instigated. Further monitoring of cats was achieved later in the
program through the occasional use of motion-detection trail cameras placed at feeding stations,
and these were particularly useful to locate apparently missing cats that had relocated to different
sites, and to monitor the return of cats to their usual feeding sites after TNR or veterinary treatment. Where indicated, veterinary care was provided for cats. In some cases, this meant that cats needed
to be trapped for transport to the veterinary practice. Because of the time required to trap sometimes
trap-shy individuals, and because of the stress this could cause these cats, the initiation of veterinary
care was only considered once other options, such as on-site provision of medication under veterinary
supervision, had been attempted. 2.1. Data Collection and Management Approximate ages of cats were recorded on the CCC database based on estimates
from veterinarians at the time of desexing after the program had commenced, whereas the ages of cats
that were already desexed were based on anecdote and the memories of those who had participated in
campus cat care prior to August 2008. The university campus is not an isolated tract of land or an island, and it was inevitable that cats
from the surrounding areas immigrated on to the campus during the course of the nine-year TNR 6 of 23 Animals 2018, 8, 77 program. Immigrant cats were typically detected by cat feeders when they appeared at cat feeding
sites, and this was confirmed where possible by the use of trail cameras. In some cases, particularly
when young kittens were found, these were reported to the CCC directly by members of the university
staff who had noticed their presence. Data on immigrant cats that were discovered on campus during the program were gathered
with as much pertinent information as possible about age, sex and desexed status. These immigrant
cats were categorized as either unsocialized (based on behavioral characteristics such as extreme
wariness, avoiding interaction with humans, hissing and aggressive vocalizations when approached)
or socialized (approachable and able to be handled). Solitary kittens that were discovered on campus
were placed in this latter category, whereas kittens born on campus were recorded separately. Unsocialized immigrant cats were absorbed permanently into the campus cat population (and
were targeted for desexing), whereas socialized immigrant cats and kittens, and kittens born on
campus, typically remained only temporarily on campus before transfer to other agencies for rehoming. Unsocialized (permanent) immigrants, socialized (temporary) immigrants, and kittens born on campus,
were reviewed separately in this paper. Because unsocialized cats were absorbed into the campus cat
population, global summaries of resident cat numbers over time included these cats as part of the
population under ongoing management on the campus. 2.2. Desexing, Feeding, Monitoring and Health Management 3.1. Audit of Campus Cat Numbers over Time
3.1. Audit of Campus Cat Numbers over Time At the start of the program in August 2008, the number of cats on campus was uncertain and
uncontrolled breeding made this a moving target. Records had not been kept previously on cat
numbers, sex, description, age, or whether cats had been desexed or microchipped. The initial CCC
estimate was between 75 and 90 campus cats at the time of preliminary negotiations with university
management, but this was subsequently modified as numbers became clearer through an intensive
ongoing audit of cats around the university campus by CCC members and other interested campus
users. In particular, cats were observed and counted during daily feedings, which were conducted at
a regular time of day to encourage cats to come to the feeding stations. On the basis of these careful
observations, a total of 74 cats was thus recorded in August 2008. At the start of the program in August 2008, the number of cats on campus was uncertain and
uncontrolled breeding made this a moving target. Records had not been kept previously on cat
numbers, sex, description, age, or whether cats had been desexed or microchipped. The initial CCC
estimate was between 75 and 90 campus cats at the time of preliminary negotiations with university
management, but this was subsequently modified as numbers became clearer through an intensive
ongoing audit of cats around the university campus by CCC members and other interested campus
users. In particular, cats were observed and counted during daily feedings, which were conducted at
a regular time of day to encourage cats to come to the feeding stations. On the basis of these careful
observations, a total of 74 cats was thus recorded in August 2008. O
th
CCC b
f
ili
ith
d
fid
t
b
t th
t
l ti Once the CCC became more familiar with, and confident about, the campus cat population
through the careful ongoing audit process, it became apparent that five cats (5/74; 7% of resident
population) either had been double-counted (n = 3) or had been local wandering domestic cats (n = 2)
at the time of the initial August 2008 population audit. The “overcount” reflects early uncertainty
about cat numbers, and similar issues are likely to occur early in any TNR program. 3.1. Audit of Campus Cat Numbers over Time
3.1. Audit of Campus Cat Numbers over Time As a result,
the overall baseline population count was adjusted to 69 cats in CCC records at December 2008, and for
all summaries in this paper a baseline population of 69 cats was used. Once the CCC became more familiar with, and confident about, the campus cat population
through the careful ongoing audit process, it became apparent that five cats (5/74; 7% of resident
population) either had been double-counted (n = 3) or had been local wandering domestic cats (n = 2)
at the time of the initial August 2008 population audit. The ‘overcount’ reflects early uncertainty
about cat numbers, and similar issues are likely to occur early in any TNR program. As a result, the
overall baseline population count was adjusted to 69 cats in CCC records at December 2008, and for
all summaries in this paper a baseline population of 69 cats was used. I
dditi
t
th
i i
l 69
t
t
t th b
i
i
f th TNR In addition to the original 69 cats present on campus at the beginning of the TNR program, a total
of 34 immigrant cats or solitary kittens were identified over the nine-year program, and 19 kittens
were born on campus to resident or immigrant cats. In addition to the original 69 cats present on campus at the beginning of the TNR program, a
total of 34 immigrant cats or solitary kittens were identified over the nine-year program, and 19
kittens were born on campus to resident or immigrant cats. 2.2. Desexing, Feeding, Monitoring and Health Management In more recent years, as cats became more socialized to their feeders,
it became increasingly possible to place certain cats in carry cages for transport to the veterinary
practice and, in some cases, for full veterinary examination to be achieved in unsedated campus cats. Usually, however, sedation was required to allow full examination. 7 of 23
7 of 23 Animals 2018, 8, 77
Animals 2018, 8, x 3.1.1. Unsocialized Immigrant Cats
3.1.1. Unsocialized Immigrant Cats Of the 34 immigrant cats, 16 cats (47%) were categorized as unsocialized based on their behavior
(wariness, fear of humans, aggression when approached) and were deemed unsuitable for rehoming. These cats were consequently managed through TNR and absorbed into the campus cat resident
population. Thus, the total resident cat population managed on the campus during the nine-year TNR
program was 85 cats (69 originals + 16 unsocialized immigrants) (Figure 1 and Appendix A). Of the 34 immigrant cats, 16 cats (47%) were categorized as unsocialized based on their behavior
(wariness, fear of humans, aggression when approached) and were deemed unsuitable for rehoming. These cats were consequently managed through TNR and absorbed into the campus cat resident
population. Thus, the total resident cat population managed on the campus during the nine-year TNR
program was 85 cats (69 originals + 16 unsocialized immigrants) (Figure 1 and Appendix A). 0
5
10
15
20
25
30
35
Aug-08 Sep-08 Sep-09 Sep-10 Sep-11 Sep-12 Sep-13 Sep-14 Sep-15 Sep-16 Sep-17
Cat numbers
Year
Male
Female
Unsure Figure 1. Resident campus male and female cats (including 69 original cats and 16 unsocialized
immigrants) at commencement of the program (August 2008) and at annual intervals from end of
September 2008 to end of September 2017. 0
5
10
15
20
25
30
35
Aug-08 Sep-08 Sep-09 Sep-10 Sep-11 Sep-12 Sep-13 Sep-14 Sep-15 Sep-16 Sep-17
Cat numbers
Year
Male
Female
Unsure
Figure 1. Resident campus male and female cats (including 69 original cats and 16 unsocialized
immigrants) at commencement of the program (August 2008) and at annual intervals from end of
September 2008 to end of September 2017. Figure 1. Resident campus male and female cats (including 69 original cats and 16 unsocialized
immigrants) at commencement of the program (August 2008) and at annual intervals from end of
September 2008 to end of September 2017. Figure 1. Resident campus male and female cats (including 69 original cats and 16 unsocialized
immigrants) at commencement of the program (August 2008) and at annual intervals from end of
September 2008 to end of September 2017. 8 of 23 Animals 2018, 8, 77 3.1.2. Socialized Immigrant Cats and Kittens 3.1.3. Kittens Born on Campus A total of 19 kittens were born on campus during the nine-year program. Five of these kittens
were born to two resident cats—the last resident kitten was born in December 2009. Both resident
mothers were trapped and desexed. In addition, a total of 14 kittens were born to three newly arrived immigrant female cats. A litter of six kittens was discovered in March 2009 with a friendly adult female. In December 2014,
an unsocialized immigrant female cat (seen occasionally over the previous two months) had a litter
of four kittens, and again in January 2017 an unsocialized immigrant female cat (noted sporadically
over the previous 2–3 months) was found with a litter of four kittens. These 14 kittens were fostered
and rehomed. The socialized mother was rehomed, and both unsocialized mothers were desexed and
returned to campus. 3.1.2. Socialized Immigrant Cats and Kittens In addition to 16 unsocialized immigrant cats absorbed permanently into the campus cat
population, a further 18 socialized immigrant adult cats or solitary kittens were present temporarily on
the campus for short periods, usually between one day and two weeks (mode 1–3 days), before their
permanent removal, typically for fostering and adoption. Socialized immigrant adult cats (n = 12) were thought to either be lost or wandering owned or
semi-owned cats, or have been abandoned by students or nearby residents, and were identified as
socialized by their approachability and friendliness to humans. Six immigrant kittens simply appeared on campus, including two kittens found in the engine bay
of a student’s car parked on the campus, three kittens found wandering by themselves on campus,
and one injured kitten brought on to campus by a student. Solitary kittens were typically fostered and
rehomed as soon as possible after their discovery. 3.2. Desexing of Campus Cats At the beginning of the program (August 2008), 10 (14%) of the 69 resident cats on campus were
of undetermined sex (Figure 1 and Appendix A). Sex and desexed status were subsequently clarified at
the time of TNR, except for one original resident that disappeared before its status could be confirmed. Based on current knowledge of the sex and desexed status of resident cats at the start of the program,
there were 33 (49%) males and 35 (51%) females, of which 48% of males (16/33) and 26% of females
(9/35) were entire (Figure 2). Socialized immigrant cats and kittens were not included in this summary of sexual status of
campus cats, because these cats and kittens were rapidly removed from the campus and thus did not
form part of the permanent resident population, and also because the majority of the kittens were
of uncertain or unrecorded sex. Cats whose status remained unknown (n = 4 through the nine-year
program), primarily because they disappeared before they could be trapped, were also not included. Immediately prior to, and on commencing the agreed management program in August 2008,
the CCC targeted fertile females for desexing (Figure 2A). By December 2009, all but one of the
26 female cats remaining on campus had been desexed—this last fertile female proved elusive but was
finally trapped and desexed in December 2010. Since then, all resident female cats on campus were
desexed, and no kittens have been born to resident cats since December 2009. Male cats were also trapped and desexed from the beginning of the program, although this
had a lower priority than female cats. By December 2009, 16 of the remaining 22 male cats (73%)
were desexed (Figure 2B). At the most recent audit, all of the remaining male cats on campus were
desexed, although the status of one long-term immigrant visitor (assumed male and desexed) has not
been confirmed. Unsocialized immigrant cats that were absorbed into the program after 2008 were immediately
targeted for TNR and 13 of these 16 cats were successfully desexed (four females, nine males). One male 9 of 23 Animals 2018, 8, 77 cat arrived on campus with an ear tip, and was assumed to have been previously desexed. Another
undesexed male disappeared before he could be trapped for desexing, and one unsocialized immigrant
visitor remains of unconfirmed status. Animals 2018, 8, x
9 of 23 Figure 2. 3.2. Desexing of Campus Cats Percentage of resident cats (including 69 originals and 16 unsocialized immigrants) desexed
versus entire (undesexed) at annual intervals during the 9-year program—(A) female; (B) male. Cats
of unknown status (n = 4) were not included
0
10
20
30
40
50
60
70
80
90
100
Aug-08 Sep-08 Sep-09 Sep-10 Sep-11 Sep-12 Sep-13 Sep-14 Sep-15 Sep-16 Sep-17
Percentage of cats
Female desexed
Female entire
A
0
10
20
30
40
50
60
70
80
90
100
Aug-08 Sep-08 Sep-09 Sep-10 Sep-11 Sep-12 Sep-13 Sep-14 Sep-15 Sep-16 Sep-17
Percentage of cats
Male desexed
Male entire
B
Figure 2. Percentage of resident cats (including 69 originals and 16 unsocialized immigrants) desexed
versus entire (undesexed) at annual intervals during the nine-year program—(A) female; (B) male. Cats of unknown status (n = 4) were not included. 0
10
20
30
40
50
60
70
80
90
100
Aug-08 Sep-08 Sep-09 Sep-10 Sep-11 Sep-12 Sep-13 Sep-14 Sep-15 Sep-16 Sep-17
Percentage of cats
Female desexed
Female entire
A Female desexed
Female entire
0
10
20
30
40
50
60
70
80
90
100
Aug-08 Sep-08 Sep-09 Sep-10 Sep-11 Sep-12 Sep-13 Sep-14 Sep-15 Sep-16 Sep-17
Percentage of cats
Male desexed
Male entire
B Figure 2. Percentage of resident cats (including 69 originals and 16 unsocialized immigrants) desexed
versus entire (undesexed) at annual intervals during the 9-year program—(A) female; (B) male. Cats
Figure 2. Percentage of resident cats (including 69 originals and 16 unsocialized immigrants) desexed
versus entire (undesexed) at annual intervals during the nine-year program—(A) female; (B) male. Cats of unknown status (n = 4) were not included. In terms of desexing, socialized adult immigrant cats (n = 12) were dealt with on a case-by-case
basis. These cats were initially scanned for a microchip, and this resulted in the return of three cats
(25%) to their owners. The owners of two other microchipped cats were not able to be contacted, or
did not want the cat returned. Cats were then checked by a veterinary surgeon for general health,
and where indicated were desexed (n = 2). All cats received vaccinations and parasite treatment,
before placement in foster care prior to rehoming. All adult socialized cats managed by this route (n
= 7) were successfully placed in adoptive homes. 3.2. Desexing of Campus Cats Two of the 12 socialized adult immigrant cats
disappeared before their status could be verified, and may in fact have been wandering local domestic
d
h
d
h i
b h
In terms of desexing, socialized adult immigrant cats (n = 12) were dealt with on a case-by-case
basis. These cats were initially scanned for a microchip, and this resulted in the return of three cats
(25%) to their owners. The owners of two other microchipped cats were not able to be contacted,
or did not want the cat returned. Cats were then checked by a veterinary surgeon for general health,
and where indicated were desexed (n = 2). All cats received vaccinations and parasite treatment,
before placement in foster care prior to rehoming. All adult socialized cats managed by this route
(n = 7) were successfully placed in adoptive homes. Two of the 12 socialized adult immigrant cats
disappeared before their status could be verified, and may in fact have been wandering local domestic
owned cats that returned to their nearby homes. 10 of 23 10 of 23 Animals 2018, 8, 77 3.3. Health Management Health of cats was monitored by the cat feeders, facilitated by daily feeding and, where necessary,
motion-detection trail cameras at feeding stations. The most common reasons for veterinary treatment
among the campus cat population were eye infections, minor injuries, abscesses, skin conditions
and dental disease. Various medications in oral form were successfully administered in food
usually as crushed tablets, including antibiotics, steroids and other treatments such as medication
for hyperthyroidism. 3.4. Colony Size over Time By December 2009, when the program was due for initial review, the number of cats on campus
had reduced from 69 to 51 cats, which represented a 26% (n = 18/69) reduction, meeting the 20%
target agreed with the university. At the 5-year time point (September 2013), there were 30 cats
remaining on campus, representing a 57% (n = 39/69) reduction, again meeting the agreed 50% target. At the time of preparing this paper (September 2017), 15 cats remain on campus, representing a 78%
(n = 54/69) reduction in the resident cat population since the TNR program was initiated in August
2008. Case studies of some of the campus cats are included in Appendix B. Nine of the remaining cats at September 2017 were present on campus at the start of the program
in August 2008, and six were cats that immigrated on to the campus during the program. Four of
these latter cats were younger than those that remained from the original cohort, most of which were
now quite elderly (estimated age of original resident cats >10 years). However, two of the remaining
six unsocialized immigrant cats were estimated to be older than 10 years when they first appeared
on campus. An overall summary of the fates of all cats managed in the program over the period August 2008
to September 2017 is presented in Table 1. An overall summary of the fates of all cats managed in the program over the period August 2008
to September 2017 is presented in Table 1. Table 1. Summary of outcomes for all cats managed in the nine-year program. Total
Rehomed
Returned to Owner
Euthanized
Died
Disappeared
Remaining
RESIDENT CATS
Original cats
69
7
0
20
12
21
9
Unsocialized immigrant adults
16
0
0
0
2
8
6
Total n (%)
85
7 (8%)
0 (0%)
20 (24%)
14 (16%)
29 (34%)
15 (18%)
NON-RESIDENT CATS
Socialized immigrant adults
12
7
3
0
0
2
0
Solitary immigrant kittens
6
5
0
0
1
0
0
Kittens born on campus
19
14
0
1
0
4
0
Total n (%)
37
26 (70%)
3 (8%)
1 (3%)
1 (3%)
6 (16%)
0 (0%)
OVERALL TOTAL CATS
Total n (%)
122
33 (27%)
3 (2%)
21 (17%)
15 (12%)
35 (29%)
15 (12%)
3.5.1. Fates of Resident Cats Table 1. Summary of outcomes for all cats managed in the nine-year program. 3.5.1. Fates of Resident Cats 3.5.1. Fates of Resident Cats Figure 3 summarizes the fates of cats from the resident (original and unsocialized immigrant)
campus cat population during the nine-year TNR program. Because many resident socialized cats on the campus had already been rehomed prior to August
2008 (in some cases to protect them from the university’s culling program), few resident campus cats
were successfully rehomed after the start of the TNR program. Over the nine-year program, seven
cats (7/85; 8% of resident population) were adopted, including two older cats (estimated 15 years old)
that were placed in permanent homes in mid-2017. These latter adoptions became possible because of
socialization of these cats through their regular daily interactions with volunteer feeders. 11 of 23
11 of 23 Animals 2018, 8, 77
Animals 2018 8 x Figure 3. Fates of resident cats (69 original and 16 unsocialized immigrant cats). 0
5
10
15
20
25
30
35
Rehomed
Euthanized
Died
Disappeared
Remaining
Number of cats
Figure 3. Fates of resident cats (69 original and 16 unsocialized immigrant cats). Figure 3. Fates of resident cats (69 original and 16 unsocialized immigrant cats). Figure 3. Fates of resident cats (69 original and 16 unsocialized immigrant cats). A total of 20 cats (20/85; 24% of resident population) were humanely euthanized during the nine-
year program (mean 2.2 resident cats per year). The primary reason for euthanasia was severe ill
health (n = 16/20, 80% of euthanized cats; Table 2). A total of 20 cats (20/85; 24% of resident population) were humanely euthanized during the
nine-year program (mean 2.2 resident cats per year). The primary reason for euthanasia was severe ill
health (n = 16/20, 80% of euthanized cats; Table 2). Table 2. Reasons for euthanasia, categorized by estimated age groups. Table 2. Reasons for euthanasia, categorized by estimated age groups. Table 2. Reasons for euthanasia, categorized by estimated age groups. Age Group < 5 Years *
Age Group 5–10 Years *
Age Group > 10 Years *
Acute renal disease
1
Intestinal sarcoma
2
Renal failure
3
Tumorous cancer on
chest
1
Saddle thrombus
1
Intestinal sarcoma
1
FIV positive **
2
Feline hypertrophic
cardiomyopathy
1
Complications of
hyperthyroidism
1
Excessive aggression
1
Aggressive oral squamous cell
carcinoma
1
Old age (estimated 20
years)—debilitation
1
Undiagnosed illness
3
Other
1
Total
9
Total
5
Total
6
Table 2. Reasons for euthanasia, categorized by estimated age groups. 3.5.1. Fates of Resident Cats Age Group <5 Years *
Age Group 5–10 Years *
Age Group >10 Years *
Hit by car
2
Heart failure **
1
Hit by car
1
Accident (caught in
machinery)
1
Severe chronic respiratory disease **
1
Accident (trapped in
demolished building)
1
Died during surgery
1
FIV+ ***—unknown cause
1
Elderly—unknown cause
2
Multiple organ lesions **
2
Unknown cause
1
Total
7
Total
3
Total
4
* Age at time of death, based on estimates—see text;
** based on necropsy;
*** positive for feline
immunodeficiency virus. which three were hit by cars on roads surrounding the campus. Based on our data on deaths and euthanasia in the resident cat population (n = 34/85), and the
approximate number of cat-years represented in this dataset (n = 419 cat-years), we estimate an
average mortality rate in our campus cat population (including adults and kittens) of approximately
8.1% per annum over the nine-year program. Table 3. Causes of death (other than euthanasia), categorized by estimated age groups. Age Group <5 Years *
Age Group 5–10 Years *
Age Group >10 Years *
Hit by car
2
Heart failure **
1
Hit by car
1
Accident (caught in
machinery)
1
Severe chronic respiratory
disease **
1
Accident (trapped in demolished
building)
1
Table 3. Causes of death (other than euthanasia), categorized by estimated age groups. Age Group <5 Years *
Age Group 5–10 Years *
Age Group >10 Years *
Hit by car
2
Heart failure **
1
Hit by car
1
Accident (caught in
machinery)
1
Severe chronic respiratory disease **
1
Accident (trapped in
demolished building)
1
Died during surgery
1
FIV+ ***—unknown cause
1
Elderly—unknown cause
2
Multiple organ lesions **
2
Unknown cause
1
Total
7
Total
3
Total
4
* Age at time of death, based on estimates—see text;
** based on necropsy;
*** positive for feline
immunodeficiency virus. 3.5.1. Fates of Resident Cats Age Group < 5 Years *
Age Group 5–10 Years *
Age Group > 10 Years *
Acute renal disease
1
Intestinal sarcoma
2
Renal failure
3
Tumorous cancer on chest
1
Saddle thrombus
1
Intestinal sarcoma
1
FIV positive **
2
Feline hypertrophic
cardiomyopathy
1
Complications of
hyperthyroidism
1
Excessive aggression
1
Aggressive oral
squamous cell carcinoma
1
Old age (estimated 20
years)—debilitation
1
Undiagnosed illness
3
Other
1
Total
9
Total
5
Total
6
* Age at time of death, based on estimates—see text; ** positive for feline immunodeficiency virus. * Age at time of death, based on estimates—see text; ** positive for feline immunodeficiency virus. Another 14 cats (14/85; 16% of resident population; mean 1.6 resident cats per year) died or were
found dead during the 9-year program (Table 3). Five of these cats met with accidental deaths, of
Another 14 cats (14/85; 16% of resident population; mean 1.6 resident cats per year) died or were
found dead during the nine-year program (Table 3). Five of these cats met with accidental deaths,
of which three were hit by cars on roads surrounding the campus. ee were hit by cars on roads surrounding the campus. d on our data on deaths and euthanasia in the resident cat population (n = 34/85)
Table 3. Causes of death (other than euthanasia), categorized by estimated age groups. e were hit by cars on roads surrounding the campus. on our data on deaths and euthanasia in the resident cat population (n = 34/85
Table 3. Causes of death (other than euthanasia), categorized by estimated age groups. which three were hit by cars on roads surrounding the campus. Based on our data on deaths and euthanasia in the resident cat population (n = 34/85), and the
approximate number of cat-years represented in this dataset (n = 419 cat-years), we estimate an
average mortality rate in our campus cat population (including adults and kittens) of approximately
8.1% per annum over the nine-year program. Table 3. Causes of death (other than euthanasia), categorized by estimated age groups. Age Group <5 Years *
Age Group 5–10 Years *
Age Group >10 Years *
Hit by car
2
Heart failure **
1
Hit by car
1
Accident (caught in
machinery)
1
Severe chronic respiratory
disease **
1
Accident (trapped in demolished
building)
1
Table 3. Causes of death (other than euthanasia), categorized by estimated age groups. 3.5.1. Fates of Resident Cats 0
5
10
15
20
25
30
Rehomed
Returned to
owner
Euthanized
Died
Disappeared
Number of cats
Figure 4. Fates of socialized immigrant adult cats (n = 12), solitary kittens (n = 6) and kittens born on
campus (n = 19). 3.5.2. Fates of Socialized Immigrant Cats, and Kittens Born on Campus
3.5.2. Fates of Socialized Immigrant Cats, and Kittens Born on Campus 2. Fates of Socialized Immigrant Cats, and Kittens Born on Campus
Fates of Socialized Immigrant Cats, and Kittens Born on Campus Most (70%; 26/37) of the socialized immigrant adult cats (7/12), solitary kittens (5/6), and kittens
born on campus (14/19) were managed rapidly through fostering followed by rehoming (Figure 4). Most (70%; 26/37) of the socialized immigrant adult cats (7/12), solitary kittens (5/6), and kittens
born on campus (14/19) were managed rapidly through fostering followed by rehoming (Figure 4). Figure 4. Fates of socialized immigrant adult cats (n = 12), solitary kittens (n = 6) and kittens born on
campus (n = 19). 0
5
10
15
20
25
30
Rehomed
Returned to
owner
Euthanized
Died
Disappeared
Number of cats
Figure 4. Fates of socialized immigrant adult cats (n = 12), solitary kittens (n = 6) and kittens born on
campus (n = 19). Figure 4. Fates of socialized immigrant adult cats (n = 12), solitary kittens (n = 6) and kittens born on
campus (n = 19). Figure 4. Fates of socialized immigrant adult cats (n = 12), solitary kittens (n = 6) and kittens born on
campus (n = 19). Five of the seven rehomed adults were removed from the campus on the same day they were
discovered, one cat took a week to capture for foster care, and another cat was rehomed after almost
three months on campus. A further three adult domestic cats with microchips were reunited on the
day of their discovery with their owners, all of whom lived within half a kilometer of the university
campus. Two socialized immigrant adult cats were briefly seen on campus on several occasions over
a 3–5 months period, then disappeared. Five of the seven rehomed adults were removed from the campus on the same day they were
discovered, one cat took a week to capture for foster care, and another cat was rehomed after almost
three months on campus. 3.5.1. Fates of Resident Cats A further three adult domestic cats with microchips were reunited on the
day of their discovery with their owners, all of whom lived within half a kilometer of the university
campus. Two socialized immigrant adult cats were briefly seen on campus on several occasions over a
3–5 months period, then disappeared. Of the six solitary immigrant kittens, five were removed and placed in adoptive homes. Three
were removed within three days of their discovery, whereas two took up to two weeks to capture
before removal for rehoming. One kitten died within three days from severe trauma sustained prior
to being brought on to campus by a concerned student. Of the six solitary immigrant kittens, five were removed and placed in adoptive homes. Three
were removed within three days of their discovery, whereas two took up to two weeks to capture
before removal for rehoming. One kitten died within three days from severe trauma sustained prior to
being brought on to campus by a concerned student. Of the 19 kittens born on campus, five kittens were born to resident cats early in the program. Four of these kittens disappeared and are thought to have died, and one kitten was euthanized due
to inoperable hernia. Of the 19 kittens born on campus, five kittens were born to resident cats early in the program. Four of these kittens disappeared and are thought to have died, and one kitten was euthanized due to
inoperable hernia. The other 14 campus-born kittens (all of which were born to immigrant pregnant cats) were
successfully placed in adoptive homes. Where necessary, kittens remained with their mothers on
campus until weaned, and then were separated at about 4–6 weeks of age for socialization in foster
care. Kittens were then transferred to a rescue/adoption agency either prior to or immediately after
early age desexing (at approximately 1 kg in weight), for further socialization and rehoming. The other 14 campus-born kittens (all of which were born to immigrant pregnant cats) were
successfully placed in adoptive homes. Where necessary, kittens remained with their mothers on
campus until weaned, and then were separated at about 4–6 weeks of age for socialization in foster
care. Kittens were then transferred to a rescue/adoption agency either prior to or immediately after
early age desexing (at approximately 1 kg in weight), for further socialization and rehoming. 3.5.1. Fates of Resident Cats g
g
y
y
Multiple organ lesions **
2
Unknown cause
1
Total
7
Total
3
Total
4
* Age at time of death, based on estimates—see text; ** based on necropsy; *** positive for feline
i
d fi i
i
Based on our data on deaths and euthanasia in the resident cat population (n = 34/85), and the
approximate number of cat-years represented in this dataset (n = 419 cat-years), we estimate an average
mortality rate in our campus cat population (including adults and kittens) of approximately 8.1% per
annum over the nine-year program. immunodeficiency virus. A total of 29 cats (29/85; 34% of resident population; mean 3.2 animals per year) simply
y
p
g
A total of 29 cats (29/85; 34% of resident population; mean 3.2 animals per year) simply
disappeared and their fates are unknown. Although some of these cats appeared to be ill or injured 12 of 23
12 of 23 Animals 2018, 8, 77
Animals 2018, 8, x prior to their disappearances (n = 4), most appeared in good health at the time they disappeared and
the reasons for their disappearances remain speculative. prior to their disappearances (n = 4), most appeared in good health at the time they disappeared and
the reasons for their disappearances remain speculative. prior to their disappearances (n = 4), most appeared in good health at the time they disappeared and
the reasons for their disappearances remain speculative. prior to their disappearances (n = 4), most appeared in good health at the time they disappeared and
the reasons for their disappearances remain speculative. p
pp
pp
g
y
pp
the reasons for their disappearances remain speculative. 3.5.2. Fates of Socialized Immigrant Cats, and Kittens Born on Campus
Most (70%; 26/37) of the socialized immigrant adult cats (7/12), solitary kittens (5/6), and kittens
born on campus (14/19) were managed rapidly through fostering followed by rehoming (Figure 4). he reasons for their disappearances remain speculative. 3.5.2. Fates of Socialized Immigrant Cats, and Kittens Born on Campus
Most (70%; 26/37) of the socialized immigrant adult cats (7/12), solitary kittens (5/6), and kittens
born on campus (14/19) were managed rapidly through fostering followed by rehoming (Figure 4). Figure 4. Fates of socialized immigrant adult cats (n = 12), solitary kittens (n = 6) and kittens born on
campus (n = 19). 4. Discussion
4. Discussion This study reports in detail the results obtained over a nine-year cat management program
conducted on the UNSW campus using a TNR protocol to manage cat numbers, combined with
adoption of socialized cats and kittens, natural attrition, and active management of immigrant cats. Over the nine-year period, 122 cats in total were managed as part of the program. These comprised
69 original residents (57%), 16 unsocialized immigrants incorporated into the resident population
(13%), 18 socialized immigrant adults and kittens (15%), and 19 kittens born on campus (16%). Socialized immigrant adults and kittens, and kittens born on campus, were targeted for rapid
removal for rehoming or return to their owners. This left 70% of the total 122 cats (69 original
residents and 16 unsocialized immigrants; total 85 cats) to be actively managed on an ongoing basis
h
This study reports in detail the results obtained over a nine-year cat management program
conducted on the UNSW campus using a TNR protocol to manage cat numbers, combined with
adoption of socialized cats and kittens, natural attrition, and active management of immigrant cats. Over the nine-year period, 122 cats in total were managed as part of the program. These comprised
69 original residents (57%), 16 unsocialized immigrants incorporated into the resident population (13%),
18 socialized immigrant adults and kittens (15%), and 19 kittens born on campus (16%). Socialized
immigrant adults and kittens, and kittens born on campus, were targeted for rapid removal for
rehoming or return to their owners. This left 70% of the total 122 cats (69 original residents and
16 unsocialized immigrants; total 85 cats) to be actively managed on an ongoing basis on the campus. 13 of 23 13 of 23 Animals 2018, 8, 77 The current campus cat population (at September 2017) comprises 15 cats, representing a 78%
reduction in resident cat numbers from the original population of 69 cats in August 2008. Therefore,
only 12% of the 122 cats managed through the course of the nine-year program remain on campus. 4.1.1. Rehoming The rehoming rate of 8% (n = 7) for the resident cat population of 85 in this program (Table 1)
was low compared to previous studies of TNR [7,18,31]. Many adult socialized resident cats had
been adopted into permanent homes prior to initiation of the TNR program, and this early phase of
adoption is not reflected in our numbers. In comparison, Levy et al. reported that 47% of cats in their
TNR program were rehomed [7]. This may reflect the much higher proportion in their population of
kittens and younger cats, which typically form the majority of cats rehomed early in a TNR program,
compared to our older campus population in which most of the resident female cats were already
desexed. However, in our study, 83% of socialized immigrant cats and kittens, and 74% of kittens born
on campus, were successfully rehomed, including three cats returned to their owner. When the overall
rehoming/return-to-owner rate in our program is considered (36/122; 30%), this is comparable to
other TNR programs that have documented rehoming rates from 21% to 32% [18,31] (Table 4). Approximately 8% of all immigrant cats (25% of socialized immigrants) were returned to their
owners who lived within 500 m of the campus. Owner contact details from microchip registration were
used to locate the owners. This wandering behavior of owned cats is consistent with GPS tracking
data for South Australian pet cats, where the average daytime range was 1 hectare, and 2 hectares at
night [32]. It is also consistent with research demonstrating that 75% of lost cats when found were
located within 500 m of their home [33]. 4.1. Reduction in Cat Population over Time For the 122 cats managed during the program, the overall reduction in cat numbers over nine
years resulted from rehoming/return to owner (36; 30%), euthanasia (21; 17%), death (15; 12%),
and unexplained disappearance (35; 29%) (Table 1). Although comparisons of these statistics with other
studies of TNR in similar populations are difficult because of differences in duration and methodology
of study, and ambiguous data recording, an attempt to compare outcome data is presented in Table 4. Table 4. Comparisons of outcome data from similar TNR studies. Table 4. Comparisons of outcome data from similar TNR studies. p
Study (1◦Author, Ref)
This Study
Levy [7]
Spehar [18]
Zaunbrecher [14]
Hughes [31]
Centonze [6] *
Duration (years)
9
11
9
3
2
1.5 **
Original cohort (n)
69
155
195
41
158
920
Immigration (n)
34
NR
NR
6
NR
(103)
Born on site (n)
19
NR
NR
NR
NR
(498)
Total managed (n)
122
155
195
47
158
920
Remaining (n)
15
23
44
36
95
678
Remaining (%)
12
15
23
77
60
74
Rehomed *** (%)
30
47
32
0
21
(26)
Euthanasia (%)
17
11
3
2
15
(0)
Death (%)
12
6
7
11
4
(16)
Disappeared (%)
29
21
34
11
0
(16)
Other (%)
0
0
2
0
0
(0)
NR = not reported. * ambiguous numbers (note that numbers in brackets from the Centonze and Levy paper [6] are
unreliable by authors’ admission). ** median—range from two weeks to 15 years. *** includes “returned to owner”. NR = not reported. * ambiguous numbers (note that numbers in brackets from the Centonze and Levy paper [6] are
unreliable by authors’ admission). ** median—range from two weeks to 15 years. *** includes “returned to owner”. 4.1.2. Euthanasia and Death The overall euthanasia rate amongst the resident cat population was 24% (20/85) over a nine-year
period, or 17% (21/122) for the total cat population (Table 1). These cases were primarily due to severe
and terminal illnesses in the original adult resident cat population (Table 2). There was a higher rate
of euthanasia earlier in the program. Overall, one-third of cases of euthanasia occurred during the 14 of 23 Animals 2018, 8, 77 first 18 months of the program, primarily involving younger cats. Later cases of euthanasia related to
terminal but relatively common cat diseases such as renal disease and neoplasia, involving older cats. The overall euthanasia rate is higher than Levy’s study [7] of 11% over 11 years, but differences
may reflect that Levy’s study population comprised younger cats compared to our older campus
cat population. Our euthanasia rate is also significantly higher than the 3% reported by Spehar
and Wolf [18], but comparable to other reports [31]. This variability in euthanasia rates may reflect
differences in program duration, and methodological and demographic differences between studies. It
could also be argued that the close monitoring of our campus population meant that sick cats were
identified quickly and euthanized following terminal diagnosis, rather than disappearing or being
found dead later due to their illness. Furthermore, in most calculations of overall euthanasia rate,
rehomed cats are assumed to remain alive, further confounding comparisons between studies with
different rehoming rates. Our philosophy relating to euthanasia developed during the course of the program as a result of
early experiences. For example, early in the program, some younger cats were euthanized because
they were found to be FIV-positive, but this practice subsequently ceased as the CCC became more
knowledgeable about this condition. Specifically, the risk of false positive test results, the high cost of
testing, and the low incidence and late occurrence of clinical disease were felt to argue against testing
and euthanasia. Furthermore, advice from other organizations such as Alley Cat Allies [34] suggested
that a better way to limit FIV in the campus population was through desexing of male cats to reduce
fighting and bite wounds, the main route of transmission of this virus. A further 16% of the resident cats, or 12% of the overall cat population in our program, died or
were found dead (Tables 1 and 3). 4.1.2. Euthanasia and Death Reported percentages of cats found dead in other TNR studies
vary from 4% to 16% (Table 4), but differences in study duration and methodology make comparisons
difficult. Rehoming of cats may also confound these estimates as these cats are effectively lost to follow
up and are assumed to remain alive in calculations of overall death rates. The average mortality rate based on deaths and euthanasia in our campus cat population
(including adults and kittens) of approximately 8.1% per annum is consistent with the calculated
death rate for pet cats of 7% per annum, based on the reported average age at death of 14 years [35]. Our rates of euthanasia and death are also comparable with those that have been reported in pet cat
populations; for example, the estimated annual pet cat mortality rate was 8.3% based on a telephone
survey of North American cat owners [36]. The main causes for death or euthanasia in our population, where this could be determined
with reasonable confidence, included trauma, neoplasia and renal disease—the latter two causes were
predominant in cats over five years of age (Tables 2 and 3). Because of the campus-based rather
than veterinary practice-based nature of this program, it is not surprising that some deaths were of
unknown cause or for unspecified reasons. Studies in several countries have reported the most common diseases leading to death or
euthanasia in pet cats. Cause of death/euthanasia in cats younger than five years are predominantly
due to trauma or viral and respiratory disease, whereas death/euthanasia in older cats, such as
our cohort, are mainly related to renal disease, neoplasia, cardiovascular disorders, or non-specific
illness [37,38]. Thus, our findings of rate and causes of cat mortality in our unowned urban cat
population managed with TNR differ little from those commonly cited in relation to owned cat
populations. This is consistent with previous findings that TNR programs may be associated with
comparable or even increased longevity compared with owned cats [39], and that unowned urban cats
in TNR programs show similar levels of health and profiles of disease as owned pet cats [40,41]. 4.1.4. Summary of Reasons for Reduction in Cat Population 4.1.4. Summary of Reasons for Reduction in Cat Population In summary, in common with other studies of TNR in targeted populations, the main reasons
for reductions in the campus cat population in our program were rehoming, death, euthanasia and
disappearance. Although concerns relating to the welfare of released cats are often cited by opponents
of TNR programs, the death, euthanasia and disappearance rates in our campus cat population were
similar to those reported for pet cats. Active identification and management of immigrant cats also played an essential role in control of
the campus population. Immigrant cats (n = 34) represented a potential increase of 50% on the original
resident population of 69 cats during the nine-year program and, if not appropriately managed, could
have contributed significantly to population growth by approximately 4% per year. The birth of kittens
to immigrant cats further increased the potential for population growth. At the same time, it must
be acknowledged that our program reduced overall cat numbers (see Figure 1 and Appendix A),
potentially creating the circumstances in which a “vacuum effect” may have encouraged the influx of
immigrant cats. We cannot know what would have happened had the resident population remained
stable and resources to support the cat population had remained constant. Nevertheless, failure of
some TNR programs to control cat populations because of uncontrolled immigration of sexually intact
animals from surrounding areas, and the dumping of unwanted cats or kittens, has been reported
by others [29,30]. Furthermore, our program did not have the capacity to address management of
the source of immigrant cats through community desexing programs for owned cats and promotion
of responsible pet ownership in the areas surrounding the campus. The potential impact of such
strategies on immigration of owned cats on to the campus is thus unclear. Nevertheless, a critical factor responsible for the gradual reduction in resident cat numbers during
the nine-year program was the rigorous application of desexing to limit cat reproduction, which
otherwise may have maintained or even increased the cat population. Cats have a large reproductive
potential. A single queen may produce an average of ten kittens per year, and female kittens can
become pregnant at four months of age [43,44]. Failure to desex all (or nearly all) female cats in a colony
is one of the reasons that some TNR programs have failed to reduce cat numbers [28,29]. 4.1.3. Disappearance The most common cause for loss of cats in our study population of 122 cats was unexplained
disappearance, accounting for 29% of cats and representing a mean of 3.9 disappearances per year
across the overall population (Table 1). Reported disappearance rates from similar TNR studies 15 of 23 Animals 2018, 8, 77 range from 11% to 34% (Table 4), but as discussed previously, these comparisons are confounded by
differences in study duration and methodology [6,7,14,18,31]. Furthermore, these statistics assume
that adopted cats do not disappear after rehoming. However, 15% of pet cat owners lose their cat at
least once in a 5-year period, and 30% of pet cats go missing at least once in their lifetime [42]. The fates of cats in our program that disappeared are unknown. Likely reasons include relocation
(emigration), adoption, or death (although the majority were apparently healthy at the time of
disappearance). Because some cats were becoming increasingly socialized due to their regular
interactions with campus cat feeding volunteers, it is certainly possible that some of these more
socialized cats were simply adopted informally by students, staff or campus visitors without the
knowledge of the CCC team. Others may have left the campus to join nearby urban cat colonies at
the racing stables or local shopping strip, possibly in response to competitive behavior from fellow
campus cats, or because they were undesexed males looking for mates. In our study, the total number of cats that disappeared (or emigrated, 35) almost exactly balanced
the number of cats that appeared (immigrated, 34). This is in contrast to the findings from a one-year
Israeli study that compared numbers in desexed and entire colonies of unowned urban cats. In that
study, despite high desexing rates for females in the desexed colonies, overall numbers increased
compared to the entire cat colonies. Sexually intact cats more readily immigrated into the desexed
colonies, whereas desexed cats were less likely to emigrate [29]. 4.1.4. Summary of Reasons for Reduction in Cat Population In our campus
population, 95% of females were desexed within the first 15 months of the program, and no kittens
were born to resident cats thereafter. However, despite this early application of TNR, three pregnant
females immigrated on to campus and gave birth to kittens, highlighting the ongoing need to monitor
immigrants, manage new kittens born, and desex immigrant cats as quickly as possible. 16 of 23 Animals 2018, 8, 77 Without active management of immigrant cats, combined with effective suppression of
reproduction through TNR, it is likely that the campus cat population would have increased, as was
apparent before initiation of the TNR program in August 2008. This emphasizes our view that
effective TNR management cannot occur as a “one-off” intervention, but necessitates ongoing
management of colonies to minimize the impacts of immigration. It should also be noted that ongoing
management is also essential when culling is used to reduce cat populations, because the birth of
new kittens and immigration into the culling area due to a “vacuum” effect will act to increase the cat
population [2,12–14]. 4.2. Health Management and Maintenance One of the key elements of the university-approved TNR trial reported here was the maintenance
of health of the cat population through daily feeding and regular monitoring of cat welfare and health
status. Under the original university agreement, veterinary care was also to be provided where this
was indicated. Best practice for daily feeding of unowned urban cats usually involves the removal of any left-over
food after about 30 min, to reduce attraction of vermin and also to minimize complaints [1]. Because
we relied heavily on student volunteers to place food for our resident cats, this approach was not
logistically feasible because of students’ other commitments. Furthermore, because of the high volume
of campus foot traffic at the end of each day as students left classes, cats frequently remained hidden
until later in the evening, after student volunteer feeders had left campus themselves. Our approach
therefore was to prescribe carefully the amount of food left per cat, and in recent years premeasured
bags of dry kibble were supplied for student feeders to emphasize limitations on the amounts of food
provided per cat. The most common reasons for veterinary treatment among the campus cat population such as
dental disease, eye infections, minor injuries, abscesses, and skin conditions, differed little from the
spectrum of minor illnesses seen in the domestic cat population. In our experience, tablet medications
including antibiotics, steroids and even treatment for hyperthyroidism (maintained over a 12-month
period) could be successfully administered crushed in the food of the affected cats. Various strategies
to ensure that medications were targeted to the cats receiving treatment were adopted, including
staged and separate feeding to prevent other cats in the colony from accessing the medication. 4.3. Arguments for and against TNR Management of Cats on Campus 4.3. Arguments for and against TNR Management of Cats on Campus 4.3. Arguments for and against TNR Management of Cats on Campus The two major arguments against maintaining a population of cats on the campus are the potential
threat to campus wildlife, and the potential spread of disease from cats to humans. The UNSW campus is an urban campus surrounded by residential housing, other institutions,
a shopping precinct and a racecourse. The campus environment could be described as very highly
disturbed, with many buildings, paved and concreted areas to expedite student traffic, some large
spaces of lawn for recreation, and some garden plantings. The wildlife on the campus consists of some
native and introduced bird life, mammals such as rodents (in particular non-native rats), possums and
fruit bats, and some populations of small lizards and frogs, although we are not aware of any census
of campus wildlife. It is beyond the scope of this study to estimate with any certainty the potential
impact of the campus cats on these animal populations. Nevertheless, an argument can be made that
by reducing the resident cat population through TNR, and through regular feeding of resident cats,
the impact of predation by campus cats is likely to be substantially mitigated. Although it is clear that feral cat populations have impacted on native wildlife in rural and wild
areas of Australia [15], there is no published study that establishes that cats have a significant negative
impact on native wildlife populations in an Australian urban environment. A study in Perth, Western
Australia [45], found that medium-sized native mammals such as possums and bandicoots were not
affected by the unregulated presence of cats compared to areas where cats were prohibited or kept
indoors at night. The small mardo Antechinus flavipes, which is considered to be highly susceptible to 17 of 23 Animals 2018, 8, 77 cat predation, was actually found in higher numbers in areas where cats were unregulated. Another
Perth study [46] concluded that urbanization with associated habitat degradation had a significant
negative influence on passerine bird populations, whereas there was no measurable effect of cats. In a
study of 24 forest patches in the Sydney metropolitan area, black rats, possums and other birds were
found to be the main predators of bird nests, and nest predation reduced when cats were present [47]. 4.3. Arguments for and against TNR Management of Cats on Campus In an Australian study, prey items caught by pet cats were, in decreasing order, mice, rats, small lizards,
and then common species of birds [48]. These studies indicate that our population of campus cats
probably had minimal effect on the population of native campus wildlife. Opponents of TNR typically raise concerns about released cats killing wildlife, but there is little
recognition or acknowledgement that killing healthy cats and kittens damages the health of the humans
involved. Research in Australia and elsewhere has found that many shelter staff directly involved
with euthanasia of animals develop perpetration-induced post-traumatic stress, and other shelter staff
frequently develop compassion fatigue [22–24,49]. These conditions damage mental health and can
lead to depression and increase the risk of suicide. In the US, the suicide rate for animal rescue workers
is amongst the highest in American workers, with a rate equal to firefighters and police officers [50]. These adverse effects on human mental health deserve to be considered when choosing urban cat
management strategies, because traditional complaint-based programs involving trapping and killing
cats, as occurred initially on the UNSW campus, result in more cats and kittens entering shelters and
their resultant euthanasia compared to TNR programs [25]. A number of diseases are spread from cats to humans, but most are spread by direct contact or
by fleas, and therefore pet cats pose a greater risk to humans [51]. Toxoplasmosis is known to be
carried by some cats, and can cause disease in humans, wildlife and cats, but is not spread by direct
contact [52]. There is evidence that, compared to trap and cull management programs, TNR may
reduce environmental contamination by toxoplasmosis oocysts, thus mitigating risks of transmission
to other species. In trap and cull programs, new susceptible kittens are in higher proportions due to
unchecked reproduction, and these immunologically naïve kittens are more likely to become infected
by the Toxoplasma parasite and to shed oocysts in the environment [53,54]. On the other hand,
TNR programs, which achieve high desexing rates and actively rehome young kittens, result over time
in a population of more mature cats that have a higher probability of being immune to toxoplasmosis
and are not shedding oocysts. Furthermore, adult cats that are not yet immune shed fewer cysts than
younger cats when infected with Toxoplasma [52,54]. The reduction in overall numbers of free-living
cats through TNR also minimizes risks of Toxoplasma infection [55]. 4.3. Arguments for and against TNR Management of Cats on Campus Nevertheless, the CCC was conscious of the potential for disease transmission to our volunteer
feeders. Although infection occurs through ingestion of oocysts which only become infectious several
days after being shed in cat feces, to mitigate the potential risk of toxoplasmosis infection during
management of campus cats we recommended the use of plastic gloves when feeding or handling the
cats, and emphasized the importance of personal hygiene after engaging in feeding activities. We also
discussed this potential risk with university management, so that they could appropriately inform and
instruct ground staff who may work in areas frequented by the cats. 4.5. Institutional Attitudes University management was initially skeptical about the potential of a TNR program to successfully
manage and reduce cat numbers, but the rapid reduction in the resident cat population and the
significantly reduced level of complaints from the campus community, with no formal complaints
recorded after 2009, quickly changed institutional attitudes towards the program. This was enhanced by
eliminating costs sustained by the university in managing the resident cats, such as costs associated with
previous culling of cats, and with complaint management and resolution. Furthermore, the ongoing
direct costs relating to feeding, veterinary care and desexing were borne by the animal welfare charity
and volunteers. Regular meetings between CCC and university management expedited increased cooperation. There was a noticeable difference in practical support from university divisions such as facilities/estate
management, security and grounds/landscaping teams, as a result of the clear and apparent
improvement in the management of the campus cat population, the improved health of the resident
cats, and the manifest reduction in numbers. These advantages of TNR programs are also likely to be apparent in community-based TNR
programs to manage unowned urban cats. Much of the cost to local councils and governments of
trapping and holding cats for minimum periods mandated by Australian state legislation, prior to
adoption or killing, can be minimized not only through reduction in the unowned cat population
through TNR, but also because of volunteer support and input from local community groups [1]. Furthermore, based on our campus experience, a significantly reduced level of complaints and
enhanced local community support for more humane management strategies for unowned urban cats
are likely to result from the application of TNR principles. 4.4. Financial Support An estimate of the annual costs associated with this TNR program is not possible because records
of expenditure were not kept until later years of the program, and because a majority of the costs
associated with the program, in particular food, were met by informal out-of-pocket contributions
from feeders and other members of the CCC. Food for the campus cats was supplied as a donation
by those feeders who were in full-time or part-time employment, whereas food was supplied on a
complimentary basis by the CCC to enrolled university student feeders, from a central campus location. This latter food was funded through donations. Similarly, all veterinary care including desexing, examination and treatment of ill cats, and costs of
euthanasia, was funded through donations, supplemented by generous discounts from local veterinary 18 of 23 Animals 2018, 8, 77 practices. Early in the program, most veterinary costs were related to desexing, whereas later in the
program most of these costs were associated with health maintenance and management of an aging
cat population. In the event of substantial individual veterinary care bills, email appeals to CCC
supporters were usually successful in raising the necessary funding to cover costs. Although the
university initially provided some limited funds (approximately $9000 AUD) for cat desexing at the
start of the program, the CCC was subsequently self-supporting as a registered animal welfare charity
since 2009, through regular donations from supporters and fundraising activities. 4.6. Limitations This study has a number of limitations, as would be expected from a retrospective study. The TNR
program on the university campus was not initiated as a formal scientific study, but to provide humane
management of a free-living unowned urban cat population. At the outset of the program, there was no
intention that program outcomes might be formally reported in this paper. No scientific protocol was
developed before commencing the program in terms of cat management, nor was there a predetermined
schedule of checking or trapping the cats. Management of the cats was developed informally by a
small group of untrained cat lovers and citizen scientists, rather than by veterinary scientists. Although the enumeration of cats on the campus during the nine-year program was carried out
with diligence and care, it is possible that some cats, particularly if present only transiently on campus,
may not have been recorded. No clear data on age of cats was available, because of the absence of
records before initiation of the TNR program in August 2008. Records of costs of the TNR program
were not maintained, and in any case many of the costs were “hidden”, having been met informally
by donations from members of the CCC and other supporters of the program. In-kind contributions,
in particular discounts from veterinary practices, were also not readily quantifiable. These factors
precluded comparisons with the costs that might have been incurred if a trap and cull approach, as
initially pursued by the university, had been continued. The applicability of our findings outside the limited and defined range of a university campus
or similar site is unclear. Immigration might constitute a larger component where colonies are 19 of 23 Animals 2018, 8, 77 immediately adjacent to houses with free-roaming owned cats, given the limited range of most pet
cats [32]. Furthermore, in common with many previous reports of TNR programs, no control group
was included to document and compare changes in cat numbers in an unmanaged population. Further
prospective research is therefore recommended to confirm outcomes of this TNR program. 5. Conclusions In summary, a nine-year cat management program conducted on the UNSW Kensington university
campus in Sydney, Australia, successfully reduced the campus cat population through a combination
of TNR and rehoming. At the beginning of the program, there were 69 resident cats. A further
34 immigrant cats (16 unsocialized and 18 socialized), and 19 kittens born on campus, were documented
on the campus during the program. At the time of reporting (end of September 2017), the campus has
a resident population of 15 cats. All current resident cats are desexed, clinically healthy and managed
on a daily basis by volunteer feeders. Many of these cats are now elderly (>10 years of age), and it
is anticipated that resident cat numbers will decline over the next few years due to natural attrition. At the same time, an inevitable continuing influx of immigrant cats is expected to supplement current
population levels, necessitating continuing active management through ongoing TNR and rehoming. Author Contributions: H.S. collected and summarized the data presented in the paper, and wrote the paper. J.R. contributed substantially to the preparation and writing of the paper. Funding: No external funding was provided for the study presented here. A minimal contribution to support the
initiation of the TNR program ($9000 AUD) was received from the University of New South Wales, but otherwise
the program was supported by donations to Campus Cats NSW. Acknowledgments: The authors sincerely acknowledge the significant practical support for the UNSW TNR
program from Colleen Ringe, Lisa Lifman and Beverley Lawless, and from the many other volunteer supporters
and cat feeders. The veterinary support of Struggletown Veterinary Hospital and Maroubra Junction Veterinary
Clinic is also gratefully acknowledged. The contributions of Maddie's Fund®, Found Animals, and The Humane
Society of the United States (HSUS) towards processing charges for this paper are gratefully acknowledged. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Appendix A Table A1. Numbers of cats on campus at commencement of the program in August 2008, and in
three-monthly steps from September 2008 to September 2017, stratified by sex and desexed status. Table A1. Numbers of cats on campus at commencement of the program in August 2008, and in
three-monthly steps from September 2008 to September 2017, stratified by sex and desexed status. Table A1. Numbers of cats on campus at commencement of the program in August 2008, and in
three-monthly steps from September 2008 to September 2017, stratified by sex and desexed status. End of Month
Male
Total
Male
Desexed
Male
Entire
Female
Total
Female
Desexed
Female
Entire
Sex/Status
Uncertain
Total Cats
August 2008
27
15
12
32
24
8
10
69
September 2008
30
17
13
30
24
6
6
66
December 2008
31
18
13
27
24
3
3
61
March 2009
28
19
9
26
25
1
3
57
June 2009
26
17
9
26
25
1
3
55
September 2009
23
16
7
26
25
1
3
52
December 2009
22
16
6
26
25
1
3
51
March 2010
19
15
4
25
24
1
3
47
June 2010
19
15
4
25
24
1
3
47
September 2010
19
16
3
24
23
1
2
45
December 2010
19
16
3
24
24
0
2
45
March 2011
17
15
2
21
21
0
2
40
June 2011
16
14
2
20
20
0
2
38
September 2011
15
13
2
18
18
0
2
35
December 2011
16
13
3
18
18
0
2
36
March 2012
16
13
3
18
18
0
2
36
June 2012
15
12
3
18
18
0
2
35
September 2012
15
13
2
17
17
0
2
34
December 2012
15
13
2
14
14
0
2
31
March 2013
13
11
2
13
13
0
2
28
June 2013
12
10
2
13
13
0
2
27 20 of 23 Animals 2018, 8, 77 Table A1. Cont. 1.
Tan, K.; Rand, J.; Morton, J. Trap-neuter-return activities in urban stray cat colonies in Australia. Animals
2017, 7, 46. [CrossRef] [PubMed]
2.
Doherty, T.S.; Dickman, C.R.; Johnson, C.N.; Legge, S.M.; Ritchie, E.G.; Woinarski, J.C.Z. Impacts and
management of feral cats Felis catus in Australia. Mamm. Rev. 2017, 47, 83–97. [CrossRef] Appendix B Case studies: The following case studies are included to illustrate the variety of campus cat histories. Pearl was one of the original campus cats at the start of the TNR program. She is a ginger desexed
female cat, born in approximately 2002. Over time, she became socialized due to regular contact with
her feeders, and enjoyed increasing interactions with students and staff on campus. She appeared
regularly in Facebook posts around the world as a result of her friendliness and approachability,
and was featured by Alley Cat Allies in one of their annual Feral Cat Day programs. In July 2017, at an
estimated age of 15 years, Pearl was successfully rehomed to an indoor living situation with two other
rescue cats, and has settled well into this new domestic environment. Basil, an undesexed male black cat, appeared on campus in December 2014, but was initially
extremely wary of humans so was categorized as an unsocialized immigrant. In January 2016, he was
eventually trapped, desexed and returned to his campus home. During surgery, he was found to be
microchipped, and his original owner was contacted. She had given him away as a kitten in 2010,
and had lost track of his whereabouts. She did not want him returned to her, but has continued to
financially support his on-campus care. Soon after desexing, Basil began to interact more with his
feeders, and has become well-socialized to certain carers. Kisa was a young undesexed female tabby when the TNR program began, and was a regular
breeding cat. In December 2008, she was trapped in advanced pregnancy, and gave birth to five
kittens in an off-campus foster placement in January 2009. Because of her unsocialized nature, she was
desexed soon after the birth of her kittens and was returned to campus, and the kittens were successfully
adopted. Five years later in 2014, Kisa inexplicably disappeared from campus. Early in 2017, the CCC
was contacted by a suburban veterinary practice, which advised that Kisa had been brought into the
practice, several kilometers distant, after having been hit by a car. She had been identified from her
microchip. Unfortunately, she required euthanasia because of the extent of her injuries. We have no
explanation why she was found so far from her original campus home, or how she survived during
the 3 years following her disappearance from campus. Appendix A End of Month
Male
Total
Male
Desexed
Male
Entire
Female
Total
Female
Desexed
Female
Entire
Sex/Status
Uncertain
Total Cats
September 2013
15
13
2
13
13
0
2
30
December 2013
13
11
2
14
12
2
3
30
March 2014
12
11
1
14
13
1
3
29
June 2014
12
11
1
14
13
1
3
29
September 2014
12
10
2
14
14
0
4
30
December 2014
12
10
2
13
13
0
2
27
March 2015
12
9
3
13
13
0
1
26
June 2015
11
8
3
12
12
0
1
24
September 2015
11
8
3
11
11
0
1
23
December 2015
11
8
3
11
11
0
1
23
March 2016
11
9
2
10
10
0
1
22
June 2016
11
9
2
9
9
0
2
22
September 2016
9
9
0
9
9
0
2
20
December 2016
9
9
0
9
9
0
2
20
March 2017
9
8
1
8
8
0
1
18
June 2017
9
9
0
8
8
0
1
18
September 2017
7
7
0
7
7
0
1
15 References Dickman, C. Impact of exotic generalist predators on the native fauna of Australia. Wildl. Biol. 1996, 2, 185–195. 16
N t li E M
li
L C
i l
G F i i A B
i R C f
S F
ti i C M
t
f f
l 15. Dickman, C. Impact of exotic generalist predators on the native fauna of Australia. Wildl. Biol. 1996, 2, 185–195. 16. Natoli, E.; Maragliano, L.; Cariola, G.; Faini, A.; Bonanni, R.; Cafazzo, S.; Fantini, C. Management of feral 15. Dickman, C. Impact of exotic generalist predators on the native fauna of Australia. Wildl. Biol. 1996, 2, 185–195. 16. Natoli, E.; Maragliano, L.; Cariola, G.; Faini, A.; Bonanni, R.; Cafazzo, S.; Fantini, C. Management of feral
domestic cats in the urban environment of Rome (Italy). Prev. Vet. Sci. 2006, 77, 180–185. [CrossRef] [PubMed]
17. Spehar, D.D.; Wolf, P.J. An examination of an iconic trap-neuter-return program: The Newburyport, 15. Dickman, C. Impact of exotic generalist predators on the native fauna of Australia. Wildl. Biol. 1996, 2, 185 195. 16. Natoli, E.; Maragliano, L.; Cariola, G.; Faini, A.; Bonanni, R.; Cafazzo, S.; Fantini, C. Management of feral
domestic cats in the urban environment of Rome (Italy). Prev. Vet. Sci. 2006, 77, 180–185. [CrossRef] [PubMed]
17. Spehar, D.D.; Wolf, P.J. An examination of an iconic trap-neuter-return program: The Newburyport,
Massachusetts case study. Animals 2017, 7, 81. [CrossRef] [PubMed] 6. Natoli, E.; Maragliano, L.; Cariola, G.; Faini, A.; Bonanni, R.; Cafazzo, S.; Fantini, C. Management of f
domestic cats in the urban environment of Rome (Italy). Prev. Vet. Sci. 2006, 77, 180–185. [CrossRef] [PubM y
17. Spehar, D.D.; Wolf, P.J. An examination of an iconic trap-neuter-return program: The Newburyport,
Massachusetts case study. Animals 2017, 7, 81. [CrossRef] [PubMed] 18. Spehar, D.D.; Wolf, P.J. A case study in citizen science: The effectiveness of a trap-neuter-return program in a
Chicago neighbourhood. Animals 2018, 8, 5. [CrossRef] [PubMed] 19. Andersen, M.C.; Martin, B.J.; Roemer, G.W. Use of matrix population models to estimate the efficacy of
euthanasia versus trap-neuter-return for management of free-roaming cats. JAVMA 2004, 225, 1871–1876. [CrossRef] [PubMed] 20. Miller, P.S.; Boone, J.D.; Briggs, J.R.; Lawler, D.F.; Levy, J.K.; Nutter, F.B.; Slater, M.; Zawistowski, S. Simulating
free-roaming cat population management options in open demographic environments. PLoS ONE 2014, 9,
e113553. [CrossRef] [PubMed] 21. Frommer, S.S.; Arluke, A. Loving them to death: Blame-displacing strategies of animal shelter workers and
surrenderers. References 21 of 23 Animals 2018, 8, 77 3. Gunther, I.; Terkel, J. Regulation of free-roaming cat (Felis silvestris catus) populations: A survey of the
literature and its application to Israel. Anim. Welf. 2002, 11, 171–188. 4. Zito, S.; Vankan, D.; Bennett, P.; Paterson, M.; Phillips, C.J. Cat ownership perception and caretaking explored
in an internet survey of people associated with cats. PLoS ONE 2015, 10, e0133293. [CrossRef] [PubMed] 4. Zito, S.; Vankan, D.; Bennett, P.; Paterson, M.; Phillips, C.J. Cat ownership perception and caretaking explored
in an internet survey of people associated with cats. PLoS ONE 2015, 10, e0133293. [CrossRef] [PubMed]
5. Toukhsati, S.R.; Bennett, P.C.; Coleman, G.J. Behaviors and attitudes towards semi-owned cats. Anthrozoos in an internet survey of people associated with cats. PLoS ONE 2015, 10, e0133293. [CrossRef] [PubMed]
5. Toukhsati, S.R.; Bennett, P.C.; Coleman, G.J. Behaviors and attitudes towards semi-owned cats. Anthrozoos
2007, 20, 131–142. [CrossRef] 5. Toukhsati, S.R.; Bennett, P.C.; Coleman, G.J. Behaviors and attitudes towards semi-owned cats. Anthrozoos
2007, 20, 131–142. [CrossRef] 6. Centonze, L.A.; Levy, J.K. Characteristics of free-roaming cats and their caretakers. JAVMA 2002, 220, 1627–1633. [CrossRef] [PubMed] 7. Levy, J.K.; Gale, D.W.; Gale, L.A. Evaluation of the effect of a long-term trap-neuter-return and adoption
program on a free-roaming cat population. JAVMA 2003, 222, 42–46. [CrossRef] [PubMed] 8. Zito, S.; Morton, J.; Paterson, M.; Vankan, D.; Bennett, P.C.; Rand, J.; Phillips, C.J.C. Cross-sectional study of
characteristics of owners and nonowners surrendering cats to four Australian animal shelters. J. Appl. Anim. Welf. Sci. 2015. [CrossRef] [PubMed] 9. Alberthsen, C.; Rand, J.; Bennett, P.; Paterson, M.; Lawrie, M.; Morton, J. Cat admissions to RSPCA shelters
in Queensland, Australia: Description of cats and risk factors for euthanasia after entry. Aust. Vet. J. 2013, 91,
35–42. [CrossRef] [PubMed] 0. Patronek, G.J. Free-roaming and feral cats—Their impact on wildlife and human beings. JAVMA 1998,
218–226. [PubMed] 11. Mahlow, J.C.; Slater, M.R. Current issues in the control of stray and feral cats. JAVMA 1996, 209, 2016–2020. [PubMed] 12. Neville, P.F.; Remfry, J. Effect of neutering on two groups of feral cats. Vet. Rec. 1984, 114, 447–450. [CrossRef]
[PubMed] 13. Neville, P.F. Feral cats: Management of urban populations and pest problems by neutering. In Mammals as
Pests; Putman, R.J., Ed.; Chapman and Hall: London, UK, 1989; pp. 261–267. 14. Zaunbrecher, K.I.; Smith, R.E. Neutering of feral cats as an alternative to eradication programs. JAVMA 1993,
203, 449–452. [PubMed] 15. References Soc. Anim. 1999, 7, 1–16. [CrossRef] 22. Reeve, C.L.; Rogelberg, S.G.; Spitzmuller, C.; Digiacomo, N. The caring-killing paradox: Euthanasia-related
strain among animal-shelter workers. J. Appl. Soc. Psychol. 2005, 35, 119–143. [CrossRef] 23. Rohlf, V.; Bennett, P.C. Perpetration induced traumatic stress in persons who euthanize nonhuman animals
in surgeries, animal shelters and laboratories. Soc. Anim. 2005, 13, 201–220. [CrossRef] [PubMed] 24. Baran, B.E.; Allen, J.A.; Rogelberg, S.G.; Spitzmuller, C.; Digiacomo, N.A.; Webb, J.B.; Carter, N.T.; Clark, O.L.;
Teeter, L.A.; Walker, A.G. Euthanasia-related strain and coping strategies in animal shelter employees. JAVMA 2009, 235, 83–88. [CrossRef] [PubMed] 25. Spehar, D.D.; Wolf, P.J. The impact of an integrated program of return-to-field and targeted trap-neuter-return
on feline intake and euthanasia at a municipal animal shelter. Animals 2018, 8, 55. [CrossRef] [PubMed] 25. Spehar, D.D.; Wolf, P.J. The impact of an integrated program of return-to-field and targeted trap-neuter-return
on feline intake and euthanasia at a municipal animal shelter. Animals 2018, 8, 55. [CrossRef] [PubMed] p
, ,
[
] [
]
26. Levy, J.K.; Isaza, N.M.; Scott, K.C. Effect of high-impact targeted trap-neuter-return and adoption of
community cats on cat intake to a shelter. Vet. J. 2014, 21, 269–274. [CrossRef] [PubMed] 26. Levy, J.K.; Isaza, N.M.; Scott, K.C. Effect of high-impact targeted trap-neuter-return and adoption of
community cats on cat intake to a shelter. Vet. J. 2014, 21, 269–274. [CrossRef] [PubMed] 22 of 23 Animals 2018, 8, 77 27. Boone, J.D.; Slater, M. A Generalized Population Monitoring Program to Inform the Management of Free-Roaming
Cats. Alliance for Contraception in Cats and Dogs. 2014. Available online: http://www.acc-d.org/docs/default-
source/think-tanks/frc-monitoring-revised-nov-2014.pdf?sfvrsn=2 (accessed on 29 April 2018). 8. Foley, P.; Foley, J.E.; Levy, J.K.; Paik, T. Analysis of the impact of trap-neuter-return programs on populat
of feral cats. JAVMA 2005, 227, 1775–1781. [CrossRef] [PubMed] 29. Gunther, I.; Finkler, H.; Terkel, J. Demographic differences between urban feeding groups of neutered and
sexually intact free-roaming cats following a trap-neuter-return procedure. JAVMA 2011, 238, 1134–1140. [CrossRef] [PubMed] 30. Castillo, D.; Clarke, A.L. Trap/neuter/release methods ineffective in controlling domestic cat “colonies” on
public lands. Nat. Areas J. 2003, 23, 247–253. 31. Hughes, K.L.; Slater, M.L. Implementation of a feral cat management program on a university campus. J. Appl. Anim. Welf. Sci. 2002, 5, 15–28. [CrossRef] [PubMed] 32. Roetman, P.; Tindle, H.; Litchfield, C.; Chiera, B.; Quinton, G.; Kikillus, H.; Bruce, D.; Kays, R. References Cat Tracker
South Australia: Understanding Pet Cats through Citizen Science; Discovery Circle Initiative, University of South
Australia: Adelaide, Australia, 2017. 33. Huang, L.; Coradini, M.; Rand, J.; Morton, J.; Albrecht, K.; Wasson, B.; Robertson, D. Search methods used to
locate missing cats and locations where missing cats are found. Animals 2018, 8, 5. [CrossRef] [PubMed] 34. Alley Cat Allies. Feline Immunodeficiency Virus. Available online: https://www.alleycat.org/resources/
feline-immunodeficiency-virus-fiv/ (accessed on 16 May 2018). 35. O’Neill, D.G.; Church, D.B.; McGreevy, P.D.; Thomson, P.C.; Brodbelt, D.C. Longevity and mortality of cats
attending primary care veterinary practices in England. J. Feline Med. Surg. 2015, 17, 125–133. [CrossRef]
[PubMed] 36. New, J.C., Jr.; Kelch, W.J.; Hutchinson, J.M.; Salman, M.D.; King, M.; Scarlett, J.M.; Kass, P.H. Birth and death
rate estimates of cats and dogs in U.S. households and related factors. J. Appl. Anim. Welf. Sci. 2004, 7, 229–241. [PubMed] 37. Huang, W.; Liao, A.T.; Chu, P.; Yen, I.; Liu, C. A real-time reporting system of causes of death or reasons for
euthanasia: A model for monitoring mortality in domesticated cats in Taiwan. Prev. Vet. Med. 2017, 137,
59–68. [CrossRef] [PubMed] 38. Egenvall, A.; Nodtvedt, A.; Haggstrom, J.; Strom Holst, B.; Moller, L.; Bonnett, B.N. Mortality of life-insured
Swedish cats during 1999–2006: Age, breed, sex, and diagnosis. J. Vet. Intern. Med. 2009, 23, 1175–1183. [CrossRef] [PubMed] 39. Levy, J.K.; Crawford, P.C. Humane strategies for controlling feral cat populations. JAVMA 2004, 225,
1354–1360. [CrossRef] [PubMed] 40. Scott, K.C.; Levy, J.K.; Gorman, S.P.; Newell, S.M. Body condition of feral cats, and the effect of neutering. J. Appl. Anim. Welf. Sci. 2002, 5, 203–213. [CrossRef] [PubMed] 41. Dale, A. The comparative welfare status of owned, managed stray and unmanaged stray cats. In Proceedings
of the 6th National Getting 2 Zero Summit and Workshops, Gold Coast, Australia, 14–19 September 2015. 42. Weiss, E.; Slater, M.; Lord, L. Frequency of lost dogs and cats in the United States and the methods used to
locate them. Animals 2012, 2, 301–315. [CrossRef] [PubMed] 43. Griffin, B. Prolific cats: The impact of their fertility on the welfare of the species. Compendium 2001, 23,
1058–1069. Joyce, A.; Yates, D. Early age neutering in cats. J. Feline Med. Surg. 2011, 13, 3–10. [CrossRef] [PubMed] 45. Lilith, M.; Calver, M.C.; Garkaklis, M. Do cat restrictions lead to increased species diversity or abundance
of small and medium sized mammals in remnant urban Bushland? Pacif. Conserv. Biol. 2010, 16, 162–172. © 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/). References [CrossRef] 46. Grayson, J.; Calver, M.; Lymbery, A. Species richness and community composition of passerine birds
in suburban Perth: Is predation by pet cats the most important factor? In Pest or Guest: The Zoology of
Overabundance; Lunney, D., Eby, P., Hutchings, P., Burgin, S., Eds.; Royal Zoological Society of New South
Wales: Mosman, Australia, 2007; pp. 195–207. 47. Matthews, A.; Dickman, C.R.; Major, R.E. The influence of fragment size and edge on nest predation in urban
bushland. Ecography 1999, 22, 349–356. [CrossRef] 48. Franklin, M.; Rand, J.; Marston, L.; Morton, J. Prey captured by owned cats and dogs in Australia. Animals
2018, submitted for publication. 23 of 23 Animals 2018, 8, 77 49. Anderson, K.A.; Brandt, J.C.; Lord, L.K.; Miles, E.A. Euthanasia in animal shelters: Management’s perspective
on staff reactions and support programs. Anthrozoos 2013, 26, 569–578. [CrossRef] 50. Tiesman, H.M.; Konda, S.; Hartley, D.; Chaumont Menendez, C.; Ridenour, M.; Hendricks, S. Suicide in US
workplaces, 2003–2010: A comparison with non-workplace suicides. Am. J. Prev. Med. 2015, 48, 674–682. [CrossRef] [PubMed] 51. Centers for Disease Control and Prevention. Healthy Pets, Healthy People: Cats. Available online: https:
//www.cdc.go/healthypets/pets/cats.html (accessed on 10 February 2018). 52. VanWormer, E.; Fritz, H.; Shapiro, K.; Mazet, J.A.K.; Conrad, P.A. Molecules to modelling: Toxoplasma gondii
oocysts at the human-animal-environment interface. Comp. Immunol. Microbiol. Infect. Dis. 2013, 36, 217–231. [CrossRef] [PubMed] 53. Dubey, J.P.; Hoover, E.A.; Walls, K.W. Effect of age and sex on the acquisition of immunity to toxoplasmosis
in cats. J. Protozool. 1977, 24, 184–186. [CrossRef] [PubMed] 54. Dubey, J.P. Duration of immunity of shedding of Toxoplasma gondii oocysts by cats. J. Parasitol. 1995, 81,
410–415. [CrossRef] [PubMed] 55. Torrey, E.F.; Yolken, R.H. Toxoplasma oocysts as a public health problem. Trends Parasitol. 2013, 29, 380–384. [CrossRef] [PubMed] © 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/).
|
https://openalex.org/W2474948059
|
https://www.biodiversitylibrary.org/partpdf/319776
|
English
| null |
The Structure of Podocarpus spinulosus, (Smith) R. Br.
|
Annals of botany
| 1,910
|
public-domain
| 7,327
|
The
Structure
of
Podocarpus
spinulosus,
(Smith)
R.
Br. The
Structure
of
Podocarpus
spinulosus,
(Smith)
R. Br. BY
F. T. BROOKS, M.A. Senior Demonstrator of Botany, Cambridge University. BY
F. T. BROOKS, M.A. Senior Demonstrator of Botany, Cambridge University. AND
WALTER
STILES,
B.A. A ssistant- Lecturer in Botany in the University of Leeds. A ssistant- Lecturer in Botany in the University of Leeds. [Annals of Botany, Vol. XXIV. No. XCIV. April, 1910.] Introduction. JL HE following account of the structure of Podocarpus is based mainly
JL upon material of Podocarpus spinulosus , (Smith) R. Br., which was
obtained from the Botanic Gardens, Sydney, New South Wales. The
material was sent to Professor Seward by Mr. Maiden, and was handed
over to us for investigation. Material of Podocarpus alpimts obtained from the Royal Gardens, Kew,
and of one or two other species of which only a limited stock was available,
were examined at the same time for the sake of comparison. Pilger (13) in his monograph of the Taxaceae places Podocarptts and
four other genera — Pherosphaera , Microcachrys , Saxegothaea , Dacrydium —
in the sub-family Podocarpoideae, the other sub-families of the group being
the Phyllocladoideae, containing the single genus Phyllocladtts , and the
Taxoideae, comprising Taxus , Torreya , Cephalotaxus . Pilger evidently
then considers the Podocarpoideae to be very closely related to the
Taxoideae. Later work, however, would appear to necessitate a modifica¬
tion of this view. The systematic position of P odocarpus and closely allied genera is
discussed in the latter part of this paper. The genus Podocarpus , as described in Pilger’ s monograph, contains no
less than sixty-three species, the large majority of which are found in the
Southern Hemisphere. New Zealand, Australia, and the Malayan islands
are at present the home par excellence of the genus, while smaller centres
of distribution are such different regions as East Africa (Sao Thome), Cape
Colony and Natal, Assam, Japan, China, Central America, West Indies,
and Chili. Where the genus is present within the tropics it is generally [Annals of Botany, Vol. XXIV. No. XCIV. April, 1910.] and
Stiles. —
The
Structure
of 306 found on mountain ranges at a considerable elevation. Just as Pinus may
be considered the dominant Coniferous genus of the Northern Hemisphere,
so Podocarpus occupies a similar position among the Conifers of the
Southern regions. In regard to the distribution of the genus in geological times, most
of the specimens referred to Podocarpus or closely allied genera have been
determined from leaves only, and it is well known that leaf determinations
are frequently untrustworthy. In this connexion mention may be made
of the striking similarity in external features between the leaves of some
species of Agathis and those of the Nageia section of Podocarpus . Introduction. Seward
and Ford (16) found, on cutting sections of a species originally referred to
Dammar a Motleyi , that the plant so named is really a species of Podocarpus. Assuming that some at least of the specimens are correctly deter¬
mined, then Podocarpus , like Araucaria, must have been widely distributed
in the North temperate zone during the later Mesozoic and Tertiary times. Pilger states that the leaves from the Potomac or later Mesozoic flora of
the United States are to be referred to the Nageia section of the genus on
account of the plurinerved form of venation, while leaves from the Eocene
and Miocene strata of Europe belong to the uninerved sections of the genus. Podocarpus shows a considerable range of habit among its many
species. Several of them are large trees ; e. g. P. amarus attains a height
of sixty metres in Java, and P. dacrydioides forms practically pure forests
in New Zealand. On the other hand, P. alpinus , found on mountains in
Tasmania, is a low shrub, and P. nivalis , on exposed places in the alpine
regions of New Zealand, becomes a dwarf shrub with prostrate branches. Stem. The structure of the wood of P. spimdosus is very similar to that of
P. macrophyllus described by Penhallow. Resin canals are absent in the
wood, but there is a ring of them present outside the phloem. Their place
in the wood is taken by resin cells, which are present in considerable
numbers. The bordered pits of the secondary tracheides are uniseriate ;
no trace of the Araucarian type of pitting could be discerned, such as has
been described by Gothan (8) for Dacrydium spp. and for Saxegothaea by
Beust (3), Gothan (8), and Stiles (17). The medullary rays are invariably
uniseriate, and are from one to three cells deep. The cells of the rays
contiguous to the pith frequently contain resinous material, but apart from
this all the cells of the rays are alike, there being no tracheidal cells such
as are met with in the rays of Pinus and some other Conifers. There is
a small central pith which consists mainly of resin cells and groups of stone
cells ; the latter are very conspicuous. Resin cells are also extremely
abundant in the cortex. Podocarpus
spinulosus
,
(Smith)
R. 307 Reproductive
Organs. With regard to the reproductive organs Podocarpus is generally
dioecious, though very rarely monoecious. This distribution of the sexes
is the same as that in Dacrydium and in Microcachrys , but Saxegothaea is
monoecious. The reproductive organs in Taxus are also dioecious, and in
Araucaria and Agathis they are generally dioecious. Leaf. The leaves of P. spinulosus , as indeed is the case in the large majority
of the species, are arranged spirally on the stem axis. It is interesting
to note, however, that in the Nageia section of the genus the leaves have
a decussate or subdecussate arrangement. The leaf of P. spinulosus is
about 4-6 cm. long by 3-4 mm. broad, and ends in an acute point. The anatomy of the leaf of P. spinulosus is very similar to that of
P. chilinus described by Worsdell (29), and that of P. neriifolius and
P. falcaUts examined by Bernard (1). As in all the narrow-leaved species
of the genus, there is a single vascular bundle ; this is accompanied on its
phloem side by a resin canal. Placed laterally to the vascular bundle is
the transfusion tissue, which is extremely well developed. The transfusion
tracheides show numerous reticulations on their walls when the leaf is cut
at right angles to the midrib. On the side of the transfusion tissue remote from the bundle, there is
a layer of parenchymatous cells, beyond which is the ‘ accessory transfusion
tissue’, such as is described by Worsdell for P. chilinus . This tissue runs
outwards almost to the margin of the leaf. This ‘ accessory transfusion
tissue ’ of Worsdell corresponds to the ‘ hydrostereome transversal ’ of
Bernard. The cells composing this tissue are long and lignified, and, as
Worsdell points out, they have the appearance of stone cells rather than of
tracheides. Their function presumably is primarily that of strengthening
the lamina of the leaf. and
Stiles
.
—
The
Structure
of and
Stiles
. —
The
Structure
of The axis of the cone, as seen in transverse section, contains a ring of
collateral vascular bundles of the usual structure. No trace of centripetal
xylem could be found, such as is present in the axis of the male cone of
Saxegothaea (17). Each vascular bundle is accompanied by a single resin
/
canal outside the phloem, but it seems to be the general rule that these
resin canals are not functional, for in most of them no distinct epithelial
layer could be seen. On the other hand, resin cells are frequent throughout
the fundamental tissue. A single vascular bundle, consisting of a few
tracheides and phloem elements, passes out to each sporophyll. It arises
by division of one of the axial bundles, the plane of division being radial ;
the two derived bundles lie side by side before one of them passes out to
the sporophyll. The tracheides in the sporophyll are somewhat scattered,
and some of them may represent centripetal xylem. The phloem dies out
before the xylem, as it ordinarily does in the termination of the vascular
bundles of the leaves. A small resin canal, which appears to be non¬
functional, accompanies the single vascular bundle in its passage from the
cone axis, but this canal dies out very quickly. In Saxegothaea , on the
other hand, the resin canal of the sporophyll is a much more conspicuous
structure, and passes out practically as far as the xylem elements extend. The microsporangia of our material were either ready for dehiscence
or else dehiscence had already taken place. Remains of two layers of cells
within the sporangium wall could be made out. The wall of the sporangium
is very much like that of Saxegothaea (10, 17) and Araucaria (16). It is one
cell wide except on the outside, and the vertical cell-walls are very thick,
and have numerous strengthening bands on them running at right angles
to the surface (cf. PI. XXI, Fig. i). The line of dehiscence of the
sporangium is oblique, as can be seen by an examination of PI. XXI,
Fig. 2. Pilger (13) states that the dehiscence of the sporangium in the
Taxaceae is longitudinal, but this is without doubt incorrect for P. spinu-
tosus. Thibout (20) correctly figures the dehiscence of the microsporangia
of several species of Podocarpus as being oblique. and
Stiles
.
—
The
Structure
of In Saxegothaea (17)
the line of dehiscence is transverse. (1) Male Cone. The male cones are borne either singly on a very short axis which
arises in the axil of an ordinary foliage leaf, or more often they are arranged
in clusters of two to four which are borne on a similar abbreviated axis,
each cone being attached by means of a short stalk. The male cone is
from 1 cm. to 1*5 cm. long and about 2*5 mm. in diameter. At the base
of each male cone are a few tiny bracts which are spirally arranged along
the short stalk of the cone. The axis of the cone bears a number of
spirally arranged sporophylls, each one of which consists of a pedicel which
becomes flattened distally to a lamina on either side. Each sporophyll
bears on its under surface two ovoid microsporangia. 308 (2) Pollen-grains. As is well known, the pollen-grains of Podocarpus are winged like
those of Pinus. The material of P. spinulosus available contained only
pollen-grains which were mature. The material had been fixed in a dilute
solution of formalin, and apparently had all been collected at one time. Several observers have recently related the occurrence of as many as eight
prothallial cells in the pollen-grains of different species of Podocaipiis. Thibout (20) describes the presence of several prothallial cells in P. poly -
stachyus. Coker (5) mentions the division in P. coriacea of the two original
prothallial cells, but he considers that these divisions are abnormal. Podocarpus
spinulosus
,
{Smith)
R. 309 Jeffrey and Chrysler (9) found that in two other species, P. fernigineiis
and P. dacrydioides , there were as many as eight prothallial cells present,
and that the generative cell also had divided. They and Miss Young (31)
also record several prothallial cells in species of Dacrydium. Jeffrey and
Chrysler incline to the view that these divisions of the prothallial cells and
the generative cell cannot be regarded as a primitive feature. Burlingame (4)
reports the occurrence of a similar group of prothallial cells in P. totarra
Hallii. Noren (10) records a similar complex of prothallial cells in
Saxegothaea , and Thomson (23) describes the occurrence of the same in
Microcachrys. The latter (21) has also found that in Agathis as many as
thirty to forty prothallial cells may be present. It was thought advisable to compare the structure of the male
gametophyte of P. spinulosus with that of other species of Podocarpus
previously described. Microtome sections of male cones were cut whose
microsporangia had not dehisced, and were stained with Heidenhain’s Iron
Alum Haematoxylin with a background stain of Orange G. It will be
seen on examination of PI. XXI, Figs. 3-8, that the contents of the pollen-
grains are almost exactly the same as those figured by Jeffrey and Chrysler
for P. ferrugineus and P. dacrydioides. In Figs. 3 and 4 both prothallial
cells have divided in a plane at right angles to that of the section, but in
Figs. 5 and 6 divisions in the first and second prothallial cells respectively
have not occurred in this plane. From an examination of Fig. (2) Pollen-grains. 7, which
is a section of a pollen-grain cut tangentially at its prothallial end, it is
apparent that there may be another plane of division of the prothallial cells
at right angles to that indicated in Figs. 3 and 4, &c. Thus as many as
eight prothallial cells may be present. The generative cell is generally
conspicuous with its aggregation of protoplasm around the central nucleus. It is evident from Fig. 5 that the generative cell sometimes divides
before the liberation of the pollen in a manner which is probably the
same as that described for the first time by Jeffrey and Chrysler (9) as
occurring in the pollen of P. ferrugineus and P. dacrydioides. The tube-
nucleus is the largest nucleus present in the pollen-grain. All the nuclei
of our material were in the resting condition, as indicated by the large
nucleolus and absence of a reticulum. After a time the walls of the
prothallial cells break down, and the nuclei come to lie free in the
cytoplasm, as will be seen on examination of Fig. 8. Jeffrey and Chrysler
record a similar phenomenon in P. polystachyus. It is apparent then from this examination of P. spinulosus , and from
the resume of work done on other species given above, that the multi¬
cellular condition of the male gametophyte of Podocarpus must be con¬
sidered a characteristic of the genus, especially as the species investigated
come from widely different regions (New Zealand, Australia, Cuba, Java). The fact that a similar condition of the male gametophyte exists in and
Stiles. —
The
Structure
of 310 Dacrydium, Microcachrys , and Saxegothaea is another indication that the
Podocarpeae are a natural group. It is impossible, with our present meagre knowledge of the inter-relation-*
ships of the Conifers, to say whether this multicellular condition of the male
gametophyte of Podocarpus is primitive or derived. Jeffrey and Chrysler (9)
consider that it is not a primitive feature, but it seems to us that data for
deciding the question are not at present available. The fact that a similar
prothallial complex is present in Saxegothaea and Microcachrys— two
genera which on other grounds are considered to be relatively primitive-^
is a point in favour of considering the prothallial complex of Podocarpus
primitive. (2) Pollen-grains. The fact that the divisions of the nuclei of the prothallial cells
are karyokinetic is in keeping with the interpretation that the occurrence
of several such cells is a normal feature for the genus. The presence of
wings in the pollen-grains of Podocarpus does not point to any necessary
relationship with the Abietineae, a group from which we consider the
Podocarpeae to be considerably removed, for in the Podocarpeae the wings
on the pollen-grains appear to have been evolved within the group.1
Jeffrey and Chrysler (9) consider, on the other hand, that the ground
plan of the male gametophyte of Podocarpus is very similar to that
of the Abietineae, but we cannot gather from their statements what
adequate grounds they have for this conclusion. Jeffrey and Chrysler
bring the proliferation of the prothallial cells in Araucarieae into line with
the peculiar protosiphonogamic method of fertilization in this group
described by Thomson (22). But this does not account for the occurrence of
several prothallial cells in Podocarpus .2 On examining our material it was
seen that the pollen-grains alight in the micropyle, and so presumably the
growth of the pollen-tubes is a normal one through the nucellus. (3) Female Fructification. Of the female fructification we had material of three species, Podo -
carpus spimdosus , P. alpinus , and a third species. We had the first in
largest quantity, so this species will be described more fully and the others
compared with it. The female fructification is borne laterally on the stem in the axil of
a bract about six millimetres long, resembling a small foliage leaf. In
the axil of the bract is the peduncle, about six or seven millimetres long
and nearly a millimetre wide, and somewhat flattened in the vertical
plane at right angles to that containing the main axis and the peduncle. At its upper end it bears three pairs of bracts decussately arranged
(cf. PI. XXI, Fig. 9). The lowest pair are small, about one and a half milli¬
metres long, and are recurved and more or less leaf-like. They lie in the
plane containing both stem and pedicel. The other two pairs of bracts* Burlingame (4). 1 Cf. Thomson Podocarpus
spinulosus
,
{Smith)
R. 1
1 which are inserted close together, appear more or less swollen and fleshy ;
they are about five or six millimetres long, and fused together except at
the tip. The two lower bracts are about a millimetre longer than the
upper pair; each bears, just above the point where the four bracts become
free from one another, a single stalked anatropous ovule. Occasionally
one of the two lower bracts is sterile, while the upper pair of bracts in all
cases examined were both sterile. Generally one of these is developed
more strongly than the other. Pilger (13) notes a good deal of variation
in the bracts of P. spinulosus. Sometimes the lowest pair may be missing,
while the other four may vary a great deal in size relatively to one
another. The following account of the internal structure is of the normal
case where the two lowest bracts are present, and the next two bracts are
fertile and of approximately equal size, while the uppermost pair are also
fairly well developed. The peduncle contains in transverse section a somewhat elliptical
ring of endarch collateral bundles which anastomose somewhat. Each
bundle is accompanied on its phloem side by a single resin canal. A section through the base of the fused bracts shows a similar
structure (PI. XXI, Fig. 10). (3) Female Fructification. At the chalazal end of the ovule the two lateral bundles bend round
through about a right angle and descend, one on each side of the ovule, for
a short distance towards the micropyle ; as seen in transverse section they
lie on the diameter of the ovule (PL XXI, Fig. 17). The central bundle
continues its course over the top of the ovule, and in so doing divides into
two bundles (PL XXI, Fig. 18) which descend a short distance towards the
micropyle (PL XXI, Fig. 17). All these descending bundles soon die out,
and all show a tendency to amphivasal structure (PL XXI, Fig. 20). The nucellus of the ovule is surrounded by two integuments. The
inner is fused for about half its length with the nucellus. The outer
integument or epimatium is free from the inner for a short distance at the
micropyle end, and on the side remote from the ovular stalk; on the other
side it is either not present or is completely fused with the stalk.1 Where free, the innermost layer of the inner integument often con¬
sists largely of resin cells. These are apparently continued into the part of
the ovule where the inner integument and nucellus become fused, and
so apparently mark the line of division between these. A zone containing
larger and more numerous resin cells also seems to mark the line of
division between the inner and outer integuments, but these do not extend
far enough towards the chalaza, and are not definite enough to enable one
to state whether the descending bundles belong to any particular integument
or to the nucellus. In a species of Podocarpus grown in the Cambridge Botanic Garden,
the structure was found, on the whole, to be similar. The peduncle is borne
in the axil of an ordinary foliage leaf. In the stage examined the peduncle
is about six millimetres long and nearly a millimetre broad, and, as in
P. spinulosus , is flattened in a vertical plane at right angles to that con¬
taining the peduncle and the branch bearing it. In the few specimens
available for examination the lowest pair of leaf-like bracts were not
present, and the whole fructification consisted of two pairs of bracts fused
together, of which one or both of the lower pair bore ovules. The
arrangement of the bundles is practically the same as in P. spinulosus. (3) Female Fructification. The bundles divide occasionally and reunite,
the resin canals sometimes dividing as well as the bundle ; in other cases
the canal arises de novo outside one of the bundles, and occasionally it may
be absent altogether from one of the bundles. This behaviour points to
the canals being functionless, and there is no epithelium properly developed. Resin cells are extremely abundant throughout the whole of the parenchyma
of the bracts. At a higher level the ring of bundles widens out in one plane, and the
bundles then come to arrange themselves in two groups of three at opposite
sides of the wider diameter with two bundles between them (PI. XXI,
Figs. 11 and 13). The two groups at each end belong to the fertile scales and ovules,
the bundles between them belonging one to each of the sterile scales. This
state of affairs is intermediate between that figured by Van Tieghem (27)
for Podocarpus sinensis , where the sterile bracts also contain each a group of
three bundles, and that figured by Strasburger (18) for Podocarpiis chiueusisy
Wall., apparently the same species as that described by Van Tieghem,
where the sterile bracts have no vascular supply. In one place the stronger
of two sterile bract bundles had two small groups of xylem elements some
little distance away from it on the xylem side (PI. XXI, Figs. 12 and 19) ;
these apparently correspond to the two inner bundles of one of the groups
in Van Tieghem’s figures. As the bundles of the sterile bracts die out,
some transfusion tracheides appear. Of the group of three bundles serving the fertile scale, the middle one,
passes out into the bract subtending the ovule, the other two approach and
Stiles. —
The
S
'true
hire
of 312 one another and fuse by their margins to form a V-shaped bundle with its
xylem and phloem inversely orientated with regard to the xylem and
phloem of the subtending bract bundle (PI. XXI, Fig. 13). This V-shaped
bundle next straightens itself out (PI. XXI, Fig. 14) and begins to divide into
three (PI. XXI, Fig. 15), so that as the chalazal end of the ovule is approached
there are found three bundles, each, as a rule, with a small non-functional
resin canal (PI. XXI, Fig. 16). (3) Female Fructification. One bundle of the stem passes into the subtending leaf, while the two
bundles on each side of it pass into the peduncle. These almost imme¬
diately divide up into a ring of bundles. At the base of the fused bracts 1 See Postscript. Podocarpus
spinulosus
,
(
Smith
)
R. 313 two sets of three bundles are present; these belong to the fertile scales. One of these bundles on each side divides, and so gives rise to the bundles
of the two sterile scales. Additional small bundles may be present on
the inside of the sterile scale bundles as in P. spinulosus . The xylem
of the bundles tends to be semicircular in shape, more or less enclosing
the phloem on three sides. The resin canals, of which there is one on
the phloem side of each bundle, are much larger than in P. spinulosus ,
and as a rule the resin duct divides after the division of the bundle to
which it belongs. The ovular bundle, as in P. spinulosus , divides into
three ; when on a level with the nucellus, transfusion tissue begins to
appear both centripetally and laterally, and the division into three bundles
is no longer obvious. These bundles die out at the chalaza, and do not
descend on the sides of the ovule as in P. spinulosus . For the last part
of their course they are composed of transfusion-like tracheides only. In Podocarpus alpinus , material of which was obtained from the
Royal Gardens, Kew, the peduncle is very short, being less than a milli¬
metre long. The lowest pair of bracts are extremely small and scale¬
like, although Pilger (13) states they are absent altogether; of the next pair
only one bears an ovule, while of the upper sterile pair one is often aborted
altogether, and the other is small compared with those of the lower pair. The vascular system at the base of the fused bracts consists of four
bundles arranged along the sides of a square ; of these one serves the
fertile scale and two the ovule it subtends, the fourth serving the opposite
sterile scale. The upper sterile scale has usually no vascular supply. (3) Female Fructification. The two ovular bundles fuse as in the other species described, but at
first almost the whole of the xylems of the two bundles come in contact, so
that the resulting bundle is almost concentric ; further up, however, it
straightens itself out. Nearer the chalaza it divides into two bundles which
curve over the top of the ovule and descend a little way towards the micro-
pyle on the sides of the ovule in much the same way as the lateral bundles
do in P. spinulosus. In P. alpinus the resin canal accompanying each of
these two bundles extends down to the level of the nucellus where it occurs
on the inner margin of the outer integument, to which these bundles would
appear to belong. Other canals also appear on the inner margin of the
outer integument ; these presumably correspond with the middle bundle of
P. spinulosus, which bends over the ovule and then divides into two. On
the whole, it would seem from their behaviour in P. alpinus that these
bundles all belong to the outer integument, but the evidence at present is
insufficient to decide this point. As far as the female fructification is concerned, the Podocarpeae
obviously fall into two divisions ; one in which the megasporophylls are
aggregated into cones, and which comprises the monotypic genera Saxe-
got haea and Microcachrys } the other in which the fertile megasporophylls and
Stiles. —
The
Structure
of 314 are mostly either solitary or associated in pairs, and which includes the
genera Dacrydimn and Podocarpits. A remarkably different condition
exists in two species of Podocarpus, P. andinus and P. spicatus , in which
the megasporophylls bearing ovules occur at intervals along the fertile
branch. In all a single ovule is present on the upper surface of the
megasporophyll, and is borne in a more or less reversed position. Assuming that the Podocarpeae form a natural group, a conclusion
which from general considerations seems inevitable, there appears to be
every reason to suppose that the state found in Dacrydium and Podo¬
carpus is derived from that in Saxegothaea and Microcachrys by reduc¬
tion in the number of scales in the cone, from an indefinite number to
two or three pairs decussately arranged in Podocarpus. 1 But Thomson (24) states that the sporangium bundle arises in the same way as that of
Saxegothaea, by branching from the sporophyll bundle. (3) Female Fructification. Here very
occasionally both scales of two pairs may contain ovular bundles (27) j
in some species normally both bracts of the lower pair are fertile, while
in many species, e. g. P. alpimis and P. elatus , only one scale of the whole
fructification is fertile. The female fructification is then to be regarded
as a very reduced cone. In P. andinus and P. spicatus it would appear
that the axis, instead of becoming very much shortened, has lengthened
out a good deal, and the sporophylls are now separated by comparatively
long internodes. The insertion of the ovule differs markedly from that
found in Saxegothaea and Microcachrys. In these genera the single ovule
is sessile on the upper surface of the megasporophyll, whereas in Podo¬
carpus it is borne on a stalk which apparently arises from the upper
surface of the bract which bears it. Dacrydium exhibits transitions between;
the two cases, for in some species the ovule is borne on the upper surface
of the scale, in others it is more or less as in Podocarpus. The internal
structure of the megasporophylls of the Podocarpeae also shows a similar
series of stages. In Saxegothaea one bundle leaves the axis of the. cone and gives off the ovular supply by branches arising from it ; in
Microcachrys the two bundles are presumably separate from the cone axis,
but very close together (30) ; 1 in Podocarpus one bundle of the peduncle
passes up into the megasporophyll, while two others, one on each side of
the sporophyll, join up and supply the ovule. It seems fairly clear that Saxegothaea is primitive for the Podocarpeae,
and the state of things found in Podocarptts is derived from that found in
Saxegothaea. The much smaller amount of centripetal xylem and the loss
of function of the resin canals in some species support this view. The independence of the vascular supply of the ovule from the vascular
bundle of the subtending bract can be easily, and it seems to us reasonably,
explained as due to basipetal evolution of the vascular system, as the ovule
has become more and more important in relation to the subtending bract., Podocarpus
spinul-osus
,
(Smith)
R. (3) Female Fructification. 315 In Saxegothaea , where the ovule is a small appendage of the bract, the
bundles supplying it arise by branching from the bundle of the sporophylh
As the ovule has become relatively of more importance than the bract, its
vascular supply has also become of more importance, until in Podocarpus
the vascular supply of the ovule is carried down into the peduncle indepen¬
dently of the supply of the megasporophyll. This point of view has also
been emphasized by Thomson (24), in regard to the megasporophyll of
Dacrydium , in some species of which the behaviour of the bundles is very
similar to that of Podocarpus. With regard to the branching of the ovular bundles, Favre (7) has
rather vaguely described their behaviour in Podocarpus sinensis , where he
says the ovular bundle divides a little below the summit of the organ into
a large number of branches which extend downwards in the plane of
separation of the integuments and so form a vascular cupule which is
prolonged up to the point where the nucellus becomes free. In P. spinulosus
and P. alpinus the (presumably) integumentary bundles are much less
developed, and in Podocarpus sp. they are practically not developed at all. How the ovular bundles are to be homologized with those found in
the ovules of other Gymnosperms (11) there are not at present sufficient
data to decide. Havre’s species would apparently throw light on this
question, but his description is too vague to be of much help. Moreover
Strasburger’s description of P . chinensis (19), which is apparently the same
species as P. sinensis, is almost identical with our description of P. spinulosus. In P. spinulosus and P. alpinus there seems to be an outer integu¬
mentary system arising from the chalazal bundles. Whether there is an
inner integumentary system as well cannot yet be decided, but what facts
there are available point against it. General
Considerations. The relationships of Podocarpus have already been discussed at some
length in the course of this paper. The genus seems to be a recently,
modified type with close relationships to Dacrydium, Micro each rys, and
Saxegothaea. Its present wide geographical range, the wings on the
pollen -grains, the frequently functionless nature of the resin canals, and
the peculiar structure of the female fructification are all considerations
which point to this conclusion. Microcachrys and Saxegothaea are un¬
doubtedly more primitive than Dacrydium and Podocarpus. Saxegothaea
would appear to be a connecting link with the Araucarieae, and the complex
of prothallial cells in the pollen-grains of the Podocarpeae generally can
be brought into relation to the numerous prothallial cells described for
the pollen-grains of the Araucarieae. Miss Robertson (14) has pointed out that the somewhat isolated genus
Phyllocladus has definite points of resemblance to Podocarpus, on the one and
Stiles. —
The
Structure
of 316 hand, and relationships less marked, on the other, to the Taxeae. It
would seem to us that the differences between Phyllocladus and the
Taxeae are greater than Miss Robertson supposes — two of her four points
of resemblance between them being of an unsatisfactory nature. Thus
the markings of the centripetal wood of Phyllocladus are practically the
same as those in the transfusion tracheides of Podocarpus , and the presence
of a certain amount of centripetal wood in Phyllocladus is not sufficient
evidence of phylogenetic relationship. There are considerable differences in the two groups between the
structure of the male cones, and these differences become no less apparent
when we consider the male gametophytes. The only point of resemblance
between the female fructifications of the two groups is the generally fleshy
consistency of the ripe seeds. Phyllocladus is certainly near the Taxeae in its
female fructification, because its symmetrical arillus, like that of the latter
group, only develops at a late stage. There seem to be no striking resem¬
blances in anatomical characters between the Podocarpeae and the Taxeae. Thus the points of similarity between the Podocarpeae and the Taxeae
do not seem to us to be very striking, though, as has been supposed in the
past, the two groups may perhaps be connected by Phyllocladus. With regard to the relationships of the Podocarpeae with the
Abietineae, there appears to be no evidence of any recent phylogenetic
connexion between the two groups. General
Considerations. The structure of the male gameto¬
phytes in the two groups has already been discussed, and the anatomical
characters both of the female reproductive structures and of the vegetative
organs are very different in these two orders of Coniferae. Summary. 1. The structure of the stem and leaf of Podocarpus spinulosus is similar
to that of other species described respectively by Penhallow and Worsdell. 1. The structure of the stem and leaf of Podocarpus spinulosus is similar
to that of other species described respectively by Penhallow and Worsdell. 2. The structure of the microsporangium is similar to that of Saxe - to that of other species described respectively by Penhallow and Worsdell. 2. The structure of the microsporangium is similar to that of Saxe -
gothaea and Araucaria , but the line of dehiscence is oblique. 2. The structure of the microsporangium is similar to that of Saxe -
gothaea and Araucaria , but the line of dehiscence is oblique. 3. Several prothallial cells are present in the pollen-grains of P. spinu-
losus , as in other species of the Podocarpeae investigated. The walls of
the prothallial cells break down at a later stage and their nuclei come to
be free in the cytoplasm. 4. The generative cell of the pollen-grain sometimes divides in the
same way as that described by Jeffrey and Chrysler as occurring in
P. ferrugineus and P. dacrydioides. 5. The course of the vascular bundles in the female fructification is
described in detail. One bundle from the peduncle passes into the mega-
sporophyll, while a pair of bundles, one on each side of the sporophyll
trace, unite and serve the ovule. The bundle thus formed has its xylem
and phloem inversely orientated as compared with the megasporophyll d
l
{
h) Podocarpus
spinulosus
,
{Smith)
R
. 317 bundle. At the chalazal end of the ovule it divides to form ultimately
about four bundles which soon die out. 6. Podocarpus is regarded as a modified genus derived from a type
similar to Saxegothaea. 7. The Podocarpeae are a natural group with no very definite con¬
nexions with the Taxeae, though Phyllocladus may form a connecting link. There is no evident relationship between the Podocarpeae and the Abie-
tineae. It is likely that the former are connected with the Araucarieae by
means of Saxegothaea. p
8. Gothan, W. : Zur Anatomie lebender und fossiler Gymnospermen- Holzer. Berlin, 1905. Jeffrey, E. C., and Chrysler, M. A.: The Microgametophyte of the Podocarpineae.
Amer. Nat., xli, p. 355, 1907. 7. Favre, E. : Recherches sur la fleur femelle du Podocarpus sinensis. Ann. des Sci. Nat., Bot.,
s^r. 5, iii, p. 379, 1865.
h
l b
d
df
l
l
l Brooks and Stiles. — The Structure of Podocarpus. 3 1 10. Noren, C. O. : Zur Kenntnis der Entwicklung von Saxegothaea conspicua, Lindl. Sven
Botanisk Tidskrift, ii, p. ioi, 1908. p
11. Oliver, F. W. : The Ovules of the Older Gymnosperms. Ann. Bot., xvii, p. 451, 1903. 12. Penhallow, D. P. : The Anatomy of the Coniferales. Amer. Nat., xxxviii, 1904. y
A. Engler, Das Pflanzenreich, iv, 5. Taxaceae, p. 13. Pilger, R. : In A. Engler, Das Pflanzenreich, iv, 5. Taxaceae, p. 54, 1903. 14. Robertson, A. : Some Points in the Morphology of Phyllocladus alpinus, Hook. Ann. Bot.,
xx, p. 259, 1906. p
15. - : The Taxoideae: a Phylogenetic Study. New Phytologist, vi, p. 92, 1907. 16. Seward, A. C. and Ford, Miss S. O. : The Araucarieae, Recent and Extinct. Phil. Trans. Roy. Soc., ser. B, cxcviii, p. 305, 1906. 17. Stiles, W. : The Anatomy of Saxegothaea conspicua , Lindl. New Phytologist, vii, p. 209,
1
90S. 18. Strasburger, E. : Die Coniferen und die Gnetaceen. Jena, 1872. 19. - : Die Angiospermen und die Gymnospermen. Jena, 1879. ’ 20. Thibout, E. : Recherches sur l’appareil male des Gymnospermes. Lille, 1896. 21. Thomson, R. B. : Preliminary note on the Araucarieae. Science, N.S., xxii, p. 88, 1905. 22. - : The Araucarieae — a Protosiphonogamic Method of Fertilization. Science,
N.S., xxv, p. 271, 1907. p
23. - : On the Pollen of Microcachrys tetragona. Bot. Gaz., xlvii, p. 26, 1909. 24. - : The Megasporophyll of Saxegothaea and Microcachrys. Bot. Gaz., xlvii,
P- 345* i9°9- 25. Tison, A. : Lanucelle stigmatifere et la pollinisation chez le Saxe-Gothaea conspicua. Compt
Rendus, cxlvii, p. 137, 1908. p
26. - : Sur le Saxe-Gothaea conspicua , Lindl. Mem. de la Soc. Linn, de Normandie,
xxiii, p. 139, 1909. p
27. Van Tieghem, P. : Anatomie comparee de la fleur femelle et du fruit des Cycadees, des
Coniferes et des Gnetacees. Ann. des Sci. Nat., s^r. 5, x, p. 269, 1869. p
28. Veitch : Manual of the Coniferae. Edited by A. Kent, London, 1900. 29. Worsdell, W. C. : On Transfusion Tissue. Trans. Linn. Soc., ser. 2, v, p. 301, 1897. ,
,
,
, p
,
30. - : Observations on the Vascular System of the Female Flowers of Coniferae. Ann. Bot., xviii, p. 527, 1899. 31. Young, Miss M. S. : The Male Gametophyte of Dacrydium. Bot. Gaz., xliv, p. 189, 1907. Botany School, Cambridge,
October , 1809. Postscript. — Since this paper was sent in for publication we have
received Tison’s second paper on Saxegothaea (26). This author considers
the epimatium and ovular stalk in Podocarpus as together equivalent
to the epimatium in Saxegothaea. He also refers to two figures of the
ovule of Saxegothaea published by one of us (17), which both show an
epimatium ‘ qui enveloppe completement l’ovule et son tegument a la fagon
de celui des Dacrydium et des Podocarpus .’ One of these figures was
a diagram to show the ovular supply arising as a branch from the sporophyll
bundle and was not intended to show details of ovular structure. Never¬
theless, inasmuch as it represented the epimatium as present on the under
side of the ovule, it was incorrect. The other figure showed a section of
the ovule cut more or less parallel to the cone scale, and so showed the
epimatium present on both sides ; it did not show an epimatium completely
surrounding the ovule. Moreover, only on Tison’s view can it be con¬
sidered as proved that the epimatium completely surrounds the ovule in
Podocarpus. 1. Bernard, C. : Le Bois centripete dans les feuilles des Coniferes. Beihefte zum Botanischen
Centralblatt, xvii, p. 293, 1904. p
2. Bertrand, C. E. : Anatomie des Gn^tac^es et des Coniferes. Ann. des Sci. Nat., Bot., ser. 5,
xx, p. 5, 1874. 3. Beust. : Untersuchungen iiber fossile Holzer aus Gronland. Allgem. Schweiz. Gesellschaft,
neue Denkschrift, xxix, 1884. 4. Burlingame, L. L. : The Staminate Cone and Male Gametophyte of Podocarpus. Bot. Gaz.,
xlvi, p. 161, 1908. p
5. Coker, W. C. : Notes on the Gametophytes and Embryo of Podocarpus. Bot. Gaz., xxxiii,
p. 89, 1902. 6. Eichler, A. W. : Excursus morphologicus de formatione florum Gymnospermarum. Ann. des
Sci. Nat., Bot., sdr. 4, xix, p. 257, 1863. 7. Favre, E. : Recherches sur la fleur femelle du Podocarpus sinensis. Ann. des Sci. Nat., Bot.,
s^r. 5, iii, p. 379, 1865. p
8. Gothan, W. : Zur Anatomie lebender und fossiler Gymnospermen- Holzer. Berlin, 1905 9. Jeffrey, E. C., and Chrysler, M. A.: The Microgametophyte of the Podocarpineae. Amer. Nat., xli, p. 355, 1907. EXPLANATION
OF
PLATE
XXI. Illustrating Messrs. Brooks and Stiles’s Paper on Podocarpus. Illustrating Messrs. Brooks and Stiles’s Paper on Podocarpus. All the figures are of Podocarpus spinulosus. All the figures are of Podocarpus spinulosus. Brooks, F. T. and Stiles, Walter. 1910. "The structure of Podocarpus
spinulosus, (Smith) R. Br." Annals of botany 24, 305–318. https://doi.org/10.1093/oxfordjournals.aob.a089268. View This Item Online: https://www.biodiversitylibrary.org/item/262605
DOI: https://doi.org/10.1093/oxfordjournals.aob.a089268
Permalink: https://www.biodiversitylibrary.org/partpdf/319776 All the figures are of Podocarpus spinulosus. Fig. 1. Microsporophyll as seen in radial section through the cone, x Fig. 2. Microsporophyll showing oblique dehiscence of sporangia, x 63. Fig. 2. Microsporophyll showing oblique dehiscence of sporangia, x 63. Figs. 3-8. Sections of pollen-grains showing contents. Stained with Heidenhain’s Iron Alum
aematoxylin and Orange G. x 725. Haematoxylin and Orange G. x 725. Fig. 9. Diagram of female fructification indicating the position of the sections shown in the
ollowing
diagrams. Figs. 10-18. Diagrams representing sections through the female fructification at different
levels. Figs. 10-18. Diagrams representing sections through the female fructification at different
levels. Fig. 19. Transverse section of the vascular bundle of a sterile scale of the female fructification
showing two small isolated groups of xylem outside the main bundles. x 300. Fig. 19. Transverse section of the vascular bundle of a sterile scale of the female fructification
showing two small isolated groups of xylem outside the main bundles. x 300. Fig. 20. Transverse section of, presumably, an integumentary vascular bundle showing
a tendency to amphivasal structure, x 300. Fig. 20. Transverse section of, presumably, an integumentary vascular bundle showing
a tendency to amphivasal structure, x 300. c/huvaZs of Botany, c/huvaZs of Botany, Y. T.B. esn d W.S. del. BROOKS Y. T.B. esn d W.S. del. Y. T.B. esn d W.S. del. BROOKS
&
STILES
-
PODOCARPU5
SPINULOSUS. Vol. XXIV. PL. XXI. Vol. XXIV. PL. XXI. Hirth litih et imp. Hirth litih et imp. Holding Institution New York Botanical Garden, LuEsther T. Mertz Library This file was generated 30 March 2024 at 18:48 UTC 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 30 March 2024 at 18:48 UTC
|
https://openalex.org/W2015118259
|
https://zenodo.org/records/577109/files/ZK_article_2653.pdf
|
English
| null |
Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae) from karst poljes in Eastern Herzegovina and Dubrovnik littoral (Bosnia and Herzegovina and Croatia)
|
ZooKeys
| 2,012
|
cc-by
| 13,536
|
Two new freshw
ZooKeys 180: 53–80 (2012)
doi: 10.3897/zookeys.180.2127
www.zookeys.org Two new freshw
ZooKeys 180: 53–80 (2012)
doi: 10.3897/zookeys.180.2127
www.zookeys.org Two new freshw
ZooKeys 180: 53–80 (2012)
doi: 10.3897/zookeys.180.2127
www.zookeys.org s of the genus Telestes
Research article Two new freshwater fish species of the genus Telestes
(Actinopterygii, Cyprinidae) from karst poljes in
Eastern Herzegovina and Dubrovnik littoral
(Bosnia and Herzegovina and Croatia) G. Bogutskaya1,†, Primož Zupančič2,‡, Ivan Bogut3,§, Alexander M. Naseka1,| 1 Zoological Institute, Russian Academy of Sciences, 1 Universitetskaya Emb., St Petersburg 199034, Russia
2 Dolsko 14, 1262 Slovenia 3 Agronomski fakultet u Sveučilišta u Mostaru, Zavod za ribarstvo, zoologiju i
zaštitu voda, Biskupa Čule 10, 88000 Mostar, Bosnia and Hercegovina † urn:lsid:zoobank.org:author:F6957EF1-366E-484E-BF40-211E17A93A1B
‡ urn:lsid:zoobank.org:author:0DC4A949-8AF7-4E80-8305-FDF271318A36
§ urn:lsid:zoobank.org:author:248F52CE-3AC6-4AA4-9EA2-FB5BCD95D7EF
| urn:lsid:zoobank.org:pub:D89D42DE-5B28-48B7-9431-2D603C985CAF Corresponding author: Nina G. Bogutskaya (nbogutskaya@rambler.ru) Academic editor: C. Baldwin | Received 23 September 2011 | Accepted 19 March 2012 | Published 5 April 2012
urn:lsid:zoobank.org:pub:D89D42DE-5B28-48B7-9431-2D603C985CAF Citation: Bogutskaya NG, Zupančič P, Bogut I, Naseka AM (2012) Two new freshwater fish species of the genus Telestes
(Actinopterygii, Cyprinidae) from karst poljes in Eastern Herzegovina and Dubrovnik littoral (Bosnia and Herzegovina
and Croatia). ZooKeys 180: 53–80. doi: 10.3897/zookeys.180.2127 Copyright Nina G. Bogutskaya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0
(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Keywords Cypriniformes, West Balkans, Alpha Taxonomy and new taxa Abstract Two new species, Telestes dabar and T. miloradi, are described on the basis of morphological comparisons
of isolated geographical populations of fishes identified earlier as T. metohiensis. A lectotype is designated
for T. metohiensis, whose range is shown to include waters of Gatačko, Cerničko, and Nevesinjsko poljes
in Eastern Herzegovina. Telestes dabar from Dabarsko Polje (Eastern Herzegovina) and T. miloradi from
Konavosko Polje (south Croatia) share with T. metohiensis the following combination of characters that
distinguish them from the rest of the genus Telestes: pharyngeal teeth in one row, usually 5–4; preoperculo-
mandibular canal not communicating with the infraorbital canal; mouth subterminal, the tip of the mouth
cleft on or below the level of the ventral margin of the eye; postcleithrum minute or absent; ventral por-
tion of the trunk with a dark stripe on a pale background; and dorsal portion of trunk uniformly dark
and bordered ventrally by a dark midlateral stripe. Telestes dabar and T. miloradi are distinguishable from
T. metohiensis in usually having 8½ branched dorsal-fin rays (vs. usually 7½), 9 or 10 gill rakers (vs. 7–10, Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 54 usually 8), and the dark stripe on the ventral portion of the trunk below the main pigmented area of the
back narrow and usually not reaching posteriorly to the caudal peduncle (vs. dark stripe wide and extending
posteriorly to the caudal peduncle). Telestes dabar is distinguished from T. miloradi by having scales on most
of the body situated close to one another and overlapping in a region behind the pectoral girdle and usually
on the caudal peduncle (vs. overlapping scales on most of the body); the lateral line usually incomplete
and interrupted, with 24–69, usually 54–65, total scales (vs. lateral line usually complete, with 55–67 total
scales); scales above and below the lateral line slightly smaller than lateral-line scales (vs. of about equal
size); head width 43–52% HL (vs. 48–58% HL); and lower jaw length 10–12% SL or 36–41% HL (vs. 8–10% SL or 33–38% HL). Telestes miloradi, a very local endemic species, is known only by historical sam-
ples. Telestes dabar is an abundant fish in Dabarsko Polje, but its range is critically restricted during the dry
season by a few permanent sources. Nothing is known about its occurrence in underground karst waters. Introduction Karst basins in Croatia and Bosnia and Herzegovina are populated by many local en-
demic species most of them belonging to the cyprinid subfamily Leuciscinae (Mrakovčić
et al. 2006; Kottelat and Freyhof 2007; Jelić et al. 2008; Zupančič 2008). Among leu-
ciscine cyprinids of the Dinaric karst, most numerous is a group of species that was
formerly assigned to the genus Phoxinellus Heckel, 1843. In a revision of this group
(Zupančič and Bogutskaya 2002; Bogutskaya and Zupančič 2003) ten Phoxinellus spe-
cies were divided into two groups, one with two subgroups. Later (Freyhof et al. 2006),
based on nuclear and mtDNA sequences, Phoxinellus was found to be paraphyletic
because it includes three unrelated monophyletic units. The scientific name Phoxinellus
was therefore restricted to species having plain colouration, a small or absent post-
cleithrum, no genital papilla, and an almost entirely naked body (species included: type
species P. alepidotus Heckel, 1843, P. dalmaticus Zupančič & Bogutskaya, 2000, and P. pseudalepidotus Bogutskaya & Zupančič, 2003). Species having an irregularly spotted
colour pattern, a large postcleithrum, an increased number of precaudal anal-fin ptery-
giophores, and a large female genital papilla were assigned to a new genus, Delminichthys
Freyhof, Lieckfeldt, Bogutskaya, Pitra & Ludwig, 2006 (species included: type species
D. adspersus (Heckel, 1843), D. ghetaldii (Steindachner, 1882), D. jadovensis Zupančič
& Bogutskaya, 2002, and D. krbavensis Zupančič & Bogutskaya, 2002). Finally, species
having a small or absent postcleithrum, no genital papilla, a dark midlateral stripe from
the head to the caudal peduncle, and non-overlapping scales were assigned to Telestes (T. croaticus (Steindachner, 1866), T. fontinalis (Karaman, 1972), and T. metohiensis (Stein-
dachner, 1901)), bringing the total number of species of Telestes to 11. These generic
assignments were later supported by a molecular phylogenetic study based on the same
mitochondrial but different nuclear markers (Perea et al. 2010). Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 55 Zupančič and Bogutskaya (2002) and Bogutskaya and Zupančič (2003) rede-
scribed T. metohiensis based on 251 specimens (including syntypes) from Nevesinjsko
Polje, Gatačko Polje, Cerničko Polje, and Dabarsko Polje karsts in Bosnia and Her-
zegovina, and from Ljuta River in Konavle Region [Konavosko Polje] in southern
Croatia. Bogutskaya and Zupančič (2003: 375) mentioned that the syntypes from
the Ljuta River were distinguished by having 8½ branched anal-fin rays (vs. usually
7½ in most other syntypes). Introduction Specimens from Dabarsko Polje (Vrijeka and Opačica
rivers) were also only tentatively identified as metohiensis because of having overlap-
ping scales (vs. non-overlapping, narrowly to widely spaced) and usually 8½ branched
anal-fin rays (Zupančič and Bogutskaya 2002: 417). Since 2003, we have examined
additional material of the entire T. metohiensis species complex, and two species – one
from Ljuta River in Konavosko Polje and one from Dabarsko Polje – are described
here as new. Methods Measurements were made point to point to the nearest 0.1 mm and follow methods
used by Bogutskaya and Zupančič (2010) for Squalius species. In most aspects, they
coincide with the scheme by Kottelat and Freyhof (2007: fig. 1), but a few clarifica-
tions of the latter are as follows. Standard length (SL) was measured from the anteri-
ormost point of the upper lip (not of the snout) to the end of the hypural complex. Head length (HL) was measured from the anteriormost point of the upper lip to the
posteriormost point of the opercular membrane. Interorbital width was measured
including the skin fold. The term ‘length of dorsal fin’ is used for the length of the
dorsal-fin base, and the term ‘depth of dorsal fin’ is used for the length of the longest
ray of the dorsal fin. Respective terms are used for the measurements of the anal fin. The last two branched rays articulating on a single pterygiophore in the dorsal and
anal fins are noted as “1½”. Total lateral-line scale count includes all pored scales,
from the first one just behind the posttemporal bone to the posteriormost one located
on the bases of the caudal-fin rays. Total number of lateral-row scales includes all
scales, pored and non-pored, from the first one just behind the posttemporal bone to
the posteriormost one located on the bases of the caudal-fin rays. Osteological char-
acters were examined from dissections and from radiographs. Statistical analyses were
performed with Microsoft Excel and Statistica 6.0 packages. Abbreviations used: NMW, Naturhistorisches Museum, Wien; SMNH, Slovenian
Museum of Natural History; PZC, Collection of P. Zupančič, Dolsko (Slovenia); SMF,
Senckenberg Museum, Frankfurt a. Main; ZISP, Zoological Institute, Russian Acad-
emy of Sciences, St Petersburg; ZMH, Zoologisches Museum und Institut, Universität
Hamburg; (cephalic sensory canals) CIO, infraorbital canal; CPM, preoperculo-man-
dibular canal; CSO, supraorbital canal; and CST, supratemporal canal. Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 56 Results Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a Holotype. NMW 95295, 79.1 mm SL, BOSNIA & HERZEGOVINA: Dabarsko
Polje, Opačica River at Potkom, 43°5.9'N, 18°7.6'E, 15 Sept. 2006, coll. Zupančič. Paratypes. NMW 95300, 6, 55.2–71.1 mm SL, same data as holotype; PZC 525,
72, 32.5–51.3 mm SL, same data as holotype; PZC 526, 13, 48.0–73.8 mm SL, same
data as holotype; PZC 565, 21, 35.5–56.7 mm SL, same locality, 8 July 2011; ZISP
54995, 15, 38.1–58.9 mm SL, same locality, 8 July 2011; SMNH 444, 35.5–60.8 mm
SL, same locality, 8 July 2011; PZC 279, 12, 44.2–71.9 mm SL, BOSNIA & HER-
ZEGOVINA: Dabarsko Polje, Vrijeka River, 24 May 2001; PZC 521, 13, 54.0–62.6
mm SL, same locality as 279, 15 Sept. 2004; PZC 575, 18, 40.5–69.8 mm SL, same
locality as 279, 15 Sept. 2006. Paratypes. NMW 95300, 6, 55.2–71.1 mm SL, same data as holotype; PZC 525,
72, 32.5–51.3 mm SL, same data as holotype; PZC 526, 13, 48.0–73.8 mm SL, same
data as holotype; PZC 565, 21, 35.5–56.7 mm SL, same locality, 8 July 2011; ZISP
54995, 15, 38.1–58.9 mm SL, same locality, 8 July 2011; SMNH 444, 35.5–60.8 mm
SL, same locality, 8 July 2011; PZC 279, 12, 44.2–71.9 mm SL, BOSNIA & HER-
ZEGOVINA: Dabarsko Polje, Vrijeka River, 24 May 2001; PZC 521, 13, 54.0–62.6
mm SL, same locality as 279, 15 Sept. 2004; PZC 575, 18, 40.5–69.8 mm SL, same
locality as 279, 15 Sept. 2006. Diagnosis. Telestes dabar is distinguished from T. metohiensis and T. miloradi by
having the following combination of characters: slightly curved dark stripe (obvious in
live and preserved specimens) present from just behind operculum to vertical just ante-
rior to origin of anal fin, this stripe narrow and separated from dark pigmented area on
back along its entire length; scales on most of body not overlapping but situated close
to one another; scales overlapping behind pectoral girdle along lateral line and usually Figure 1. a Telestes dabar, holotype, female, 79.1 mm SL, NMW 95295, Bosnia & Herzegovina:
Opačica River, Dabarsko Polje b Telestes metohiensis, female, 82.1 mm SL, PZC 293, Bosnia & Herzego-
vina: Zovidolka River (Zalomka River system), Nevesinjsko Polje. a
b a b Figure 1. a Telestes dabar, holotype, female, 79.1 mm SL, NMW 95295, Bosnia & Herzegovina:
Opačica River, Dabarsko Polje b Telestes metohiensis, female, 82.1 mm SL, PZC 293, Bosnia & Herzego-
vina: Zovidolka River (Zalomka River system), Nevesinjsko Polje. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 57 Figure 2. a Telestes dabar, male paratype, live specimen, ZISP 54995, 58.9 mm SL, Bosnia & Herzegovina:
Opačica River, Dabarsko Polje b Telestes metohiensis, live specimen, male, 86.2 mm SL, PZC 567, Bosnia &
Herzegovina: spring Ljeskovik in Zalomka River, Nevesinjsko Polje c Telestes metohiensis, live specimen, male,
84.5 mm SL, PZC 566, Bosnia & Herzegovina: Zovidolka River (Zalomka River system), Nevesinjsko Polje. c
a
b a a b b c c c Figure 2. a Telestes dabar, male paratype, live specimen, ZISP 54995, 58.9 mm SL, Bosnia & Herzegovina:
Opačica River, Dabarsko Polje b Telestes metohiensis, live specimen, male, 86.2 mm SL, PZC 567, Bosnia &
Herzegovina: spring Ljeskovik in Zalomka River, Nevesinjsko Polje c Telestes metohiensis, live specimen, male,
84.5 mm SL, PZC 566, Bosnia & Herzegovina: Zovidolka River (Zalomka River system), Nevesinjsko Polje. on caudal peduncle; snout with fleshy tip projecting over upper lip; mouth subtermi-
nal with tip of mouth cleft at or below level of ventral margin of eye; lateral line usually
interrupted, with 24–69 total lateral-line scales; branched dorsal-fin rays usually 8½;
branched anal-fin rays usually 8½; gill rakers 9 or 10; total vertebrae 39–41, mode 40;
abdominal vertebrae 22–24, mode 22; caudal vertebrae 16–18, mode 17; head width
43–52% HL; and lower jaw long, length 10–12% SL. Description. Morphometric data are summarised in Table 1a, selected counts in
Tables 2–4. General appearance can be seen in Figs 1a and 2a. Body compressed, elon- Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 58 Figure 3. a Telestes dabar, alizarin stained specimen, PZC 575, 63.7 mm SL, Bosnia & Herzegovina:
Vrijeka River, Dabarsko Polje b Telestes metohiensis, alizarin stained specimen, 63.3 mm SL, PZC 312,
Bosnia & Herzegovina: spring Ljeskovik in Zalomka River, Nevesinjsko Polje. a
b a
b a Figure 3. a Telestes dabar, alizarin stained specimen, PZC 575, 63.7 mm SL, Bosnia & Herzegovina:
Vrijeka River, Dabarsko Polje b Telestes metohiensis, alizarin stained specimen, 63.3 mm SL, PZC 312,
Bosnia & Herzegovina: spring Ljeskovik in Zalomka River, Nevesinjsko Polje. gate. Caudal-peduncle depth only slightly less than half maximum body depth; head
length greater than maximum body depth. Eye small, its diameter smaller than snout
length. Snout fleshy, slightly to markedly projecting beyond upper lip (similar to a
feature Kottelat and Freyhof [2007: fig. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a 39] called the “rostral cap,” which covers all or
part of upper lip); snout terminating laterally in prominent crease along anterior edge
of first infraorbital. Mouth subterminal, tip of mouth cleft at level of ventral margin
of eye or, more frequently, below it. Lower jaw-quadrate junction at vertical through
anterior half of eye. Length of lower jaw 10–12% SL or 36–41% HL, or 102–132%
depth of operculum. Dorsal fin with 7½ (9 specimens), 8½ (151) or 9½ (1) branched rays, 8½ in holo-
type. Dorsal-fin origin above posterior end of pelvic-fin base. Anal fin with 8½ (153)
or 9½ (8) branched rays, 8½ in holotype. Outer margin of anal fin slightly concave or
almost straight. Caudal fin moderately forked, lobes weakly pointed, with 9+8 princi-
pal branched rays. Total gill rakers (Table 2) 9 (20) or 10 (20), 10 in holotype. Pharyngeal teeth 5–4,
hooked, slightly serrated (examined in 5 specimens). Scales covering entire body including pre-pectoral area and abdomen, non-over-
lapping on most parts of body but overlapping in triangular-shaped area just behind
pectoral girdle and usually on caudal peduncle at least behind anal fin (Fig. 3a); lateral-
line scales always overlapping, sometimes a few posteriormost scales not overlapping. Scales irregularly set but close to one another. Most flank scales oval, somewhat deeper
than long; scales on caudal peduncle more elongated (longer than deep) having promi-
nent posterior attenuation. Trunk scales smaller than lateral-line scales but not con- Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 59 Table 1a. Morphometric data of T. dabar. Table 1a. Morphometric data of T. dabar. T. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 60 Table 1b. Morphometric data of T. miloradi. Table 1b. Morphometric data of T. miloradi. T. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a dabar
Holo-
type
females, n=26
males, n=13
min
max
mean
SD
min
max
M
SD
SL, mm
79.1
60.3
81.8
67.8
55.5
69.0
61.1
Maximum body depth (% SL)
21.6
21.6
26.0
23.6
1.3
21.7
25.0
22.6
0.9
Depth of caudal peduncle (% SL)
9.8
9.8
12.4
11.0
0.7
10.0
12.4
11.2
0.7
Depth of caudal peduncle
(% length of caudal peduncle)
47.9
47.7
59.6
53.6
4.3
44.6
62.2
52.9
5.2
Maximum body width (% SL)
13.4
11.4
16.0
13.7
1.2
12.0
22.0
14.2
2.6
Predorsal length (% SL)
55.3
53.5
58.7
56.9
1.1
53.8
57.3
55.5
1.2
Postdorsal length (% SL)
36.3
32.8
36.6
35.2
1.0
34.4
37.2
35.8
1.0
Prepelvic length (% SL)
51.6
50.4
55.5
52.7
1.4
50.6
54.4
52.2
1.0
Preanal length (% SL)
71.2
68.3
73.2
70.8
1.3
68.6
71.9
69.9
1.0
Pectoral – pelvic-fin origin length (% SL)
24.5
24.5
27.7
25.6
0.9
22.3
26.5
24.1
1.2
Pelvic – anal-fin origin length (% SL)
20.2
17.2
20.2
19.0
0.6
16.6
20.3
18.4
1.3
Length of caudal peduncle (% SL)
20.6
18.5
21.9
20.5
0.8
19.2
23.5
21.2
1.1
Dorsal-fin base length (% SL)
10.3
9.3
12.3
10.9
0.8
9.9
12.2
11.2
0.7
Dorsal fin depth (% SL)
18.9
16.6
20.2
18.6
1.4
18.5
21.8
19.7
0.9
Anal-fin base length (% SL)
12.1
9.2
12.1
10.6
0.8
10.3
12.4
11.2
0.5
Anal fin depth (% SL)
12.3
11.9
14.8
13.2
0.8
13.4
17.2
15.9
1.1
Pectoral fin length (% SL)
19.9
18.1
21.2
19.9
0.8
22.5
26.7
24.2
1.3
Pelvic fin length (% SL)
14.8
12.9
16.3
14.9
0.9
16.3
17.7
16.9
0.5
Head length (% SL)
27.1
25.6
28.7
27.1
0.9
26.6
29.6
28.1
1.0
Head length (% body depth)
125.1 104.0 125.1 115.3
5.9
110.8 135.1 124.3
7.6
Head depth at nape (% SL)
17.4
17.1
19.3
18.1
0.7
17.0
18.8
18.2
0.5
Head depth at nape (% HL)
64.2
62.4
70.7
66.8
2.3
60.6
69.3
65.0
2.7
Maximum head width (% SL)
13.5
11.6
14.6
13.6
0.7
12.4
14.5
13.6
0.6
Maximum head width (% HL)
50.0
42.8
52.3
50.1
2.4
44.7
52.1
48.3
2.4
Maximum cranial width
(% cranium roof length)
68.9
61.9
72.6
65.8
3.1
59.7
68.8
64.4
2.5
Snout length (% SL)
8.5
7.4
9.2
8.1
0.5
7.8
9.0
8.5
0.4
Snout length (% HL)
31.4
27.7
32.8
29.7
1.3
28.5
31.7
30.3
1.1
Eye horizontal diameter (% SL)
6.6
5.9
7.2
6.6
0.4
6.3
7.8
6.8
0.4
Eye horizontal diameter (% HL)
24.4
22.1
26.8
24.5
1.5
22.1
26.8
24.3
1.4
Eye horizontal diameter
(% interorbital width)
74.5
66.3
81.9
73.3
4.7
69.5
82.0
74.4
4.7
Postorbital distance (% HL)
53.0
48.8
54.0
52.2
1.4
47.1
53.0
50.2
1.9
Interorbital width (% SL)
8.9
8.2
9.6
9.1
0.4
8.5
9.9
9.2
0.4
Interorbital width (% HL)
32.8
31.3
36.0
33.4
1.2
29.0
35.6
32.7
1.9
Length of upper jaw (% HL)
28.6
24.5
30.5
27.8
1.9
26.2
29.5
27.7
1.1
Length of upper jaw (% SL)
7.7
6.6
8.5
7.5
0.6
7.0
8.6
7.8
0.5
Length of lower jaw (% SL)
10.6
9.5
11.2
10.2
0.5
9.7
11.5
10.9
0.5
Length of lower jaw (% HL)
39.2
35.8
40.7
37.6
1.3
35.6
41.1
38.6
1.6
Length of lower jaw (% interorbital width) 119.7 106.4 123.4 112.6
4.4
110.2 129.8 120.2
6.4
Length of lower jaw
(% depth of operculum)
110.2 102.4 120.0 108.2
4.5
105.7 131.7 116.6
8.2 Nina G. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a 61 Table 1c. Morphometric data of T. metohiensis. Table 1c. Morphometric data of T. metohiensis. T. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a miloradi
Holo-
type
females, n=9
males, n=3
min
max
mean
SD
min
max
Mean
SD
SL, mm
66.7
34.1
66.7
57.9
61.8
Maximum body depth (% SL)
22.4
21.7
25.8
23.5
1.5
19.6
24.6
23.0
2.9
Depth of caudal peduncle (% SL)
11.4
10.4
11.6
11.2
0.4
9.6
11.3
10.8
1.0
Depth of caudal peduncle
(% length of caudal peduncle)
53.8
49.8
54.8
52.4
2.1
41.8
52.8
49.1
6.4
Maximum body width (% SL)
14.3
12.9
15.3
14.6
0.7
11.4
16.8
15.0
3.1
Predorsal length (% SL)
55.9
55.9
57.7
57.0
0.7
54.3
54.9
54.5
0.3
Postdorsal length (% SL)
35.2
32.8
35.3
34.3
1.0
35.4
35.6
35.5
0.1
Prepelvic length (% SL)
51.6
51.5
53.3
52.3
0.9
50.0
50.2
50.1
0.1
Preanal length (% SL)
72.8
68.2
72.8
70.3
1.9
68.2
68.4
68.3
0.1
Pectoral – pelvic-fin origin length (% SL)
25.1
25.1
27.6
26.2
1.1
23.3
25.0
24.4
1.0
Pelvic – anal-fin origin length (% SL)
20.8
16.6
20.8
18.3
1.7
17.6
18.4
18.2
0.5
Length of caudal peduncle (% SL)
21.2
20.5
22.1
21.3
0.7
21.4
23.0
22.0
0.9
Dorsal-fin base length (% SL)
11.6
9.9
11.6
10.9
0.7
11.2
12.6
12.2
0.8
Dorsal fin depth (% SL)
19.6
19.2
20.9
19.8
0.8
20.9
22.1
21.4
0.6
Anal-fin base length (% SL)
12.4
10.7
12.4
11.9
0.6
11.8
11.9
11.8
0.1
Anal fin depth (% SL)
13.2
0.0
15.0
12.5
4.7
13.7
15.5
14.3
1.0
Pectoral fin length (% SL)
21.7
18.5
21.7
20.8
1.0
23.5
25.4
24.8
1.1
Pelvic fin length (% SL)
15.6
13.6
15.6
14.6
0.8
16.9
17.6
17.2
0.4
Head length (% SL)
26.8
25.4
28.0
27.0
0.8
26.5
29.0
28.2
1.4
Head length (% body depth)
119.9 105.0 119.9 115.0
6.0
117.6 135.4 123.5
10.3
Head depth at nape (% SL)
18.3
16.9
18.3
17.6
0.5
16.7
19.0
18.2
1.4
Head depth at nape (% HL)
68.1
62.5
68.1
65.1
2.3
62.9
65.7
64.7
1.6
Maximum head width (% SL)
14.1
12.3
14.5
14.0
0.7
12.6
16.9
15.4
2.5
Maximum head width (% HL)
52.5
48.7
53.6
52.0
1.5
47.5
58.2
54.6
6.2
Maximum cranial width
(% cranium roof length)
62.8
59.6
73.9
66.0
5.0
58.3
65.2
62.9
4.0
Snout length (% SL)
9.0
8.0
9.7
8.9
0.5
8.5
8.6
8.6
0.0
Snout length (% HL)
33.7
31.4
34.7
33.0
1.2
29.5
32.4
30.5
1.7
Eye horizontal diameter (% SL)
6.5
6.3
6.9
6.5
0.2
6.6
6.7
6.7
0.0
Eye horizontal diameter (% HL)
24.4
22.5
26.4
24.2
1.4
22.9
25.2
23.7
1.3
Eye horizontal diameter
(% interorbital width)
70.2
64.7
83.0
71.3
5.9
69.4
83.4
74.1
8.1
Postorbital distance (% HL)
49.1
48.9
51.6
50.2
1.2
50.2
51.0
50.5
0.5
Interorbital width (% SL)
9.3
8.1
9.7
9.2
0.5
8.0
9.6
9.1
0.9
Interorbital width (% HL)
34.8
31.9
34.8
34.1
0.9
30.2
33.1
32.1
1.7
Length of upper jaw (% HL)
27.9
26.0
28.1
27.2
0.9
23.5
25.1
24.0
0.9
Length of upper jaw (% SL)
7.5
6.6
7.9
7.3
0.4
6.7
6.8
6.8
0.1
Length of lower jaw (% SL)
9.3
8.4
10.4
9.5
0.6
8.7
10.4
9.8
1.0
Length of lower jaw (% HL)
34.7
33.1
38.4
35.3
1.8
32.9
35.9
34.9
1.7
Length of lower jaw (% interorbital width)
99.8
99.8
114.3 103.7
6.1
108.5 109.1 108.7
0.4
Length of lower jaw
(% depth of operculum)
104.0 95.6
104.0 101.1
2.9
95.6
107.1 103.3
6.7 Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 62 Table 1d. Morphometric data of T. metohiensis. Table 1d. Morphometric data of T. metohiensis. T. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a metohiensis, Gatačko and Cerničko poljes
lectotype
females, n=25
males, n=10
min
max
mean
SD
min
max
mean
SD
SL, mm
87.9
60.26 102.1
74.9
54.4
75.7
63.1
Maximum body depth (% SL)
23.9
19.9
25.1
22.5
1.4
20.7
24.1
22.5
1.0
Depth of caudal peduncle (% SL)
11.3
9.7
11.9
10.7
0.5
10.4
11.8
11.2
0.5
Depth of caudal peduncle
(% length of caudal peduncle)
61.5
46.7
61.5
53.0
4.1
46.7
55.7
51.0
3.1
Maximum body width (% SL)
16.0
11.1
16.4
14.3
1.7
14.1
15.6
14.8
0.6
Predorsal length (% SL)
57.6
49.4
60.1
56.5
2.2
53.7
57.7
55.7
1.3
Postdorsal length (% SL)
33.4
31.0
36.5
33.7
1.6
32.5
38.0
35.2
1.6
Prepelvic length (% SL)
52.0
46.0
55.9
52.7
1.9
50.4
53.8
51.9
1.3
Preanal length (% SL)
69.1
65.6
73.6
70.2
1.9
67.1
69.7
68.5
0.9
Pectoral – pelvic-fin origin length
(% SL)
27.5
24.7
29.7
27.0
1.3
24.1
27.2
25.5
1.1
Pelvic – anal-fin origin length (% SL)
19.8
16.4
20.1
18.5
1.1
15.6
18.2
17.0
0.8
Length of caudal peduncle (% SL)
18.3
19.1
22.3
20.3
1.2
20.9
22.8
22.0
0.6
Dorsal-fin base length (% SL)
11.1
9.5
11.5
10.8
0.6
10.8
13.6
12.0
1.0
Dorsal fin depth (% SL)
17.6
14.9
18.3
17.2
0.8
16.3
19.6
18.5
1.1
Anal-fin base length (% SL)
11.3
9.1
12.0
10.9
0.7
10.0
13.2
11.9
1.1
Anal fin depth (% SL)
12.3
12.4
15.3
13.7
0.8
12.8
15.4
14.3
1.0
Pectoral fin length (% SL)
18.4
17.0
19.8
18.4
0.8
22.7
25.3
23.6
0.8
Pelvic fin length (% SL)
12.9
12.4
15.3
13.7
0.8
15.4
17.7
16.6
0.7
Head length (% SL)
27.0
23.8
28.2
26.6
1.1
26.1
28.2
27.3
0.6
Head length (% body depth)
112.9
111.0 132.5 120.4
6.6
114.4 131.8 122.8
5.2
Head depth at nape (% SL)
17.4
15.5
18.7
17.3
0.8
15.9
18.1
17.3
0.7
Head depth at nape (% HL)
64.6
62.9
71.8
65.1
2.2
58.5
66.1
63.5
2.7
Maximum head width (% SL)
14.8
12.9
15.7
14.8
0.7
14.5
15.7
14.9
0.4
Maximum head width (% HL)
54.9
50.6
59.3
55.7
2.7
52.8
56.9
54.7
1.6
Maximum cranial width
(% cranium roof length)
63.5
62.9
78.6
68.6
4.4
65.4
71.7
68.2
2.7
Snout length (% SL)
8.1
7.3
8.8
8.2
0.4
8.5
9.4
8.9
0.4
Snout length (% HL)
30.0
27.9
33.1
30.8
1.6
31.2
34.2
32.5
1.1
Eye horizontal diameter (% SL)
6.0
4.6
6.9
5.9
0.5
5.3
6.8
6.0
0.4
Eye horizontal diameter (% HL)
22.4
19.4
25.2
22.1
1.5
19.5
24.8
22.1
1.8
Eye horizontal diameter
(% interorbital width)
70.7
57.0
80.2
68.2
6.9
60.2
71.8
65.1
4.3
Postorbital distance (% HL)
51.9
47.9
55.6
51.9
2.4
46.2
53.1
50.8
2.3
Interorbital width (% SL)
8.5
7.7
9.2
8.7
0.4
8.4
9.8
9.3
0.5
Interorbital width (% HL)
31.6
29.6
34.8
32.6
1.7
30.8
35.8
34.0
1.5
Length of upper jaw (% HL)
27.8
27.0
31.0
29.2
1.0
27.0
30.9
29.0
1.1
Length of upper jaw (% SL)
7.5
6.4
8.3
7.8
0.4
7.5
8.4
7.9
0.3
Length of lower jaw (% SL)
9.9
8.3
11.3
10.3
0.8
9.5
11.0
10.3
0.6
Length of lower jaw (% HL)
36.7
35.0
42.2
38.7
2.2
34.8
41.2
37.8
2.1
Length of lower jaw
(% interorbital width)
116.0
106.5 132.5 120.6
6.4
101.6 121.9 113.7
6.9
Length of lower jaw
(% depth of operculum)
107.2
106.5 117.9 112.5
3.0
109.6 119.2 112.7
3.5 Nina G. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a 63 Table 2. Gill-raker counts in Telestes dabar, T. miloradi and T. metohiensis. 7
8
9
10
mean
SD
T. dabar
20
20
9.5
0.51
T. miloradi
1
11
6
9.3
0.57
T. metohiensis, Gatačko Polje
2
33
15
1
8.3
0.58
T. metohiensis, Nevesinjsko
Polje
6
28
12
1
8.2
0.67 Table 2. Gill-raker counts in Telestes dabar, T. miloradi and T. metohiensis. siderably so. All scales well ossified, usually visible without staining. In live specimens,
scales clearly visible because of some silver highlights (Fig. 2a). Lateral line complete (2
specimens), long but incomplete (5) or interrupted (33) as in specimen in Fig. 3a; if
interrupted, gaps typically comprising absence of a few scales in a few places, 24–69 in
total (Table 3), 65 with one interruption in holotype. Lateral line making clear curva-
ture above anal-fin origin. Number of scales in total lateral series 62–69 (modal range
65–67), 68 in holotype. Parietal segment of CSO lacking. CPM not communicating with CIO, terminat-
ing over the upper margin of opercular antedorsal process. CSO complete with 8,
rarely 7 or 9, pores. CIO complete with 14–17 pores and with 4 canal openings on
first infraorbital. CPM complete or interrupted between the angulo-articular and pre-
operculum and/or between preoperculum and operculum, CPM with 14–17 pores (4,
rarely 5, canal openings on dentary, and 7–9, usually 8, canal openings on preopercu-
lum). CST complete, with 5–7 pores or narrowly interrupted in middle. p
p
y
p
Total vertebrae (Table 4) 39 (49), 40 (101) or 41 (11), 40 in holotype; abdominal
vertebrae 22 (101), 23 (58) or 24 (2), 22 in holotype; caudal vertebrae 16 (9), 17 (82)
or 18 (70), 18 in holotype; predorsal vertebrae 13 (24), 14 (126) or 15 (11), 14 in
holotype; intermediate vertebrae 3 (123) or 4 (38), 3 in holotype. Most frequent verte-
bral formulae 22+17 (41), 22+18 (60) and 23+17 (40), 22+18 in holotype. p
Colouration. In live specimens, dark back contrasting sharply with pale area
below lateral midline, even in small specimens. Black midlateral stripe extending
from head to caudal peduncle forming ventral border of darkly pigmented area on Table 3. Total lateral-line scale and total lateral-series scales counts in Telestes dabar, T. miloradi and
T. metohiensis. Total lateral-line scales
Total lateral-series
scales
0–53 54–57 58–61 62–65 66–69 70–73 mean
SD
range
mean
SD
T. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a metohiensis (affinis nomen museale), Nevesinjsko Polje
lectotype
females, n=33
males, n=14
min
max
mean
SD
min
max
M
sd
SL, mm
87.9
57.8
113.8
79.5
57.7
86.2
70.4
Maximum body depth (% SL)
23.9
19.3
24.1
21.8
1.1
19.8
23.8
21.6
1.4
Depth of caudal peduncle (% SL)
11.3
9.0
12.0
10.7
0.7
9.6
12.3
11.1
0.7
Depth of caudal peduncle
(% length of caudal peduncle)
61.5
42.7
60.0
54.2
4.5
43.6
59.9
51.8
4.2
Maximum body width (% SL)
16.0
11.6
17.6
14.5
1.4
11.0
16.6
14.3
1.7
Predorsal length (% SL)
57.6
54.2
59.4
57.1
1.3
54.4
58.4
56.3
1.0
Postdorsal length (% SL)
33.4
29.9
36.4
34.0
1.3
33.8
36.5
35.2
0.7
Prepelvic length (% SL)
52.0
51.6
55.1
53.3
1.0
50.1
54.6
51.6
1.2
Preanal length (% SL)
69.1
67.8
73.4
70.9
1.3
65.9
70.3
68.8
1.3
Pectoral – pelvic-fin origin length (%
SL)
27.5
24.2
30.0
25.9
1.2
21.7
25.4
23.8
1.0
Pelvic – anal-fin origin length (% SL)
19.8
16.7
44.1
19.4
4.6
15.6
19.9
17.5
1.3
Length of caudal peduncle (% SL)
18.3
18.0
21.3
19.7
0.9
20.0
23.2
21.4
0.8
Dorsal-fin base length (% SL)
11.1
9.4
12.9
11.1
0.8
8.1
12.1
10.4
1.2
Dorsal fin depth (% SL)
17.6
15.8
20.4
17.9
1.4
15.9
21.8
18.0
1.5
Anal-fin base length (% SL)
11.3
9.4
12.0
10.9
0.6
10.5
13.2
11.4
0.7
Anal fin depth (% SL)
12.3
10.6
15.0
13.1
1.1
12.0
16.2
14.0
1.4
Pectoral fin length (% SL)
18.4
18.0
21.9
20.2
1.0
21.8
26.4
23.8
1.4
Pelvic fin length (% SL)
12.9
12.5
15.6
14.1
0.8
14.2
17.5
15.7
0.9
Head length (% SL)
27.0
26.7
29.4
27.8
0.8
26.3
30.1
28.0
1.1
Head length (% body depth)
112.9
114.5 142.6 127.4
6.6
116.5 142.9 130.0
8.5
Head depth at nape (% SL)
17.4
16.5
18.7
17.6
0.6
15.7
18.8
17.3
0.9
Head depth at nape (% HL)
64.6
60.0
67.9
63.4
1.8
57.1
65.9
62.0
2.9
Maximum head width (% SL)
14.8
13.7
16.6
14.9
0.6
13.3
16.2
14.7
0.8
Maximum head width (% HL)
54.9
50.5
61.2
53.7
2.3
51.0
57.4
53.0
2.4
Maximum cranial width
(% cranium roof length)
63.5
64.9
79.6
72.5
4.3
66.3
75.8
69.1
2.8
Snout length (% SL)
8.1
7.9
9.8
8.7
0.5
7.7
9.3
8.6
0.4
Snout length (% HL)
30.0
29.4
33.4
31.3
1.1
27.2
33.0
30.8
1.6
Eye horizontal diameter (% SL)
6.0
4.7
7.7
6.1
0.7
5.4
8.0
6.3
0.7
Eye horizontal diameter (% HL)
22.4
17.3
27.3
21.9
2.5
20.6
26.4
22.5
2.0
Eye horizontal diameter
(% interorbital width)
70.7
52.0
86.7
67.5
8.3
59.8
92.5
73.1
11.9
Postorbital distance (% HL)
51.9
49.7
57.2
53.3
1.9
48.1
54.4
51.2
1.8
Interorbital width (% SL)
8.5
8.0
10.0
9.0
0.6
7.5
9.9
8.7
0.8
Interorbital width (% HL)
31.6
29.7
35.9
32.5
1.9
26.2
34.8
31.2
2.7
Length of upper jaw (% HL)
27.8
26.7
30.5
28.7
0.9
27.4
29.8
28.5
0.8
Length of upper jaw (% SL)
7.5
7.2
8.7
8.0
0.4
7.3
8.9
8.0
0.5
Length of lower jaw (% SL)
9.9
10.0
12.2
10.7
0.5
10.0
12.5
11.0
0.9
Length of lower jaw (% HL)
36.7
36.3
43.8
38.5
1.5
36.3
44.0
38.9
2.5
Length of lower jaw
(% interorbital width)
116.0
110.0 133.8 121.2
6.0
109.1 138.7 123.0
9.8
Length of lower jaw
(% depth of operculum)
116.5
110.8 133.8 116.1
4.9
111.5 132.3 118.5
7.0 Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a Another black lateral stripe occurring more ventrally, on otherwise pale ventral
portion of trunk; this stripe extending from eye or opercle (or just behind oper-
cle) to at least vertical through point halfway between origins of pelvic and anal
fins, sometimes extending as poorly coalesced spots onto caudal peduncle. Dash-like
black marking present along internal procurrent rays of caudal-fin dorsal lobe, and
elongate black blotch present at bases of 3rd–7th branched rays of dorsal fin. Black
pigment also occurring on rays of dorsal and caudal fins, but its intensity varying
among individuals. Peritoneum black. This general pattern of pigmentation retained
in formaldehyde-fixed and ethanol-preserved specimens. Live specimens collected
from May through September, both males and females, exhibiting yellowish-orange
pigment at bases of all fins, especially pectoral and anal fins, and yellowish pigment
on iris and along anterior, dorsal and posterior margins of operculum. Colouration
of specimens in cold season unknown. back. Another black lateral stripe occurring more ventrally, on otherwise pale ventral
portion of trunk; this stripe extending from eye or opercle (or just behind oper-
cle) to at least vertical through point halfway between origins of pelvic and anal
fins, sometimes extending as poorly coalesced spots onto caudal peduncle. Dash-like
black marking present along internal procurrent rays of caudal-fin dorsal lobe, and
elongate black blotch present at bases of 3rd–7th branched rays of dorsal fin. Black
pigment also occurring on rays of dorsal and caudal fins, but its intensity varying
among individuals. Peritoneum black. This general pattern of pigmentation retained
in formaldehyde-fixed and ethanol-preserved specimens. Live specimens collected
from May through September, both males and females, exhibiting yellowish-orange
pigment at bases of all fins, especially pectoral and anal fins, and yellowish pigment
on iris and along anterior, dorsal and posterior margins of operculum. Colouration
of specimens in cold season unknown. back. Another black lateral stripe occurring more ventrally, on otherwise pale ventral
portion of trunk; this stripe extending from eye or opercle (or just behind oper-
cle) to at least vertical through point halfway between origins of pelvic and anal
fins, sometimes extending as poorly coalesced spots onto caudal peduncle. Dash-like
black marking present along internal procurrent rays of caudal-fin dorsal lobe, and
elongate black blotch present at bases of 3rd–7th branched rays of dorsal fin. Black
pigment also occurring on rays of dorsal and caudal fins, but its intensity varying
among individuals. Peritoneum black. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a dabar
5
17
8
7
3
54.2
9.58
62–69
66.6
2.41
T. miloradi
6
5
5
2
60.3
3.91
58–69
62.8
2.93
T. metohiensis,
Gatačko Polje
11
10
25
5
61.0
3.92
56–71
63.5
3.74
T. metohiensis,
Nevesinjsko Polje
1
9
11
19
5
2
61.1
5.32
60–71
65.0
3.07 e 3. Total lateral-line scale and total lateral-series scales counts in Telestes dabar, T. miloradi an
etohiensis. Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 64 Table 4a. Vertebral counts in Telestes species endemic in Croatia and Bosnia and Herzegovina. Total
vertebrae. Table 4a. Vertebral counts in Telestes species endemic in Croatia and Bosnia and Herzegovina. Total
vertebrae. 38
39
40
41
42
mean
SD
T. dabar
49
101
11
39.8
0.56
T. miloradi
1
10
11
40.5
0.60
T. metohiensis, Gatačko Polje
6
33
12
2
39.2
0.68
T. metohiensis, Nevesinjsko Polje
10
112
35
39.2
0.52
T. croaticus
5
21
38.8
0.40
T. fontinalis
1
7
3
39.2
0.60
T. karsticus
2
2
3
41.1
0.89
T. polylepis
2
9
6
41.2
0.66
T. turskyi
2
9
6
1
40.3
0.77
T. ukliva
8
42.0
0
Table 4b. Vertebral counts in Telestes species endemic in Croatia and Bosnia and Herzegovina. Vertebral
formulae. 38
39
40
41
42
mean
SD
T. dabar
49
101
11
39.8
0.56
T. miloradi
1
10
11
40.5
0.60
T. metohiensis, Gatačko Polje
6
33
12
2
39.2
0.68
T. metohiensis, Nevesinjsko Polje
10
112
35
39.2
0.52
T. croaticus
5
21
38.8
0.40
T. fontinalis
1
7
3
39.2
0.60
T. karsticus
2
2
3
41.1
0.89
T. polylepis
2
9
6
41.2
0.66
T. turskyi
2
9
6
1
40.3
0.77
T. ukliva
8
42.0
0 Table 4b. Vertebral counts in Telestes species endemic in Croatia and Bosnia and Herzegovina. Vertebral
formulae. formulae. 21+17 22+16 22+17 22+18 22+19 23+16 23+17 23+18 23+19 24+16 24+17 24+18 24+19
T. dabar
41
60
8
40
10
1
1
T. miloradi
1
6
1
4
10
T. metohiensis,
Gatačko Polje
6
28
2
5
10
2
T. metohiensis,
Nevesinjsko
Polje
7
58
12
33
24
T. croaticus
2
3
16
5
T. fontinalis
1
1
7
3
T. karsticus
1
1
2
1
2
T. polylepis
1
1
8
4
1
2
T. turskyi
1
4
1
5
6
1
T. ukliva
3
5 back. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a This general pattern of pigmentation retained
in formaldehyde-fixed and ethanol-preserved specimens. Live specimens collected
from May through September, both males and females, exhibiting yellowish-orange
pigment at bases of all fins, especially pectoral and anal fins, and yellowish pigment
on iris and along anterior, dorsal and posterior margins of operculum. Colouration
of specimens in cold season unknown. Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 65 Table 4c. Vertebral counts in Telestes species endemic in Croatia and Bosnia and Herzegovina. Abdominal, caudal and predorsal vertebrae in Telestes dabar, T. mi-
loradi and T. metohiensis. Abdominal vertebrae
Caudal vertebrae
Predorsal vertebrae
21
22
23
24
Mean
SD
15
16
17
18
19
mean
SD
12
13
14
15
mean
SD
T. dabar
101
58
2
22.4
0.51
9
82
70
17.4
0.59
24
126
11
13.9
0.46
T. miloradi
8
14
22.6
0.49
5
16
1
17.8
0.50
15
7
14.3
0.5
T. metohiensis,
Gatačko Polje
36
17
22.3
0.47
7
42
4
16.9
0.46
3
38
12
14.2
0.50
T. metohiensis,
Nevesinjsko Polje
1
92
64
22.4
0.51
2
43
105
7
16.7
0.56
2
124
31
14.2
0.42
T. croaticus
2
19
5
22.1
0.52
8
18
16.7
0.47
21
5
14.2
0.40
T. fontinalis
2
9
22.8
0.40
7
4
16.4
0.50
2
9
14.8
0.40
T. karsticus
1
4
2
23.1
0.69
1
5
1
18.0
0.58
4
3
13.4
0.53
T. polylepis
1
13
3
23.1
0.49
2
11
4
18.1
0.60
1
16
13.9
0.24
T. turskyi
5
13
22.7
0.46
1
6
10
1
17.6
0.62
16
2
13.1
0.32
T. ukliva
3
5
23.6
0.52
5
3
18.4
0.52
2
5
1
12.9
0.64 Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 66 Figure 4. Map of distribution of Telestes dabar (diamond), T. miloradi (star) and T. metohiensis (square)
in karst fields of Eastern Herzegovina and Dubrovnik littoral; white circles show ponors and white circles
with black dots - springs. Figure 4. Map of distribution of Telestes dabar (diamond), T. miloradi (star) and T. metohiensis (square)
in karst fields of Eastern Herzegovina and Dubrovnik littoral; white circles show ponors and white circles
with black dots - springs. Sexual dimorphism. Genital papilla absent in both males and females. Most mor-
phometric characters not significantly different between males and females (Table 1a)
with five exceptions. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a In males, distance between origins of pectoral and pelvic fins long-
er than in females (P<0.0001), dorsal fin deeper (P<0.02), anal fin deeper (P<0.0001),
pectoral fin longer (P<0.0001), pectoral fin often reaching pelvic-fin origin in males,
and pelvic fin longer (P<0.0001), pelvic fin often reaching anal-fin origin in males. Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 67 Figure 5. a Habitat of Telestes dabar: Opačica River at Potkom, Dabarsko Polje (type locality) b Opačica
River 100 m away from the spring (8 July 2011) c–d habitat of Telestes metohiensis: Nevesinjsko Polje;
c – spring Ljeskovik in Zalomka River (8 July 2011) d Zovidolka River at Udbine (8 July 2011). All in
Bosnia and Herzegovina. a
c
d
b b b d Figure 5. a Habitat of Telestes dabar: Opačica River at Potkom, Dabarsko Polje (type locality) b Opačica
River 100 m away from the spring (8 July 2011) c–d habitat of Telestes metohiensis: Nevesinjsko Polje;
c – spring Ljeskovik in Zalomka River (8 July 2011) d Zovidolka River at Udbine (8 July 2011). All in
Bosnia and Herzegovina. In samples collected in May, ripe males with small but prominent conical breeding
tubercles. Tubercles regularly covering entire body, including dorsal and ventral sur-
faces of caudal peduncle, except for ventralmost surface of head. Single tubercle located
on each scale. On all fins (except for caudal fin), tubercles present on both sides along
all rays and on fin membrane, being particularly dense along marginal rays. Tubercles
forming rows along outer margins of operculum and pectoral fin; tubercles in those
rows larger than others on body. Degree of tubercle development varying between
males with regard to both size of tubercles and their location. Tubercles always present
on head, back, and pectoral fin. Males retaining tubercles, though reduced in size and
density, until September. Distribution. The new species is known from two rivers, Vrijeka and Opačica, in
the Dabarsko Polje of Eastern Herzegovina in Bosnia and Herzegovina (Fig. 4). Habitat and biology. From May through September T. dabar is found in shal-
low water of those river sections that are adjacent to and filled from underground Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 68 springs. There is no current, and the water is clean (Fig. 5a). Telestes miloradi sp. n.
l id
b
k Telestes miloradi sp. n. urn:lsid:zoobank.org:act:9A45C6CE-B7BF-4593-BAF6-0D10C45C78EA
http://species-id.net/wiki/Telestes_miloradi
Fig. 7 Holotype. NMW 95296 (ex 51169), 66.7 mm SL; CROATIA: stream Ljuta at Gruda
[misspelt Grinda in Steindachner (1901: 197)), 1901, coll. Kolombatovič. Holotype. NMW 95296 (ex 51169), 66.7 mm SL; CROATIA: stream Ljuta at Gruda
[misspelt Grinda in Steindachner (1901: 197)), 1901, coll. Kolombatovič. Paratypes. NMW 51169, 13 (? syntypes [now paralectotypes] of Paraphoxinus
metohiensis), 31.4–62.6 mm SL, same data as holotype; NMW 51170, 4 (syntypes
[now paralectotypes] of Paraphoxinus metohiensis), 57.9–66.4 mm SL, same data as
holotype; NMW 51171, 3 syntypes [now paralectotypes] of Paraphoxinus metohiensis,
74.6–83.1 mm SL, same data as holotype; NMW 51173, 1 syntype [paralectotype] of
Paraphoxinus metohiensis, 119.3 mm SL, same data as holotype. Diagnosis. Telestes miloradi is distinguished from T. metohiensis and T. dabar
by having the following combination of characters: slightly curved, relatively nar-
row dark stripe (obvious in most preserved specimens) present on ventral portion of
trunk from just behind operculum to vertical at or anterior to origin of anal fin, this
stripe separated from dark pigmented area on back along its entire length; scales on
most of body not overlapping; mouth subterminal with tip of mouth cleft at or be-
low level of ventral margin of eye; snout not fleshy; lateral line complete with 55–67
total scales; branched dorsal-fin rays 8½; branched anal-fin rays 8½; gill rakers usu-
ally 8–10, mode 9; total vertebrae usually 40 or 41; abdominal vertebrae 22–23,
mode 23; caudal vertebrae 16–18, mode 18; head width 48–58% HL, and lower jaw
short, length 8–10% SL. Diagnosis. Telestes miloradi is distinguished from T. metohiensis and T. dabar
by having the following combination of characters: slightly curved, relatively nar-
row dark stripe (obvious in most preserved specimens) present on ventral portion of
trunk from just behind operculum to vertical at or anterior to origin of anal fin, this
stripe separated from dark pigmented area on back along its entire length; scales on
most of body not overlapping; mouth subterminal with tip of mouth cleft at or be-
low level of ventral margin of eye; snout not fleshy; lateral line complete with 55–67
total scales; branched dorsal-fin rays 8½; branched anal-fin rays 8½; gill rakers usu-
ally 8–10, mode 9; total vertebrae usually 40 or 41; abdominal vertebrae 22–23,
mode 23; caudal vertebrae 16–18, mode 18; head width 48–58% HL, and lower jaw
short, length 8–10% SL. Description. Telestes dabar sp. n.
urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A
http://species-id.net/wiki/Telestes_dabar
Figs 1a, 2a, 3a Females with eggs and
just-spent females were caught on 24 May 2001 in Vrijeka River and mature males
and just-spent females on 31 May 2000 in Opačica River. The size of the ripe eggs
was 1.3–1.7 mm in diameter. In all examined samples females predominate. The
smallest spent female was 45.0 mm SL, and the smallest ripe male 43.7 mm SL. No
other fishes were caught in Opačica together with T. dabar while D. ghetaldii were
collected in Vrijeka. Etymology. The specific name, dabar, refers to the type locality, Dabarsko, or Da-
bar Polje; it is a noun in apposition. Telestes miloradi sp. n.
l id
b
k Morphometric data are summarised in Table 1b, selected counts
in Tables 2–4. General appearance can be seen in Figs 7a and 7b. Body compressed,
elongate. Caudal peduncle depth equal to or only slightly less than half maximum
body depth; head length greater than maximum body depth. Eye small, its diam-
eter smaller than snout length. Snout not fleshy, rostral cap covering only part of
upper lip, at least in preserved specimens. Mouth subterminal, tip of mouth cleft Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 69 at level of ventral margin of eye or, more frequently, below it. Lower jaw-quadrate
junction at vertical through anterior half of eye. Length of lower jaw 8–10% SL
or 33–38% HL, or 96–107% depth of operculum (equal to depth of operculum
on average). Dorsal fin with 8½ branched rays. Dorsal-fin origin above posterior end of pelvic-
fin base. Anal fin with 8½ branched rays. Outer margin of anal fin slightly concave. Caudal fin moderately forked, lobes weakly pointed, with 9+8 principal branched rays. Total gill rakers (Table 2) 8 (1 specimen), 9 (11) or 10 (6), 10 in holotype. Pharyngeal
teeth 5–4, hooked, slightly serrated (examined in 5 specimens). Scales covering entire body including pre-pectoral area and abdomen, overlap-
ping on most parts of body. Scales regularly set; lateral-line scales and scales above and
below it of about equal size. Lateral line complete (Table 3), 55–67 scales in total, 61
in holotype. Lateral line not curving above anal-fin origin. Number of scales in total
lateral series 58–67 (modal range 62–64), 63 in holotype. Parietal segment of CSO lacking. CPM not communicating with CIO, terminat-
ing over upper margin of opercular antedorsal process or communicating with CIO
(on one side in 3 specimens). CSO complete with 8, rarely 7 or 9, pores. CIO com-
plete with 14–16 pores and with 4 canal openings on first infraorbital. CPM complete,
with 14–16 pores (4 canal openings on dentary, and 7–9, usually 8, on preoperculum). CST complete, with 5 or 7 pores. Total vertebrae (Table 4) 39 (1), 40 (10) or 41 (11), 41 in holotype; abdominal
vertebrae 22 (8) or 23 (14), 23 in holotype; caudal vertebrae 17 (5), 18 (16) or 19 (1),
18 in holotype; predorsal vertebrae 13 (15) or 14 (7), 14 in holotype; intermediate ver-
tebrae 3 (12) or 4 (9), 3 in holotype. Comparative remarks Telestes dabar, T. miloradi, T. metohiensis, T. croaticus, and T. fontinalis are distinguished
from all congeners by having the pharyngeal teeth in one row, 5–4 or 5–5 (vs. usually
2.5–5.2 or 2.5–4.2), having the preoperculo-mandibular canal terminating in a free
pore at the upper margin of the opercular antedorsal process and not communicating
with the infraorbital canal (vs. communicating), and in lacking a postcleithrum (vs. postcleithrum present). All five species possess a dark midlateral stripe from the head
to the caudal peduncle that forms the ventral border of the darkly pigmented area on
the back. This feature is similar to that in some other Telestes species (Kottelat and
Freyhof 2007: 282–289). Within this group, T. dabar, T. miloradi, and T. metohiensis
differ from T. croaticus and T. fontinalis in having an additional black lateral stripe
occurring on the otherwise pale ventral portion of the trunk. This character was con-
sidered unique for T. metohiensis (Kottelat and Freyhof 2007: 284). In T. dabar and T. miloradi this ventral stripe (Fig. 1a, 2a, 7) is narrow and extends from just behind the
operculum maximally to a vertical through the origin of the anal fin. In T. metohiensis,
the stripe (Fig. 1b, 2b–c) is wide and usually extends posteriorly to the caudal peduncle
where it merges with the main pigmented area. The pale area between the dark area on
the back and the ventral stripe varies in length and depth, being the smallest in females
(Fig. 1b, 2b–c). Besides the presence of the ventral stripe, Telestes dabar and T. miloradi are fur-
ther distinguishable from T. croaticus and T. fontinalis by usually having 8½ branched
dorsal-fin rays (vs. usually 7½). Telestes dabar differs from T. croaticus by usually having
40 total vertebrae (vs. usually 39) (Table 4); a maximum head width of 42–52% HL
(averaging 50% HL in females and 48% HL in males), which is considerably smaller
than the head depth at nape, 61–71% HL (averaging 67% HL in females and 65% HL
in males) (vs. the maximum head width only slightly smaller than the head depth or
about equal to it); and a smaller size, up to 82 mm SL (vs. up to 160 mm). Telestes da-
bar can be further distinguished from T. fontinalis by having 5–4 pharyngeal teeth (vs. Telestes miloradi sp. n.
l id
b
k Vertebral formulae 22+17 (1), 22+18 (7), 23+17
(5) and 23+18 (10), 23+18 in holotype. Colouration. In preserved specimens, dark back contrasting sharply with pale area
below lateral midline. Dark midlateral stripe extending from head to caudal peduncle
forming ventral border of darkly pigmented region on back(faded in some specimens). Another, more conspicuous, dark lateral stripe, occurring on ventral portion of trunk, nar-
row and not extending posterior to vertical through origin of anal fin. Peritoneum dark. i
Sexual dimorphism. Genital papilla absent in both males and females. Most mor-
phometric characters not significantly different between males and females (Table 1). In the three male specimens examined, dorsal fin deeper than in females (P=0.0105);
pectoral fin longer (P<0.001), pectoral fin nearly reaching pelvic-fin origin in males
and well short of pelvic fin in females; and pelvic fin longer (P<0.0001), pelvic fin
almost reaching anal-fin origin in males, well short of anal fin in females.h ii
Distribution. The new species is known from Ljuta River in Konavosko Polje, also
called Konavoska Ljuta, of Dubrovnik littoral (Fig. 4). Only historical NMW samples
are known to us. Etymology. The species is named for Milorad Mrakovčić, Zagreb, in recognition
of his many contributions to the study of freshwater fishes in the Adriatic basin. Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 70 Comparative remarks 5–5); a usually long, though slightly incomplete and narrowly interrupted, lateral line
that reaches the posterior half of the caudal peduncle and has 24–69, usually 54–65,
total scales (vs. a short, incomplete, and widely interrupted lateral line terminating in
the area between the pectoral and anal fins with usually 23–37 total scales); usually 17
or 18 caudal vertebrae (vs. 16); usually 13 or 14 predorsal vertebrae (vs. 15) (Table 4);
3 or 4 intermediate vertebrae (vs. 5); and a moderately compressed body without any
ventral keel (vs. a markedly laterally compressed body and a scaled ventral keel in front
of the pelvic fins). i
Telestes miloradi further differs from T. croaticus and T. fontinalis in usually having
a long, complete lateral line with 55–67 scales vs. an often incomplete and interrupted
lateral line with (18–45)51–70 and (17)23–37(56) scales, respectively. The new spe-
cies further differs from T. croaticus in having 8–10, most frequently 9, gill rakers, (vs. 8–9, most frequently 8); usually 40 or 41 total vertebrae (vs. 38 or 39); 22–23, mode
23, abdominal vertebrae (vs. 21–23, mode 22); and 17–19, mode 18, caudal vertebrae Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 71 (vs. 16–17, mode 17) (Table 4). Telestes miloradi can be further distinguished from T. fontinalis by having 5–4 pharyngeal teeth (vs. 5–5); usually 40 or 41 total vertebrae (vs. 38–40, usually 39); 17–19, usually 18, caudal vertebrae (vs. 16–17, often 16) (Table
4); 3 or 4 intermediate vertebrae (vs. 5); and a moderately compressed body without a
ventral keel (vs. a markedly laterally compressed body and a scaled ventral keel in front
of the pelvic fins).f Telestes miloradi differs from T. croaticus, T. fontinalis, T. metohiensis, and T. dabar
in having comparatively well-overlapped scales, especially on the anterior part of the
flank and on the caudal peduncle; and scales of about the same size in the lateral line
and above and below it (vs. scales usually non-overlapping on most of the body, and
scales above and below the lateral line smaller than the lateral-line scales). The scale
pattern in T. miloradi is very similar to the pattern found in most leuciscine fishes, e.g. T. karsticus Marčić & Mrakovčić, 2011 (Fig. 8), T. turskyi (Heckel, 1843), T. ukliva
(Heckel, 1843). Comparative remarks The presence of overlapping scales is a plesiomorphic feature for the
Leuciscinae (Bogutskaya 1990, 1991), as is an interconnection of CPM and CIO (e.g.,
T. karsticus). In addition to overlapping scales, a few specimens of T. miloradi exhibit
interconnected CPM and CIO. In this study, we compared specimens of T. metohiensis from Gatačko, Cerničko,
and Nevesinjsko poljes and found that specimens from the Nevesinjsko Polje (labeled
as affinis by Steindachner although the name affinis was never published), though
somewhat different in usually having smaller and more scattered scales, are similar
to T. metohiensis from the Gatačko and Cerničko poljes in all other aspects examined
by us (Tables 1c–d, 2–4). We suppose that a reason for Steindachner’s opinion could
be the differences evident between the NMW specimens of T. metohiensis from the
Gatačko Polje (Mušnica River, Gacko) and those from the Nevesinjsko Polje (Zalomka
River) (Fig. 6). Additional material examined by us revealed that the differences noted
above in scales may be size-dependent: larger specimens of T. metohiensis in both poljes
usually have more densely set scales. A specimen of T. metohiensis from Gatačko Polje,
NMW 51176:3 (87.9 mm SL) is designated here as lectotype to ensure taxonomic
stability in the event that T. metohiensis from the Nevesinjsko Polje is recognised as
taxonomically distinct in the future. Both T. dabar and T. miloradi are distinguishable from T. metohiensis by usually
having 8½ branched dorsal-fin rays (vs. usually 7½: 7½ found in 192 specimens and
8½ in 22); 39–41, modes 40 and 41, total vertebrae (vs. 38–40, rarely 41, mode 39);
16–19, modes 17 and 18, caudal vertebrae (vs. 15–17, usually 16 or 17) (vs. 15–17,
usually 16 or 17) (Table 4; differences are statistically significant at P<0.0001); and
more numerous gill rakers, 9 or 10 (vs. (7)8–9(10), most frequently 8) (Table 2). Tel-
estes dabar further differs from T. metohiensis in usually having an interrupted lateral
line with 24–69, usually 54–65, total scales (vs. usually complete with 58–65 scales)
(Table 3; difference is statistically significant at P<0.0001). The scale pattern also dis-
tinguishes T. dabar from T. metohiensis. In T. dabar, the scales (Fig. 3a) are densely set,
but they do not overlap on most parts of the body except behind the pectoral girdle
and on the caudal peduncle; the scales above and below the lateral line are only slightly Nina G. Bogutskaya et al. Comparative remarks metohiensis
Th
h
T l
i
k
f
f
f h 13
i
k
lj
l
d i
a
b
Figure 7. Telestes miloradi, Croatia: Ljuta River at Gruda, Konavosko Polje a Holotype, male, 66.7 mm
SL, NMW 95296 b paratype, female, 119.3 mm SL, NMW 51173. Figure 8. Telestes karsticus, PZC 504, 62.6 mm SL, Croatia: Sušik River, Danube drainage. a
b
Figure 7. Telestes miloradi, Croatia: Ljuta River at Gruda, Konavosko Polje a Holotype, male, 66.7 mm
SL, NMW 95296 b paratype, female, 119.3 mm SL, NMW 51173. a
b
Figure 7. Telestes miloradi, Croatia: Ljuta River at Gruda, Konavosko Polje a Holotype, male, 66.7 mm
SL, NMW 95296 b paratype, female, 119.3 mm SL, NMW 51173. a a b
Figure 7. Telestes miloradi, Croatia: Ljuta River at Gruda, Konavosko Polje a Holotype, male, 66.7 mm
SL, NMW 95296 b paratype, female, 119.3 mm SL, NMW 51173. b Figure 7. Telestes miloradi, Croatia: Ljuta River at Gruda, Konavosko Polje a Holotype, male, 66.7 mm
SL, NMW 95296 b paratype, female, 119.3 mm SL, NMW 51173. Figure 8. Telestes karsticus, PZC 504, 62.6 mm SL, Croatia: Sušik River, Danube drainage. Figure 8. Telestes karsticus, PZC 504, 62.6 mm SL, Croatia: Sušik River, Danube drainage. Figure 8. Telestes karsticus, PZC 504, 62.6 mm SL, Croatia: Sušik River, Danube drainage. Comparative remarks / ZooKeys 180: 53–80 (2012) 72 Figure 6. Telestes metohiensis. a NMW 51176:3, lectotype, 87.9 mm SL, ‘Mušica’ [Mušnica] River b
NMW 51090 (labelled as affinis nomen museale), 57.9 mm SL, Zalomska [Zalomka River]. a
b Figure 6. Telestes metohiensis. a NMW 51176:3, lectotype, 87.9 mm SL, ‘Mušica’ [Mušnica] River b
NMW 51090 (labelled as affinis nomen museale), 57.9 mm SL, Zalomska [Zalomka River]. smaller than the lateral-line scales; and the scales are oval, somewhat deeper than long,
on the flanks and elongated, longer than deep with a prominent posterior attenuation,
on the caudal peduncle. In T. metohiensis the scales are more or less widely spaced and
do not overlap on the entire body except for the lateral line; this feature can be seen in
live specimens, Fig. 2b); the scales (Fig. 3b) above and below the lateral line usually are
considerably smaller than the lateral-line scales; and the scales are almost circular on
both the flanks and the caudal peduncle. Telestes dabar is further distinguished from
T. metohiensis by having a black-and-white general colouration except for yellowish-
orange pigmentation of the fin bases and yellow pigmentation in the iris and along the
operculum in adults (vs. yellowish-green or greenish-bronze pigmentation of the whole
body and fins in both young and adults, Fig. 2b–c). With regard to the morphometric
features, T. dabar is rather similar to T. metohiensis, differing only in having a narrower
head, maximum head width 12–15% SL or 43–52% HL and maximum cranial width
60–73% cranium roof length (vs. 13–17% SL or 51–59% HL, and 64–80% cranium
roof length) (Table 1; differences are statistically significant at P<0.0001).f fi
Besides the characters mentioned above, Telestes dabar differs from T. miloradi in
having the lateral-line scales larger than the scales above and below it (vs. of about
equal size); usually an interrupted lateral line making a sharp curvature upward above
the anal-fin base (vs. complete and making no sharp curvature); the length of lower jaw
10–12% SL, 36–41% HL, 102–132% depth of operculum (vs. 8–10% SL, 33–38%
HL, 96–107% depth of operculum) (Table 1; differences are statistically significant at
P<0.0001). Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 73 f
fi
p
f
g
(
p
yg ,
yp
)
7
Comments on the distribution and conservation of T. dabar, T. miloradi and T. Comments on the distribution and conservation of T. dabar, T. miloradi and T.
metohiensis The three Telestes species are known from four of the 13 main karst poljes located in
Eastern Herzegovina (Bosnia and Herzegovina) and in Dubrovnik littoral area (Croa-
tia) (Fig. 4). These karst poljes are part of a high-karst geotectonic unit known as Di-
naric Karst, which consists of Mesozoic carbonate formations. The depth of soluble and
highly karstified rocks here exceeds 3000 m (Milanović 1981, 2006). A polje (means
“field” in many Slavic languages) is a large closed depression draining underground
with a flat floor. Its streams may be permanent, intermittent and perennial, and, in
natural conditions, a polje is subject to periodic flooding and becomes a lake. In East-
ern Herzegovina and in the Dubrovnik littoral area stepwise poljes are distributed from Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 74 60 m up to 1,080 m above sea level (Fig. 4). Streams and rivers appear from temporary
or permanent springs and sink underground through swallow holes called ponors. In
general, hydro-systems of all poljes under consideration (Fig. 4), except for the Kona-
vosko Polje with direct connection to the Adriatic Sea and the Polje Gradac with no
springs or surface flows, belong to the Neretva drainage area and form a complex net of
underground flows. Within the Neretva drainage, the poljes belong to catchments of
the Buna River (Slato and Nevesinjsko), the Bregava River (Lukavačko and Dabarsko),
and the Trebišnijca River (Gatačko, Fatničko and others). At present, no one polje has
a direct groundwater (surface) flow connection with Neretva or its tributaries. His-
torically, interconnections between variable surface streams and between them and the
main Neretva course occurred during different geological epochs. Fish distributions
can provide some evidence of this. Conversely, very local distribution of some fishes
may indicate the isolation of some surface drainage systems for a long time. y
g
y
g
Slato [Zlato] Polje (1,020‒1,080 m above sea level) is situated at the highest el-
evation of all Eastern Herzegovina poljes. Ćurčić (1915a) reported finding no fishes
at Slato Polje, and no fishes are known from later literature or from museum collec-
tions. The Slato Polje is connected with Nevesinjsko Polje through a narrow valley
that is now dry. The Nevesinjsko Polje, the largest polje in Eastern Herzegovina, has a
surface area of 170 km2 and is located at an elevation from 870 m to 800 m above sea
level. Comments on the distribution and conservation of T. dabar, T. miloradi and T.
metohiensis The lowest point is Biograd Ponor, which is the terminus of the Zalomka River
that starts at Raščelica near the Gatačko Polje. This river has a permanent flow only
between Fojnica and Črni Kuk. Along the river bed downstream from Črni Kuk there
are a lot of ponors. The most prominent leakage zone is in the Rilja section where the
Zalomka is active only 213 days per year, on average (Milanović 1981, 2006). In the
warm season almost the entire river bed of the Zalomka within the Nevesinjsko Polje
is dry, and fishes are found in its upper section only. Only four species are known from
there: Salmo sp., Squalius cf. squalus, Squalius svallize Heckel & Kner, 1858, Phoxinus
sp. (PZ personal observations). Ćurčić (1915a) reported that Paraphoxinus metohienis
was the most numerous species around Fojnica, but at present only Phoxinus sp. was
found there by PZ. Further downstream, T. metohiensis occurs in those very short river
sections that are adjacent to permanent springs such as Ljeskovik. This species also oc-
curs in upper reaches of the Zavidolka River that temporarily flows to the Zalomka in
the east from the Biograd Ponor (our data). Delminichthys ghetaldii is absent from the
Nevesinjsko Polje and the Zalomka system. j
j
Dabarsko Polje, about 20 km long and 1 to 3 km wide, is located close to the
Nevesinjsko Polje but isolated from it. The Dabarsko Polje lies more than 400 m of
elevation below the Nevesinjsko Polje. At present, the Dabarsko Polje is a closed basin
without a possibility for surface runoff. All waters of the Dabarsko Polje catchment
flow through underground karst conduits toward the springs of the Bregava River,
though historically the Polje drained to the Bregava River canyon that is now dry. The
lowest point is the Ponikva Ponor (471 m above sea level), the terminus of a single
permanent stream in the polje, the Vrijeka River, which is only 2.5 km long (Milanović
1981, 2006). The Opačica River located in the northwestern part of the polje is longer Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 75 but intermittent. Only two native species occur in Dabarsko Polje: D. ghetaldii and
T. dabar (recorded earlier under the name Phoxinellus metohiensis) in Vrijeka and only
T. dabar in Opačica (our data). Comments on the distribution and conservation of T. dabar, T. miloradi and T.
metohiensis Delminichthys (as Phoxinellus) ghetaldii was first re-
corded in Sušica and Ljelješnica cave springs by Ćurčić (1915a) and then confirmed by
PZ’s findings in a stream flowing from the Ljelješnica Cave (Zupančič and Bogutskaya
2002) and in the Vrijeka. It is relatively less abundant in Vrijeka than T. dabar. A trout
has been introduced into Vrijeka. j
The small Lukavačko Polje (2.5 km2) is located at an elevation of 852–880 m east
from the Dabarsko Polje. The two poljes are divided by about 400 m of elevation. Fatničko Polje is also a small closed basin (5.6 km2) located southeast from the Dabar-
sko Polje at a much lower elevation, 462–470 m above sea level. The Fatničko Polje is
divided from the Dabarsko Polje by an extremely karstified limestone ridge which is
about 2 km wide. The most prominent karst features of the Fatničko Polje are the Obod
temporary spring and the Pasmica Ponor. About 85% to 90% of the Fatnicko Polje wa-
ter flows to the Trebišnjica springs and 10% to 15% to the Bregava springs (Milanović
1981, 2006). Ćurčić (1915a, 1915b), Taler (1953a, 1953b), Sabioncello (1967) and
Vuković (1977) reported P. metohiensis from the Lukavačko and Fatničko poljes. How-
ever, no extant samples confirm these reports. In 1998–2001, PZ found only D. ghet-
aldii in Fatničko Polje and no D. ghetaldii or T. metohiensis in the Lukavačko Polje
(Zupančič and Bogutskaya 2002). No other data have been received since then. p
g
y
Gatačko Polje (37.6 km2) consists of two geomorphologically and hydrogeologi-
cally interconnected units: Gatačko Polje itself and Small Gatačko Polje. The largest
ponor zone is situated in Small Gatačko Polje along the 8 km long tectonic contact
between flysch sediments and karstified limestone, from Srdevići to the Šabanov Ponor
(936 m above sea level). The entire Gatačko Polje belongs to the catchment area of the
Trebisnjica River, except a very small eastern part. The longest underground flow (35
km) in Eastern Herzegovina is between the Srdevići Ponor and the Trebisnjica Springs. The main flow in the Gatačko Polje is the Mušnica River with its tributary Gračanica. The Mušnica is formed by three streams, Vrba, Ulinjski Potok and Jasenički Potok. They flow from the Cemern and Lebršnik mountains. The Mušnica goes from the east-
ern to the western border of the polje and along its western border southwards before
it completely sinks in the Šabanov Ponor. Comments on the distribution and conservation of T. dabar, T. miloradi and T.
metohiensis The flow of the Mušnica River re-appears in
the Cerničko Polje where it is named the Ključka River. It originates in a large cave,
Vilina Pećina and terminates about 300 m downstream in a ponor that has a recharge
capacity of approximately 20 m3 s−l (Milanović 2006). Telestes metohiensis (in Paraph-
oxinus or Phoxinellus by earlier authors), Phoxinus sp. and Alburnus neretvae Buj, Sanda
et Perea 2010 are known to occur in Mušnica. Phoxinus sp. and, probably, Salmo sp. occur in Gračanica. Many literature sources (see in Zupančič and Bogutskaya 2002)
reported Paraphoxinus metohiensis from the Gračanica River at Gacko, but we know
of no extant samples. Only T. metohiensis was found (PZC) in short karstic streams of
the Cerničko Polje. Telestes metohiensis or close species are absent from other poljes except for the Ko-
navosko Polje. This polje is located rather far in the south from poljes inhabited by T. Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 76 metohiensis and T. dabar. The Konavosko Polje or Polje Konavli is the lowermost polje
in the region (elevation 60 m above sea level) with surface of 48 km2. It is developed
along the most important overthrust of the entire dinaric karst region (“High Karst
Overthrust”). The largest spring Konavoska Ljuta is located at this contact. In natural
conditions it was a temporary flooded polje. Main flows are Ljuta and its tributaries
Konavočica and Opačica. At present, a tunnel between the polje and the sea coast
drains the polje (Milanović 2006). No recent records exist of a Telestes species in the
Ljuta or other springs of the Konavosko Polje. However, we think that efforts are
worth trying to find T. miloradi, a new species, described here from the Konavoska
Ljuta before it is finally considered extinct. It is known that many species from Phox-
inellus, Telestes and Delminichthys genera are able to enter karstic underground waters
during droughts and dramatic water table level fluctuation (Milanović 2006; Jelić,
2008; Marčić et al. 2011). However, this phenomenon probably depends considerably
on the degree of development, size and depth of caverns, siphonic lakes and pools of
estavelles. The ranges of T. metohiensis and T. dabar are extremely small at least during the
dry season and in surface water bodies. The recognition of T. dabar as a distinct species
will require its Red List evaluation and a re-evaluation of T. Comments on the distribution and conservation of T. dabar, T. miloradi and T.
metohiensis metohiensis (now consid-
ered as Vulnerable) because of the loss of a part of its former presumed range. Telestes
dabar would probably deserve the Critically Endangered status because of its very re-
stricted distribution (IUCN 2010). Both species are threatened by habitat destruction,
in particular by construction of tunnels for the draining of poljes and controlling their
inundations, lining of river beds to obstruct water flow into ponors and reversible com-
munication with estavelles. Key to Telestes species of isolated karst river systems of the Adriatic basin in Bos-
nia and Herzegovina and Croatia, including Krbavsko Polje
1a
Pharyngeal teeth in two rows, usually 2.5–5.2 or 2.5–4.2. Preoperculo-man-
dibular canal communicating with infraorbital canal. Postcleithrum present,
of moderate size...........................................................................................2
1b
Pharyngeal teeth in one row, usually 5–4. Preoperculo-mandibular canal not
communicating with infraorbital canal. Postcleithrum minute or absent......3
2a
Branched dorsal-fin rays 8½ and branched anal-fin rays 9½. Total lateral-line
scales 60–68.................................................................................... T. ukliva
2b
Branched dorsal-fin rays 7½ and branched anal-fin rays 8½. Total lateral-line
scales 72–79....................................................................................T. turskyi
3a
Black stripe on ventral portion of trunk narrow and not reaching caudal pe-
duncle. Branched dorsal-fin rays usually 8½................................................4
3b
Black stripe on ventral portion of trunk absent or present; if present, stripe
wide anteriorly and extending posteriorly to caudal peduncle. Branched dor-
sal-fin rays usually 7½..................................................................................5 Key to Telestes species of isolated karst river systems of the Adriatic basin in Bos-
nia and Herzegovina and Croatia, including Krbavsko Polje Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 77 4a 4a
Scales overlapping on most of body, scales approximately same size in, above
and below lateral line. Lateral line not curving above anal-fin origin. Lower-
jaw length 8–10% SL..................................................................T. miloradi
4b
Scales non-overlapping on most of body, though densely set. Lateral-line
scales larger than scales above and below lateral line. Lateral line making clear
curvature above anal-fin origin. Lower-jaw length 10–12% SL........T. dabar
5a
Body strongly compressed, abdomen with weakly scaled ventral keel in front
of pelvics. Lateral line short, incomplete, terminating between pectoral and
anal fins, and usually widely interrupted, with 17–56, usually 23–37, total
scales. Branched anal-fin rays 7½............................................... T. fontinalis
5b
Body slightly compressed, abdomen rounded. Lateral line long, slightly in-
complete or complete, usually terminating on caudal peduncle, and narrowly
interrupted, with 24–70, usually 54–68, total scales. Branched anal-fin rays
usually 8½...................................................................................................6
6a
Black stripe on ventral portion of trunk present......................T. metohiensis
6b
Black stripe on vental portion of trunk absent............................ T. croaticus Comparative material Telestes karsticus. PZC 577, 7, 547–750 mm SL, CROATIA: Sušik River at Tomići,
Dobra River system (Danube drainage), 24 June 2005, coll. Zupančič. Telestes metohiensis. All from BOSNIA AND HERZEGOVINA. Gatačko Polje:
NMW 51176:3, lectotype, mm SL, 87.9 mm SL, ‘Mušica bei Imotski’ [misspelling,
should be Mušnica River, Gatačko Polje; Imotski is probably an error because Steindach-
ner (1901: 198) clearly indicated that the specimens had been received from karst waters
and streams near Gacko, a town in the north of Gatačko Polje (43°09.4'N, 18°31.8'E);
see also Zupančič & Bogutskaya 2002], 1899, coll. Redel, Sturani [Sturany]; NMW
51176:1–2, 72.2–82.5 mm SL, same data as holotype; NMW 12972, 1 paralectotype,
92.2 mm SL, same data as holotype; NMW 12973–75 (in one jar with 12972), 3 para-
lectotypes, 59.2–95.3 mm SL, same data as holotype; NMW 51172, 2 paralectotypes,
92.0–97.8 mm SL, same data as holotype; NMW 51174, 2 paralectotypes, 76.4–88.5
mm SL, same data as holotype; NMW 51175, 3 paralectotypes, 66.8–75.9 mm SL,
same data as holotype; SMF 805, 2, 53.2–65.6 mm SL, Gacko; ZMH 15137, 7, 51.2–
64.4 mm SL, Gacko, Herzegovina; Cerničko Polje: PZC 223, 9, 45.0–90.6 mm SL,
source of Ključka River in Vilina Pečina (cave), 43°5.6'N 18°29'E, 16 May 2003, coll. Zupančič; PZC 330, 9, 46.0–88.7 mm SL, same locality and collector, 23 May 2001;
PZC 337, 4, 73.7–85.4 mm SL, same locality and collector, 20 Aug. 2000; PZC 368, 4,
49.0–62.2 mm SL, spring at Ključ, 43°5.6'N 18°29.6'E, 23 May 2000, coll. Zupančič;
PZC 503, 7, same locality and collector, 3 Aug. 2000. Nevesinjsko Polje: (NMW syn-
types [now paralectotypes] of P. metohiensis labeled as Paraphoxinus affinis [handwritten
by Steindachner; never published], nomen museale) NMW 9368–9372, 5, Zalomska
[Zalomka River], 1896, coll. Hawelka; NMW 51088, 10, 47.6–57.9 mm SL, same Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 78 data as above; NMW 51089, 10, 49.6–58.1 mm SL, same data as above; NMW 51092,
5, 51.9–65.5 mm SL, same data as above; NMW 51093, 8, 47.4–58.7 mm SL, same
data as above; NMW 51094, 10, same data as above; NMW 51090, 5 (not labeled as
syntypes of P. Comparative material metohiensis though exclusion of these specimens from the type series is not
evident from the original description), 47.6–57.9 mm SL, same data as above; NMW
51091, 4 (not labeled as syntypes, as above), 63.5–87.1 mm SL, same data as above;
PZC 206, 3, 50.4–73.6 mm SL, Batuša River (Zalomka system), 43°19.3'N 18°7.1'E,
9 May 2000, coll. Zupančič; PZC 355, 17, 39.6–63.8 mm SL, same locality and collec-
tor, 21 May 2001; PZC 356, 6, 53.2–66.1 mm SL, same locality and collector, 5 May
2000; PZC 312, 19, 50.1–79.8 mm SL, spring Ljeskovik in Zalomka River near Rast
and Odžak, 43°12.1'N 18°12.4'E, 21 May 2001, coll. Zupančič; PZC 313, 14, 49.1–
78.7 mm SL, same locality and collector, 1 July 2002; PZC 567, 7, 45.1–86.2 mm
SL, same locality and collector, 8 July 2011; PZC 358, 2, 38.2–42.0 mm SL, spring
in Zalomka River at Budisavlje, 43°13.3'N 18°13.1'E, 22 May 2011, coll. Zupančič;
PZC 523, 15, 39.5–90.9 mm SL, Zovidolka [Zavodoka, Zovidolska] River at Ud-
bine, 43°8.6'N 18°14.7'E, 15 Sept. 2006, coll. Zupančič; PZC 293, 12, 49.1–99.2 mm
SL, Zovidolka [Zavodoka, Zovidolska] River at Udbine, 43°8.6'N 18°14.7'E, 29 Aug. 2006, coll. Zupančič; PZC 315, 17, 57.8–81.4 mm SL, same locality and collector, 16
July 2002; PZC 523, 15, 39.5–90.9 mm SL, same locality and collector, 15 Sept. 2006;
PZC 524, 13, 39.5–90.9 mm SL, same locality and collector, 15 Sept. 2006; PZC 566,
45, same locality and collector, 8 July 2011. y
y
Telestes polylepis. NMW 18931–41, 11, paralectotypes, 93.6–107.2 mm SL, CRO-
ATIA: Josefsthal [Josipdol], 1866; NMW 49713, 3, syntypes, 86.7–90.7 mm SL,
CROATIA: Dobra River, 1866; NMW 49714–1, lectotype, 85.9 mm SL, CROATIA:
Mresnitza [Mrežnica] River, 1866; NMW 49715, 2, paralectotypes, 80.8–84.1 mm
SL, same data as lectotype. Č yp
Telestes turskyi. NMW 49629-1, lectotype, 129.5 mm SL, CROATIA: Čicola River
[tributary of Krka], Dernis [Drniš] “Heckels Reise, 1840”; 17, paralectotypes, 55.3–
102.3 mm SL. Telestes turskyi. NMW 49629-1, lectotype, 129.5 mm SL, CROATIA: Čicola River
[tributary of Krka], Dernis [Drniš] “Heckels Reise, 1840”; 17, paralectotypes, 55.3–
102.3 mm SL. Telestes ukliva. NMW 49639-1, lectotype, 85.0 mm SL, CROATIA: Sign “[aus der
Cettina]”, “Heckels Reise, 1840”; NMW 49639-2 and 3, 2, 83.3–84.8 mm SL, same
data as lectotype; NMW 49635, 3, paralectotypes, 58.0–75.8 mm SL, same data as
lectotype; ZMH 15095, 1, CROATIA: Cetina. Telestes ukliva. Comparative material NMW 49639-1, lectotype, 85.0 mm SL, CROATIA: Sign “[aus der
Cettina]”, “Heckels Reise, 1840”; NMW 49639-2 and 3, 2, 83.3–84.8 mm SL, same
data as lectotype; NMW 49635, 3, paralectotypes, 58.0–75.8 mm SL, same data as
lectotype; ZMH 15095, 1, CROATIA: Cetina. yp
Other extensive comparative material is listed in Zupančič and Bogutskaya (2002)
and Bogutskaya and Zupančič (2003). yp
Other extensive comparative material is listed in Zupančič and Bogutskaya (2002)
and Bogutskaya and Zupančič (2003). Acknowledgements We are very thankful to N. Ančić for his assistance during field trips. Sincere appre-
ciation goes to B. Herzig, E. Mikschi, C. Prenner and H. Wellendorf (NMW) for
providing materials that have been used for comparisons from the collection under
their care. We appreciate valuable comments on an earlier version of the manuscript Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 79 and linguistic revision by two anonymous reviews, the editor Carole Baldwin, and
Brian Coad (Canadian Museum of Nature). Contribution by NB and AN to the study
was supported by a grant from the Russian Foundation for Basic Research No. 10-04-
01178, All Cypriniformes Species Inventory Project (ACSI-2), and a grant to ZISP
from Ministry of Education and Science of Russian Federation. References Bogutskaya NG (1990) Morphological fundamentals in classification of the subfamily Leucis-
cinae (Cyprinidae). Communication 1. Journal of Ichthyology 30(3): 63–77. Bogutskaya NG (1991) Morphological fundamentals in classification of the subfamily Leucis-
cinae (Cyprinidae). Communication 2. Journal of Ichthyology 31(1): 66–82. Bogutskaya NG, Zupančič P (2003) Phoxinellus pseudalepidotus (Teleostei: Cyprinidae), a
new species from the Neretva basin with an overview of the morphology of Phoxinellus
species of Ctoatia and Bosnia-Herzegovina. Ichthyological Exploration of Freshwaters
14(4): 369–383. Freyhof J, Lieckfeldt D, Bogutskaya NG, Pitra Ch, Ludwig A (2006) Phylogenetic position of
the Dalmatian genus Phoxinellus and description of the newly proposed genus Delminich-
thys (Teleostei: Cyprinidae). Molecular Phylogenetics and Evolution 37: 371–384. Ćurčić V (1915a) Narodno ribarstvo u Bosni i Hercegovini II. Hercegovina. Glasnik Zemalj-
skog Muzeja Bosne i Hercegovine 27: 37–107. [in Serbian] Ćurčić V (1915b) Narodno ribarstvo u Bosni i Hercegovini II. Hercegovina. Glasnik Zemalj-
skog Muzeja Bosne i Hercegovine 27: 313–350. [in Serbian] Heckel JJ (1843) Ichthyologie. In: von Russegger J. Reisen in Europa, Asien und Africa, mit
besonderer Rücksicht auf die naturwissenschaftlichen Verhältnisse der betreffenden Länder
unternommen in den Jahren 1835 bis 1841. Band 1. Theil 2. Reise in Griechenland, Unte-
regypten, im nördlichen Syrien und südöstlichen Kleinasien, mit besonderer Rücksicht auf
die naturwissenschaftlichen Verhältnisse der betreffenden Länder; unternommen in dem
Jahre 1836. E. Schweizerbart, Stuttgart, 991–1099. IUCN (2010) Guidelines for Using the IUCN Red List Categories and Criteria. Version 8.1. Available at http://www.iucnredlist.org/documents/RedListGuidelines.pdf [accessed on 15
August 2011] Jelić D, Duplić A, Ćaleta M, Žuntić P (2008) Endemske vrste riba jadranskog sliva. Agencija
za zaštitu okoliša, Zagreb, 78 pp. [in Croatian] Karaman MS (1972) Süsswasserfische der Türkei. 9 Teil. Revision einiger kleinwüchsiger Cy-
prinidengattungen Phoxinellus, Leucaspius, Acanthobrama usw. aus Südeuropa, Kleineasien,
Vorder-Asien und Nord Africa. Mitteilungen aus dem Hamburgischen Zoologischen Mu-
seum und Institut 69: 115–155. Kottelat M, Freyhof J (2007) Handbook of European freshwater fishes. Kottelat, Cornol, Swit-
zerland and Freyhof, Berlin, Germany, 646 pp. Marčić Z, Buj I, Duplić A, Ćaleta M, Mustafić P, Zanella D, Zupančič P, Mrakovčić M (2011) A
new endemic cyprinid species from the Danube drainage. Journal of Fish Biology 79: 418–430. Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 80 Mrakovčić M, Brigić A, Buj I, Ćaleta M, Mustafić P, Zanella D (2006) Crvena knjiga slatko-
vodnih riba. Hrvatske Ministarstvo Kulture, Državni zavod za zaštitu prirode, Zagreb, 253
pp. [In Croatian] Milanović P (1981) Karst Hydrogeology. References Water Resources Publications, Littleton, Colorado,
434 pp. Milanović PT (2006) Karst of Eastern Herzegovina and Dubrovnik Littoral. ZUHRA, Beo-
grad, 362 pp. Perea S, Böhme M, Zupančič P, Freyhof J, Šanda R, Özuluğ M, Abdoli A, Doadrio I (2010)
Phylogenetic relationships and biogeographical patterns in Circum-Mediterranean sub-
family Leuciscinae (Teleostei, Cyprinidae) inferred from both mitochondrial and nuclear
data. BMC Evolutionary Biology, 2010: 265. doi: 10.1186/1471-2148-10-265 Sabioncello I (1967) Sistematika slatkovodnih riba. In: Livojevic Z, Bojčić C (Eds) Priručnik za
slatkovodno ribarstvo. Agronomski Glasnik, posebna izdanja, 21–90. [in Croatian] Steindachner F (1866) Ichthyologische Notizen (II). Zur Flussfischfauna von Croatien. Sit-
zungsberichte der Mathematisch-Naturwissenschaftlichen Classe der Kaiserlichen Akade-
mie der Wissenschaften 52(1): 594–599. Steindachner F (1901) Bericht über ein Vorkommen einer bisher noch unbeschriebenen Pa-
raphoxinus-Art. Anzeiger der Kaiserlichen Akademie der Wissenschaften. Mathematisch-
Naturwissenschaftliche Classe 38(18): 197–198. Taler Z (1953a) Znamenite i osobite ribe u Bosni i Hercegovini. Ribarski List, Sarajevo, 28:
56–57. [in Croatian] Taler Z (1953b) Rasprostranjenje i popis slatkovodnih riba Jugoslavije. Glasnik Prirodnjačkog
muzeja Srpske Zemlje, Ser. B (Beograd) 5–6: 423–455. [in Croatian] Vuković T (1977) Ribe Bosne i Hercegovine. Svjetlost, Sarajevo, 205 pp. (in Serbian). Zupančič P, Bogutskaya NG (2000) Description of a new species, Phoxinellus dalmaticus (Cy-
prinidae: Leuciscinae), from the Cikola River in the Krka River system, Adriatic basin
(Croatia). Natura Croatia 9(2): 67–81. Zupančič P, Bogutskaya NG (2002) Description of two new species, Phoxinellus krbavensis and
P. jadovensis, re-description of P. fontinalis Karaman, 1972, and discussion of the distribu-
tion of Phoxinellus species (Teleostei: Cyprinidae) in Croatia and in Bosnia and Herzego-
vina. Natura Croatica 11(4): 411–437.
|
https://openalex.org/W2094065609
|
https://europepmc.org/articles/pmc4127975?pdf=render
|
English
| null |
Chemical modification of the 6'‐amino cyclopropyl of abacavir eliminates HLA‐B*57:01‐restricted CD8+ T‐cell activation without loss of antiviral activity
|
Clinical and translational allergy
| 2,014
|
cc-by
| 553
|
© 2014 Alhaidari 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. Authors’ details
1 1University of Liverpool, MRC Centre for Drug Safety Science, UK. 2GlaxoSmithKline, Drug Metabolism & Pharmacokinetics, UK. 1University of Liverpool, MRC Centre for Drug Safety Science, UK. 2GlaxoSmithKline, Drug Metabolism & Pharmacokinetics, UK. Published: 18 July 2014 Published: 18 July 2014 Background Abacavir, a nucleoside reverse transcriptase inhibitor, is
used within drug combination therapy to treat the HIV-1
virus. It is a guanosine-analogue pro-drug, and once meta-
bolised to carbovir, it acts as a DNA chain terminator. Exposure to abacavir is associated with a high frequency
of CD8+ T-cell-mediated hypersensitivity reactions in
individuals carrying the HLA risk allele B*57:01. To acti-
vate T-cells, abacavir interacts directly with endogenous
HLA-B*57:01, altering the repertoire of peptides displayed
on the cell surface. Docking studies show that the amino
cyclopropyl group at the 6-position of abacavir assists in
the arrangement and interaction of peptides with the
MHC binding pocket. We hypothesized that chemical
modification of abacavir at this position would provide a
new series of analogues with potent antiviral activity that
do not activate abacavir-specific CD8+ T-cells. Chemical modification of the 6’-amino cyclopropyl
of abacavir eliminates HLA-B*57:01-restricted CD8+
T-cell activation without loss of antiviral activity
Mohammad Alhaidari1*, Emma Yang1, Neil Berry1, Andrew Owen1, Steve Clarke2, Paul O’Neill1, Dean Naisbitt1,
Kevin Park1 From 6th Drug Hypersensitivity Meeting (DHM 6)
Bern, Switzerland. 9-12 April 2014 determine which analogues activated the T-cells. All of
the abacavir-responsive clones secreted IFN-, in a drug-
concentration-dependent fashion (EC50 [50% of maxi-
mal IFN- spot forming units] less than 20M for all
clones). A minimum of 5 clones were used to compare
abacavir specific responses against the analogues. Abacavir and N-propyl abacavir were equally potent at
activating clones. In contrast, two very closely related
abacavir analogues were devoid of T-cell activity. Conclusion Our findings show that it is possible to block the T-cell
activity of abacavir while maintaining anti-viral activity by
simple chemical modification. Methods Fifteen analogues were synthesized and their anti-viral
activity measured. T cell responses to these analogues
was then measured using CD8+ clones generated from
HLA-B*57:01+ donors. doi:10.1186/2045-7022-4-S3-P40
Cite this article as: Alhaidari et al.: Chemical modification of the 6’-amino
cyclopropyl of abacavir eliminates HLA-B*57:01-restricted CD8+ T-cell
activation without loss of antiviral activity. Clinical and Translational Allergy
2014 4(Suppl 3):P40. Alhaidari et al. Clinical and Translational Allergy 2014, 4(Suppl 3):P40
http://www.ctajournal.com/content/4/S3/P40 Alhaidari et al. Clinical and Translational Allergy 2014, 4(Suppl 3):P40
http://www.ctajournal.com/content/4/S3/P40 POSTER PRESENTATION Open Access 1University of Liverpool, MRC Centre for Drug Safety Science, UK
Full list of author information is available at the end of the article Results Several analogues, including the key closely related com-
pounds, retained antiviral activity, but displayed highly
divergent T-cell responses. Three HLA-B*57:01 positive
blood donors were selected to generate abacavir-responsive
T-cell clones. IFN- release was visualized using ELISpot
to measure abacavir-specific T-cell responses and to 1University of Liverpool, MRC Centre for Drug Safety Science, UK
Full list of author information is available at the end of the article
|
W4245834644.txt
|
https://www.qeios.com/read/C2E5FZ/pdf
| null |
Linseed Oil
|
Definitions
| 2,020
|
cc-by
| 63
|
Qeios · Definition, February 2, 2020
Ope n Pe e r Re v ie w on Qe ios
Linseed Oil
National Cancer Institute
Source
National Cancer Institute. Linseed Oil. NCI T hesaurus. Code C107324.
T he oil extracted from the seeds of Linum usitatissimum. Linseed oil is used as a
nutritional supplement and as a polymerizing resin.
Qeios ID: C2E5FZ · https://doi.org/10.32388/C2E5FZ
1/1
|
|
https://openalex.org/W2177526619
|
https://figshare.com/articles/journal_contribution/A_preposterior_analysis_to_predict_identifiability_in_the_experimental_calibration_of_computer_models/1496544/3/files/2193054.pdf
|
English
| null |
A preposterior analysis to predict identifiability in the experimental calibration of computer models
|
IIE transactions
| 2,015
|
cc-by
| 1,587
|
Online Supplement for "Preposterior Analysis to Predict Identifiability in Experimental
Calibration of Computer Models," by Paul D. Arendt, Daniel W. Apley, and Wei Chen Online Supplement for "Preposterior Analysis to Predict Identifiability in Experimental
Calibration of Computer Models," by Paul D. Arendt, Daniel W. Apley, and Wei Chen Online Supplement for "Preposterior Analysis to Predict Identifiability in Experimental
Calibration of Computer Models," by Paul D. Arendt, Daniel W. Apley, and Wei Chen Sensitivity to the Actual Discrepancy Function and to the Choice of σδ
2 In the MC loop of the algorithm of Figure 1, the hypothetical discrepancy functions are generated
from a GP model. In reality, however, the actual discrepancy functions can take whatever form the
physics of the problem dictate, and we used non-GP discrepancy functions in the examples in Sections
5.1 and 5.2. Here we investigate the sensitivity of the posterior covariance to the actual discrepancy
function for the sinusoidal example of Section 5.2. Specifically, we generate different sets of actual
physical experimental data via Eq. (5) with the eight different discrepancy functions {δk(x): k = 1, 2, . . .,
8} listed in Table S1, some of which are plotted in Figure S1. The first two discrepancy functions are
mathematical functions with different magnitudes but similar shape, and the remaining discrepancy
functions are realizations from GP models with the hyperparameters shown in Table S1. Figure S2 shows
results analogous to Figure 7 for the eight different discrepancy functions. Most of the scatter plots in
Figure S2 exhibit a strong linear trend, which indicates that the preposterior STD provides a reasonable
indication of the relative improvement in identifiability that one can expect from running a larger physical
experiment. Table S1. Different discrepancy functions used in the sinusoidal example. Table S1. Different discrepancy functions used in the sinusoidal example. Discrepancy function
Description
δ1(x)
2
0.25(0.1exp{ } 0.05
)
x
x
−
δ2(x)
2
0.1exp{ } 0.05
x
x
−
δ3(x)
Each δk(x) (k = 3,4,5) is a realization of a GP model with
2
{
,
,
} {0,1,9.26}
δ
δ
δ
β
σ
ω
=
δ4(x)
δ5(x)
δ6(x)
Each δk(x) (k = 6,7,8) is a realization of a GP model with
2
{
,
,
} {0,0.028,9.26}
δ
δ
δ
β
σ
ω
=
δ7(x)
δ8(x) Figure S1. Plots of selected discrepancy functions listed in Table S1. Figure S2. Plot of the posterior STD for the 8 different discrepancy functions versus the preposterior
STD (both normalized by the prior STD). In Step 3 of the MC loop for calculating the preposterior STD,
σδ
2 = 0.028 was used for all 8 cases. The numbers indicate Ne. In the vertical axis, the circles and error
bars represent the mean posterior STD and ± 1 standard deviations of the posterior STD, with respect to
the prior p(θ). Because the parameter σ 2 specified in Step 0c-i dictates the magnitude of the discrepancy Figure S1. Sensitivity to the Actual Discrepancy Function and to the Choice of σδ
2 Plots of selected discrepancy functions listed in Table S1. Figure S1. Plots of selected discrepancy functions listed in Table S1. Figure S2. Plot of the posterior STD for the 8 different discrepancy functions versus the preposterior
STD (both normalized by the prior STD). In Step 3 of the MC loop for calculating the preposterior STD,
σδ
2 = 0.028 was used for all 8 cases. The numbers indicate Ne. In the vertical axis, the circles and error
bars represent the mean posterior STD and ± 1 standard deviations of the posterior STD, with respect to
the prior p(θ). Figure S2. Plot of the posterior STD for the 8 different discrepancy functions versus the preposterior
STD (both normalized by the prior STD). In Step 3 of the MC loop for calculating the preposterior STD,
σδ
2 = 0.028 was used for all 8 cases. The numbers indicate Ne. In the vertical axis, the circles and error
bars represent the mean posterior STD and ± 1 standard deviations of the posterior STD, with respect to
the prior p(θ). page 2
Because the parameter σδ
2 specified in Step 0c-i dictates the magnitude of the discrepancy
functions that are generated when generating the hypothetical physical experimental data within the MC
loop, the sensitivity of the preposterior STD to choice of σδ
2 is of interest. For calculating the preposterior STDs in Figure S2, we used σδ
2 = 0.028 in Step 3 of the MC loop for all 8 cases (regardless of value of
σδ
2 listed in Table S1 for generating the actual discrepancy function). Figure S3 shows analogous results
but using a much larger σδ
2 = 1.0 in Step 3 of the MC loop. Relative to Figure S2, the primary difference
in Figure S3 is that the points are all shifted to the right. This is consistent with what one would expect,
since using a larger value of σδ
2 in Step 3 should result in a larger preposterior STD. Notice that there is
still a strong linear trend in most of the scatter plots in Figure S3. Figure S3. Plot of the posterior STD for the 8 different discrepancy functions versus the preposterior
STD (both normalized by the prior STD). In Step 3 of the MC loop for calculating the preposterior STD,
σδ
2 = 1.0 was used for all 8 cases. Figure S3. Sensitivity to the Actual Discrepancy Function and to the Choice of σδ
2 Plot of the posterior STD for the 8 different discrepancy functions versus the preposterior
STD (both normalized by the prior STD). In Step 3 of the MC loop for calculating the preposterior STD,
σδ
2 = 1.0 was used for all 8 cases. An Additional Example of Using the Preposterior Analysis to Select Ne The beam and sinusoidal examples were used in Section 5.3 to illustrate how to use the
preposterior analysis to select the number Ne of experimental runs. These examples involved only a single
input variable x and used uniformly spaced grid designs. Here we consider an example that uses Latin
hypercube designs with two input variable and a calibration parameter. Suppose the computer model is page 3 page 3 1
2
1
1
2
1
2
( ,
, )
sin(
)
[0,1]
[0,1]
[0,4]
m
y
x x
x
x x
x
x
θ
θ
θ
=
+
∈
∈
∈ and the physical experimental data are generated from the model and the physical experimental data are generated from the model *
1
2
1
2
1
2
2
( ,
)
( ,
,
)
( ,
)
~
(0,0.01 )
e
m
y
x x
y
x x
x x
N
θ
δ
e
e
=
+
+ *
1
2
1
2
1
2
( ,
)
( ,
,
)
( ,
)
e
m
y
x x
y
x x
x x
θ
δ
e
=
+
+ *
1
2
1
2
1
2
2
( ,
)
( ,
,
)
( ,
)
~
(0,0.01 )
e
m
y
x x
y
x x
x x
N
θ
δ
e
e
=
+
+ We began with a Latin hypercube design for the computer experiment with 40 points in three
dimensions {x1, x2, θ}. An optimal Latin hypercube design based on the maximin criterion was used. A
Gaussian process model was fit to the computer experiment data via MLE in Step 0a of Figure 1. For the
physical experimental design specified in Step 0b, we used maximin Latin hypercube designs of various
size Ne = {10, 20, 30, 40, 50} over two dimensions {x1, x2}. Because of the random nature of the Latin
hypercube designs, none of the physical experimental designs had x settings that coincided with the
computer experiment x settings. Next, in Step 0c-i, we set the GP hyperparameters of the discrepancy
function to be βδ = 0, ωδ = 3, and σδ = 0.75, which corresponds to assigning them point mass priors. The
relatively large value of σδ results in a variety of relatively large discrepancy functions being generated
within Step 3 of the preposterior algorithm. An Additional Example of Using the Preposterior Analysis to Select Ne In step 0c-ii, we specified λ = 0.012. Lastly, in Step 0c-iii, we
specified the prior for θ to be uniform over the entire range [0, 4]. From the preposterior analysis, Figure S4 shows the resulting fixed-θ preposterior standard
deviation versus θ t for the designs with different Ne, and Figure S5 shows the (normalized) preposterior
standard deviation as a function of Ne. From Figure S5, the preposterior standard deviation does not
substantially decrease beyond Ne = 30 roughly. page 4 Figure S4. Fixed-θ preposterior STD (normalized by the prior STD of θ) versus θt for Latin hypercube
designs of various size Ne (Ne is specified in the legend). 1
2
3
4
0.4
0.6
0.8
1.0
10
20
30
40
θ t
Normalized STD Normalized STD Figure S4. Fixed-θ preposterior STD (normalized by the prior STD of θ) versus θt for Latin hypercube
designs of various size Ne (Ne is specified in the legend). Figure S5. Preposterior STD (normalized by the prior STD of θ) verses Ne for the Latin hypercube
designs. 10
20
30
40
50
0.4
0.6
0.8
1.0
Ne
Preposterior STD (Normalized) 10
20
30
40
50
0.4
0.6
0.8
1.0
Ne
Preposterior STD (Normalized) Figure S5. Preposterior STD (normalized by the prior STD of θ) verses Ne for the Latin hypercube
designs. page 5
|
https://openalex.org/W4313458682
|
https://www.researchsquare.com/article/rs-2273185/latest.pdf
|
English
| null |
Detection of Microcystins in South African surface waters by high performance liquid chromatography in the light of Quality by Design statical tool.
|
Research Square (Research Square)
| 2,023
|
cc-by
| 10,107
|
Detection of Microcystins in South African surface waters by high
performance liquid chromatography in the light of Quality by Design statical
tool. Abstract Cyanobacteria, an algae bloom that is responsible for the creation of deadly toxins. These toxins have the potential to adversely impact human and animal
health. Microcystins (MC’s), are harmful toxins that are produced by cyanobacteria species. These deadly toxins are the most investigated toxins worldwide. However, in South Africa their occurrence statistics are deficient due to inaccessibility of reliable, sensitive, and precise analytical methods to investigate
them in water surfaces. Thus, a development of analytical methods for the detection and accurate quantification of algal toxins is crucial to consider the
health risk of exposure to toxins in wastewater and surface waters. This study recommends an analytical method for the detection and quantification of
algal toxins using solid phase extraction (SPE) and high-performance liquid chromatography (HPLC) coupled with photodiode detector (PDA). The
developed method was validated based on linearity which ranged from 3–2500 µg L− 1, recoveries at 99.6–113.9%, the limit of detection (LOD) ranging from
0.2–480 µg L− 1, and limit of quantification (LOQ) at 0.7–160 µg L− 1 and the regression coefficients obtained were above 0.9880 in all analytes. The
developed method was applied to Darvill Wastewater Treatment Plant, Umgeni water, Durban surface waters, University of KwaZulu-Natal (UKZN) sport
centre tap water and UKZN Westville two ponds), South Africa. MC-RR was the most detected analyte at concentration ranging from 0.069–30.521 ng L− 1
compared to other targeted MCs. The developed method is simple and cheap, which can consent developing countries to monitor these deadly MC toxins in
water surfaces. Introduction Due to the increase in the population of cyanobacteria in various water resources, recreational activities have been recently forbidden because they are toxic
to biotic components [1]. Cyanobacteria, also known as blue-green algae, occur spontaneously in freshwater [2]. There have been several reports on the
toxicity of cyanobacterial bloom in surface waters worldwide [3],[4],[5],[6],[7],[8],[9],[10]. These microbes use the energy of sunlight and fix inert atmospheric
nitrogen into usable nitrates that can be absorbed by plants [11]. Climate change was discovered to be the most common environmental factor contributing
to the rapid increase of cyanobacteria in water bodies [4],[12]. Due to climate change, heavy rain, residential detergents, sunlight, pH, fossil fuels, and
industrial pollutants were discovered to be the common factor at increasing of bloom, as water becomes rich in nutrients and trigger a rapid increase in
bloom [12],[1]. Many genera of cyanobacteria generate a toxic secondary metabolite called cyanotoxins [13]. These toxins constitute an extensive group of chemical
compounds that are different in their molecular structure and toxicological properties [14]. Amongst these toxins, Microcystins (MCs) were the most
common toxins produced by cyanobacteria in large quantities during algal blooms [1]. Microcystins are cyanotoxins released by cyanobacteria, which can
survive even under extreme conditions such as high or low temperature, sunlight, or in the dark [15]. These MCs were discovered to be the most identified
toxin produced by a vast range of cyanobacterial species [16]. Also, MCs are stable and resistant to extreme pH, even in the presence of common hydrolytic
enzymes due to their cyclic structure [17]. These toxins are cyclic peptides and are one of the most common contributors in the development of primary liver
cancer [18]. MCs’ cyclic structure are noted to be responsible for their stability in temperature and pH [19]. Several variants have been identified in waters
worldwide, such as MC-RR, MC-YR, and MC-LR, which were identified as the most toxic and prevalent worldwide [20]. Researchers described multiple ways in
which human and animal exposure to toxins has resulted in illnesses or deaths [21],[22]. Pathway exposure to toxins includes contact with contaminated water via swimming, intake of contaminated fish and shells, dietary supplements, or
inhalation [23],[22],[24]. Several illnesses or symptoms were reported, such as skin irritation, fever, and respiratory paralysis [21]. Among the three MC
variants, MC-LR was declared the most prevalent, exhibiting 46% of the total concentration of MCs in natural blooms [25]. Research Article License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/20 Introduction Due to its severe and chronic
toxicity, the WHO fixed a guideline value of 1 µg L− 1 maximum of MC-LR in drinking water [26]. The structure of MC-LR is shown in Fig. 1. The U.S Environmental Protection Agency (EPA) has recently evaluated the risk to human health from drinking water contaminated with algal toxins [27],
[16], [28]. In South Africa, no illnesses or deaths of humans and animals are reported due to cyanotoxins. Maybe we are underestimating these toxins, or the
healthcare workers have not been trained to recognise the symptoms. The global occurrence of cyanotoxins in natural water reservoirs promoted the
development of methods and approaches for detecting toxins in environmental waters. Most methods currently developed are different in terms of
sensitivity and specificity [29]. Although, several methods of MC detection were developed and optimised, there are still challenges with respect to finding a
suitable method to detect environmental samples at low concentration. Some of the reported detection methods in the literature are expensive, time consuming and require specialized personnel and other equipment, such as
Liquid Chromatogram Mass Spectrometer (LC-MS) [30]. Hence, High Performance Liquid Chromatogram (HPLC) equipped with Photo Diode Array detector
(PDA) was selected because of its ability to detect and quantify toxins in environmental waters. The SPE-HPLC/PDA method was developed and optimized
to detect and quantify MCs in natural waters and to improve the detection and quantification limits by applying a pre-concentration step in the experiment. Sample Extraction HLB cartridge was conditioned by 3 mL of methanol followed by 3 mL of Milli-Q water. Afterward, the sample was loaded at a flow rate of 15 mL min − 1. Then cartridges were dried under vacuum for a duration of 45 min. The analytes were eluted with 5 mL of methanol and filtered on a 0.45 µm syringe filter. The eluate was then transferred to an amber vial for analysis. Samples were left for 12 hours for evaporation and reconstituted with 1 mL of methanol. Sample Collection Samples were collected from different water surfaces in Durban; UKZN Westville sport centre tap water, UKZN Westville 2 ponds and from Darvill
Wastewater Treatment Plant at uMgeni water in Pietermaritzburg (PMB), all were sampled using 1 L amber bottles to prevent the analyte from degradation. Sample bottles were transported in a cooler box under control condition of 5
to the laboratory and kept in the fridge below 4
for further analysis. ℃
℃ Sample Preparation Samples from the fridge were carried out and left to reach room temperature. Then they were filtered using a gravitational method to remove all sludge and
particles. Due to sludge thickness and the large sample volume used, filtration was completed in 2 hours. The amount of sample used was 100 mL and pH
were adjusted to pH 6 and pH 9 using 0.1 M HCl and 0.1 M NaOH, respectively. Materials And Instruments Oasis HLB cartridge (3 cc, 60 mg adsorbent, 30 µm particle size, Water, from Sigma-Aldrich, (Steinheim, Germany) was optimized using ultra-pure grade
water spiked with MCs standards. Eumax ultrasonic cleaner bath obtained from LABOTEC (Elma, Singen, Germany) was used for degassing the mobile
phase. Edward 8 vacuum pump purchase from Busch (Durban, New Germany, and South Africa) was used for drying the SPE cartridges. Vacuum manifold,
Holdoph purchased from Holdoph (Germany) was used for sample pre-concentration. Different pH levels were obtained using a pH meter (Eutech)
purchased from Labotech (Durban, South Africa) and calibrated with buffers at pH 4, 7, and 10. Analyses were carried out under LC (Shimadzu technology)
purchased from (Kyoto, Japan), equipped with gradient pump. The separation of the analyte was accomplished on a Hypersil Gold C18 (Guard column 30
mm x 2.1 mm, 3 µm particle size), thermostated at 35
to 85
. Ultraviolet (UV) spectra were drawn from 238 to 254 nm. ℃
℃ Apparatus All the following apparatus used were obtained from chemistry stores in University of KwaZulu Natal (UKZN) Westville (Durban, South Africa): such as
conical flask (250 mL), measuring cylinder (100 mL – 1 L), universal bottles (250 mL – 1000 L), beakers (500 mL), glass sample tubes, pasteur pipette, SPE
tubes, filter paper 125 mm diameter and 0.45 µm syringe filter. All glasswares were washed, rinsed with acetone, and heated to a higher temperature of 150
for 24 hours to prevent emulsion. Small volumes were measured by various sizes of micropipette (5 µL – 1000 µL) plus kit Dragon lab (Shaanxi, China)
and larger volumes were measured by volumetric flask. HPLC vials (2 mL) were bought at Chemetrix (Durban, South Africa). ℃ Chemicals and reagents Liquid Chromatogram (LC) grade, methanol (MeOH), acetonitrile (ACN), trifluoroacetic acid (TFA), ≥ 99.9% including MCs standard solution (MC-RR, MC-YR
and MC-LR) were obtained from Sigma-Aldrich (Steinheim, Germany). Hydrochloric (HCl) acid 37% and sodium hydroxide (NaOH) pellets were bought from
Sigma-Aldrich (Durban, South Africa). Hydrochloric acid, HCl (0.1 M), and sodium hydroxide, NaOH (0.1 M) were prepared using an in-house method from
37% HCl and NaOH pellets. Methanol was used to prepare stock solutions of MCs. Ultra-pure water used for toxins extraction was generated by a gradient Page 2/20 Page 2/20 A10 Milli-Q system bought from Microsep (Durban, South Africa). Solutions prepared were stored in the fridge at -5 oC in an amber bottle to prevent them
from degradation. A10 Milli-Q system bought from Microsep (Durban, South Africa). Solutions prepared were stored in the fridge at -5 oC in an amber bottle to prevent them
from degradation. Spiking A 100 mL sample was spiked with different concentrations of 20 µL and 50 µL of 5000 µg L− 1 MCs, respectively. The spiked and un-spiked blank samples
were prepared in triplicate, extracted using the developed extraction method, and analysed on HPLC for matrix-matched validation. Standard Preparation A working solution of 2500 µg L− 1 of MCs was prepared from 5000 µg L− 1 stock solution of the standard by transferring 500 µL into 1 mL amber volumetric
flask and topped up to the mark with methanol. Sequential standards from 3, 5, 10, 36, 60, 120, 250, 375, 750, 1.000, and 2.500 µg L− 1 were prepared in
triplicate from the working solution. All standards for calibration curves were prepared in methanol and analysed on the HPLC instrument. The calibration
curve was established by plotting peak areas against known concentrations of MC for each analyte. Method development The MCs belong to the family of peptides made from seven amino acids [13]. They have different hydrophobicity but depend on two variant amino
acids [35]. Cyanotoxins referred to as molecules that contain an equal number of positively and negatively charged functional groups in a typical pH of 6
relevant to drinking water, making it highly water-soluble [7]. The HPLC/PDA method was developed by optimizing the instrument parameters including,
injection volume, flow rate, pH, and mobile phase. Optimisation of solvent The choice of solvent is important since the wavelength of MCs is lower, which might interfere with the detection and quantification. Methanol, water, and
acetonitrile were investigated because they are normally used as chromatographic solvents. When methanol was used, there was interference because of
the closeness of the methanol wavelength of 220 nm to 238 nm wavelength of MCs. Fig. 4 (a) shows the interference of methanol with the analyte; the red
colour is methanol, the blue and green colour are the analytes and the other colours (yellow, blue, pink) observed are interferences because of methanol
interaction with the targeted analytes. This interference is well depicted by the purity spectrum in Fig. 4 (b), which shows the baseline of methanol (red line)
above the analytes. Fig. 4 (c and d) shows the results of acetonitrile where MCs peaks were clearly visible, and the purity spectrum shows the baseline below the analytes. Acetonitrile and water were chosen as the mobile phase because they did not interfere with the analytes' wavelength. Moreover, this duo yielded better
separation [20]. Separation method The separation of MCs was achieved on the HPLC as supported by the retention times of MC-RR, MC-YR, and MC-LR, with chromatographic peaks at 6.969
min, 7.467 min, and 7.622 min, respectively. The chromatogram shown in Fig. 2 was obtained by injecting 10 µL of 2500 µg L-1 MCs standard in a C18
column. This separation is comparable with the MCs achieved by other researchers in the literature [20]. The UV detector was scanned between 200 to 300
nm, and finally, data was recorded at a 238 nm channel as this wavelength corresponds to the maximum absorption of MCs [36]. Chromatogram obtained
by injecting 10 µL of 2500 µg L-1 microcystins standard of targeted analytes the first analyte detected, this is supported by previous studies where MC-RR was reported as the first eluted Microcystin-RR was the first analyte detected, this is supported by previous studies where MC-RR was reported as the first eluted
analyte [37], [20], [38]. Microcystin-YR and MC-LR analytes were discovered to be very close to each other; therefore, MC-YR was verified by injecting 10 µL-1
of the 2500 µg L-1 MC-YR standard solution. The MC-YR peak, which corresponds to the MCs mixture solution, was found with a retention time of 7.481 min
as shown in Fig. 3. analyte [37], [20], [38]. Microcystin-YR and MC-LR analytes were discovered to be very close to each other; therefore, MC-YR was verified by injecting 10 µL-1
of the 2500 µg L-1 MC-YR standard solution. The MC-YR peak, which corresponds to the MCs mixture solution, was found with a retention time of 7.481 min
as shown in Fig. 3. Instrumental Procedure Prepared standards of MCs were analysed using HPLC to quantify and separate non-volatile samples in a solution under gradient elution mode. Mobile
phase (A) ultra-pure water consisted of 0.025% TFA acid with mobile phase (B) pure ACN consisted of 0.025% TFA acid, were prepared. The initial mobile
phase flow rate was 0.7 mL min− 1 and the injection volume of 10 µL was used for the analyte. The optimized gradient for analyte separation as followed:
20% mobile phase A was set at 10 min and kept constant until mobile phase B reached 80%. The linearity gradient was changed from 80–100% mobile
phase B until 20 min. At 30 min, the mobile phase was decreased to 30% and kept constant for 15 min to recondition the column. Three chromatographic Page 3/20 Page 3/20 peaks representing each MC were observed at less than 10 min. The retention times at which each analyte eluted were 6.969 min, 7.467 min, and 7.622 min,
respectively, for MC-RR, MC-LR, and MC-YR. peaks representing each MC were observed at less than 10 min. The retention times at which each analyte eluted were 6.969 min, 7.467 min, and 7.622 min,
respectively, for MC-RR, MC-LR, and MC-YR. Ecological Risk Determination Risk assessment for MCs have been done by previous researchers using different factors such as predicted no effect concentration (PNEC) derived from
various species and using a sensitivity distribution model toxicity data.[31–33] In this study, the hazard risk quotient (HQ), was derived from based on a
formula which involved the use of the measured environmental concentration (MEC) obtained from this study, and the no observed adverse effect level
(NOAEL) obtained from the toxicity studies done by Fawell KJ et al. 1999.[34] This was done for the ecological risk assessment of MCs (LR, RR and YR)
studied in surface waters and wastewater. HQ = MEC/NOAEL Where the HQ 1, this indicate that there is a high risk on the measured environmental samples hence, it can cause negative effects on the living organisms
on that specific area. In those areas having the HQ greater than 1, it is imperative to take some measures on managing the risk to minimise the risk. If HQ is
0.1–1, then there is a medium risk on the studied area and where HQ 0.1 there is a low potential risk in the area. Optimisation of pH Because toxins can take on different chemical species, such as nickel, mercury, arsenic etc, the pH has a significant impact on their behaviour [19]. Environmental fate is closely tied to both environmental pH and MCs hydrophobicity [19]. It was partially important to optimise the pH as MCs highly
depend on it. Due to climate change, unclean wastewater treatment plants effluents, and other factors mentioned earlier; water pH may vary due to
cyanobacterial blooms [1]. Milli-Q water samples were adjusted to 3 different pH levels (4, 5, and 6) to obtain the optimum pH and achieve better
recoveries. The effect of pH on MCs is shown in Fig. 10, where all three analytes at pH 4 showed higher recoveries above 120%, recovery at pH 4 proved to
be significantly higher than those obtained at (pH 5) and (pH 6). At pH 6, the average percentage recoveries of all targeted analyte were found within the
acceptable range by IUPAC, which is between 60% and 120% in all MC compounds [44]. Extraction method Solid-phase extraction is usually a reliable technique to extract and concentrate samples from complex matrices and sample preparation is a necessary part
of a positive chromatography [43]. Few parameters are crucial for preconcentration, such as sample elution flow rate, sample volume, selected solvent
percentage for elution, and drying duration. All these parameters were investigated in this work. The pH and extraction solvent were found to affect the
recoveries hence these two parameters were optimized. A 100 mL sample was spiked with 20 µL of 5000 µg L-1 standard containing all the targeted MCs
and prepared in triplicate in Milli-Q water. These samples were extracted and analysed as defined in sections 2.8. Optimisation of injection volume The MCs’ peaks depend on the amount of sample volume injected. MCs standard of 2500 µg L-1 was injected with varying volumes between 2 µL and 20
µL. At higher volume, the analyte peaks were not well resolved due to tailing, as observed in Fig. 6 (b). At lower volume, separation of peaks improved, with
MC-YR and MC-LR being resolved, as shown in Fig. 6 (a). However, the signal-to-noise ratio at a low injection volume of 2 µL was elevated. The injection
volume of 10 µL was chosen because it appeared to have a better resolution and symmetric peaks with a lower signal-to-noise ratio (Fig. 2). The injection
volume is normally found to affect the resolution; hence the lower volume than 20 µL is recommended [39]. Instrument stability The instrument stability is performed to ensure the instrument is stable for routine analyse. The stability of the instrument was evaluated on the method
developed by injecting 10 µL of 375 µg L-1 MC standard five times over 30 min intervals. Fig. 8 shows the plot of the peak area versus time. The linearity of
the graph over 180 min confirmed that the instrument is stable enough for routine analysis. Furthermore, to evaluate the stability of the instrument, the
retention times of the analytes over time were compared. The relative standard deviation for peak areas ranged from 1.46 to 2.80, and the obtained results
prove the instrument's stability with a confirmation of a stable signal in all analytes. Optimisation of flow rate The flow rate and other parameters, including mobile phase solvent, and injection volume, were investigated to achieve a concise and precise
chromatographic method. In this study, the mobile phase flow rate of 0.1 mL min-1, 0.5 mL min-1, and 0.7 mL min-1 were assessed by injecting 10 µL of
2500 µg L-1 into an HPLC/PDA instrument. The analytes were eluted between 21.5 to 24 min; 8 to 9.5 min; and 6.9 to 7.5 min, at 0.1 mL min-1, 0.5 mL min-
1
, and 0.7 mL min-1, respectively, as shown in Fig. 7. Retention times decreased with an increase in flow rate because the MCs interacted more with the
mobile phase than the stationary phase. Hence, MCs strongly adsorb with hydrophilic nature [40],[41],[42]. The flow rate of 0.7 mL min-1 was selected
because it yielded narrow peaks with a shorter retention time than other flow rates reported in the literature [20]. Calibration study A sensitive method is needed to detect low concentrations of MCs in environmental waters. A calibration curve was obtained by plotting analyte peak area
versus analyte concentration. The MC standards ranging from 3 to 2500 µg L-1 were prepared in triplicate and analysed as described in the experimental
section above. Fig. 9 shows the calibration curve and regression coefficient analyses for all the analytes above 0.98, with MC-LR having the highest
regression value of 0.9895. MC-RR showed more sensitivity at the 3 µg L-1 detection limit, whereas MC-YR showed the least sensitivity for the method. Development of gradient elution When 100% of water was used, there was no elution of the analytes; hence 100% of acetonitrile was introduced, and elution of analyte peaks was identified,
but there was no separation, as it is shown in Fig. 5 (b). The gradient program was changed to 50-50% of water and acetonitrile, but there was no separation
as shown in Fig. 5 (a). Hence TFA was introduced as an additive in both water and acetonitrile. Elution of peaks was observed, TFA was adjusted to 0.025%,
and peaks were separated as shown in Fig. 4 (c). Although the separation was found between 25% (0.025% TFA in water) and 75% (0.025% TFA in
acetonitrile), the percentage of 0.025% TFA in acetonitrile was increased up to 100% to remove the excess of toxins [36]. Page 4/20 Page 4/20 Intraday and interday precision Method repeatability and interday and intraday precision were validated by analysing 3 MC standards prepared in triplicate. The lowest, middle, and upper
concentrations analysed were 60, 375 and 2500 µg L-1. The obtained results were calculated as the mean percentage of the RSD of the standard solution at
the three concentration levels. Results indicated that the method developed has good precision for the analysis of MCs and is accurate for quantitative
analysis, as shown in Table 2. Instrumental method The instrument detection limit obtained was 10 µg L-1; results are shown in Table 1. Instrumental linearity and linear regression (R2) were established to be
10 µg L-1 to 2500 µg L-1 and 0.9880 to 0.9895, respectively. Limit of quantification and limit of detection The limit of quantification (LOQ) and limit of detection (LOD) are dependent on the sample matrix [51]. In this work, to study the impact of the matrix, 100
mL of drinking water, river water, Milli-Q water, and wastewater were spiked with 20 µL of 5000 µg L-1 MCs standard solution, and the final concentration
after spiking was 1 µg L-1. The results are presented in Table 5. The LOD was found to be lower in drinking water and Milli- Q water compared to river water
and wastewater due to that, in drinking and milli-Q water the matrix is purified. Matrix matched studies proved that the method can be applied in natural
surface waters but in wastewater it had a higher detection limit. Although they were high, but they were still far below the enzyme-linked immunosorbent
assay (ELISA) method. In wastewater, MCs’ recoveries were very low when using a concentration of 1 µg L-1, hence samples were further spiked with 2.5 µg
L-1 to obtain LOD and LOQ. In river waters, error margins were large, this was attributed to concentration levels of MCs already present in river water. Though
error margins were discovered low in Milli-Q and tap water, nevertheless error margins were within acceptable limits in all matrices. Method robustness The Quality by Design (QbD) process is a strategic method and development process. During this process, the capability and efficiency of the method are
ensured. The QbD process involves the systematic design and development of chromatography method. With analytical and risk-management
methodologies, Quality by Design (QbD) improves the ability of the method through a systematic approach to their design, development. The proposed
method critical method parameters (CMP’s) are identified and studied by utilizing the QbD statical tool. The CMPs are studies and proved the method robustness. Design expert software version 13 is used for the QbD approach. The robustness of an analytical
procedure, it is the ability of a method to remain stable even when small and deliberate variations in the method parameters are made, thus providing
evidence of its reliability during normal operation. The current proposed analytical procedure was studied by conducting the Design of Experiments (DoE’s)
in the approach of QbD with design expert software. The proposed method CMPs are identified flow rate (0.7 mL min-1 ± 10) pH of the mobile phase (5.0 ±
0.2) and column temperature (30 ± 5). The selected CMPs are changed deliberately and constructed the DoE and the tool had given total 19 runs by
selecting 3 center points, 2 replicates with zero blocks for 3 factors in the factorial design. The total 19 runs ran on the liquid chromatography and the
selected responses Retention time of MC-RR (R1), resolution between MC-RR and MC-YR (R2) and resolution between MC-YR and MC-LR (R3). The total runs
and responses were tabulated in Table 3. The responses R1, R2 and R3 were analysed by selecting the major effects. The Analysis of Variance (ANOVA)
table was tabulated in Table 4. The ANOVA table clearly shows that the selected design was significant, and the predicted value (P value) found below 0.05
and lack of fit was found not significant. Fig. 11 shows the contour and Pareto charts for all three responses, and it highlights the impact of flow rate on R1,
R2 and pH impact on R3. The impacts were clearly shown on Pareto charts. The variation of the responses is in the acceptable range and express the
method robustness. The model graphs for three responses Half normal plot, 3D surface, and 3D cube was shown in Fig. 12. Optimisation of solvent Due to the hydrophobicity of MCs, typically, methanol and acetonitrile were used for their extraction from an aqueous matrix [45]. Seeing that methanol is
more responsive with MCs in the SPE compared to acetonitrile [20], [46]. Both mentioned solvents were undertaken to study. Each sample was assessed at
an optimum pH of 6 and extracted with each solvent, respectively. Methanol demonstrated a good separation and identification of MCs peaks during Page 5/20 Page 5/20 chromatographic analysis. With acetonitrile as a solvent for the extraction of MCs, peaks were not identified. Hence methanol was chosen as the extraction
solvent for MCs and showed higher efficiency and was comparable to other studies in literature [47],[48],[49],[50]. chromatographic analysis. With acetonitrile as a solvent for the extraction of MCs, peaks were not identified. Hence methanol was chosen as the extraction
solvent for MCs and showed higher efficiency and was comparable to other studies in literature [47],[48],[49],[50]. Application in real water samples Some of the targeted microcystin analytes in these samples were not detected by HPLC. This might be due to that;
they were below the LOD of the developed HPLC method. The amount of MCs concentration found in this study were much lower than the WHO
recommended level of 1 µg L-1 MC-LR [52]. Application in real water samples Samples were collected from Umgeni River, UKZN Sport Centre tap water, laboratory tap water, and UKZN Westville campus ponds and analysed for MCs
using the developed method, the results are shown in Table 6. Microcystins-RR concentration obtained was from ND – 2.160 µg L-1 and MC-YR and MC-LR
were not detected in all points. There are three ponds identified within UKZN premises, named pond 1, 2 and 3 respectively in which algae was observed on
two ponds, thus were sampled to determine the presence of MCs. Pond 1 which is situated near UKZN building premises. The ponds were sampled due to
visibility of algae to investigate the presence of toxins. The results obtained are shown in Table 6 where MC-RR was detected in Pond 2. The higher Page 6/20 Page 6/20 concentration obtained in Pond 2 is a result of algae which was highly visible and moreover there were small fishes observed, which lead to determination
of toxins concentration to ensure the safety of living organisms. concentration obtained in Pond 2 is a result of algae which was highly visible and moreover there were small fishes observed, which lead to determination
of toxins concentration to ensure the safety of living organisms. Various factors contributed to a higher concentration in Pond 2 such as sunlight, pH, temperature, heavy rain, and floods since the Pond is situated next to
human activities and is more exposed to sunlight as a result of higher temperatures which contribute to the increased production of cyanotoxins. To evaluate the concentration of microcystins (µg L-1) from the Darvill wastewater treatment plant, three consecutive campaigns were undertaken for MCs
determination. Analysis was carried out on the HPLC/PDA and MC-RR was the most detected toxin with the concentration ranging from ND to 30.521 µg L-1. The digested thickened WAS from the first campaign samples, MC-YR was detected at 2.015 µg L-1 and MC-LR was not detected. On a second campaign,
the balancing tank and inlet showed a greater concentration from 4.403 – 6.470 µg L-1 MC-RR and MC-YR was detected in the inlet point at 4.938 µg L-1. On
the secondary effluent and Maturation River, MC-RR showed a lower concentration from ND – D compared to other points. On the third campaign, in contrast to MC-RR, which was the most detected across all points at concentrations ranging from ND – 30.521 µg L-1, whereas
MC-YR and MC-LR were not detected. Conclusion A sensitive and accurate analytical method of HPLC/PDA with SPE was established and optimized to pre-concentrate, purify, and detect the MC-RR, MC-YR
and MC-LR in natural water samples. Good separation and high resolution of targeted MCs analysis were achieved under the C18 Hypersil column by using
a suitable solvent (ACN and water) with 0.025% TFA. Most importantly, SPE method was worth it to remove all impurities. During the extraction method, pH
was kept constant at 6. The developed method was validated by evaluation of LOD, LOQ, linearity, accuracy, and precision to meet validation criteria for use
in drinking and surface waters. The method was then successfully employed to wastewater treatment plant samples and Umgeni River in KZN. Recoveries
ranging from 99.6 – 113.9 were obtained at pH 6. Some of the targeted MCs were not detected. Due to reduced algal visibility in winter, MCs are anticipated
to be low [55]. Low levels of toxins were caused by a variety of events, including floods. Severe flooding that recently hit KZN cleaned and drained the water
surfaces, including algae. The analytical method developed is thus applicable for monitoring MCs in an environmental sample and can replace the ELISA
method. Further research is required because these findings are significant for risk assessment. The archival results given in this study will support future
risk assessments for surface waters and assist to locate microcystin levels into a broader ecological context. Competing interest/Competing interests The authors declare that they have no competing interests/competing interests. cological Risk assessment of MCs (LR, RR and YR) in surface w Microcystins are cyanobacterial that blooms from algae as a result of various environmental factors such as light, salinity and human activities.[33, 34]
Thus, it is imperative to assess the ecological risks of these toxins possess living organisms as a results of water contamination. Moreover, the attention of
these studies are performed and stressed to the living species such as the microorganisms.[32, 34] This study uses available data on previous studies done
on the organisms, due to that this minimise the use of other organism species, hence, it is recommended. The value of NOAEL 40 µg L-1 for microcystins
used for the HQ calculations was obtained from the study done by Fawell KJ et al. 1999[34] using mouse, in realistic water conditions. The NOAEL with the
MECs from the study qualified the ecological risk assessment in the points selected on the environment, the results are shown in Table 4. Only the sampling
areas were certain microcystins were quantified, the risk assessment was done. The hazard quotients for the studies areas ranged from 0.0503 - 0.764,
showing the low risk and medium risk. The was no point having high risk, although there on the Inlet, 3rd campaign- Inlet (MC-RR) the HQ is close to one,
this indicates that there is a potential to risk. Comparison of the current study with literature To compare the analytical methods, factors including the column, the mobile phase, the extraction method, the limit of detection, and the concentration level
reported in previous studies were evaluated, as shown in Table 8. Acknowledgements Authors are thankful to the University of KwaZulu-Natal, School of Chemistry and Physics, University Road, Westville 3630, South Africa, for supporting this
work and guidance for the complete research. Declarations Competing interest/Competing interests Ethics approval This article does not contain any studies with animals performed by any of the authors. This article does not contain any studies with animals performed by any of the authors. We authorize to publish the article without any conflict. We authorize to publish the article without any conflict. We authorize to publish the article without any conflict. References 1. de la Cruz, A.A., et al., Introduction to cyanobacteria and cyanotoxins. Water treatment for purification from cyanobacteria and cyanotoxins, 2020: p. 1-
35. 1. de la Cruz, A.A., et al., Introduction to cyanobacteria and cyanotoxins. Water treatment for purification from cyanobacteria and cyanotoxins, 2020: p. 1-
35. 2. Chorus, I. and M. Welker, Toxic cyanobacteria in water: a guide to their public health consequences, monitorin 3. Codd, G.A., et al., Cyanobacteria, Cyanotoxins, and Human Health. Water Treatment for Purification from Cyanobacteria and Cyanotoxins, 2020: p. 37-
68. 3. Codd, G.A., et al., Cyanobacteria, Cyanotoxins, and Human Health. Water Treatment for Purification from Cyanobacteria and Cyanotoxins, 2020: p. 37-
68. 3. Codd, G.A., et al., Cyanobacteria, Cyanotoxins, and Human Health. Water Treatment for Purification from Cyan
68. 4. Sukharevich, V.I. and Y.M. Polyak, Global Occurrence of Cyanobacteria: Causes and Effects (Review). Inland W 4. Sukharevich, V.I. and Y.M. Polyak, Global Occurrence of Cyanobacteria: Causes and Effects (Review). Inland Water Biology, 2020. 13(4): p. 566-575. 4. Sukharevich, V.I. and Y.M. Polyak, Global Occurrence of Cyanobacteria: Causes and Effects (Review). Inland Water Biology, 2020. 13(4): p. 566-575. 5. Czyżewska, W., M. Piontek, and K. Łuszczyńska, The Occurrence of Potential Harmful Cyanobacteria and Cya
Source of Drinking Water. Toxins, 2020. 12(5): p. 284. 5. Czyżewska, W., M. Piontek, and K. Łuszczyńska, The Occurrence of Potential Harmful Cyanobacteria and Cyanotoxins in the Obrzyca River (Poland), a
Source of Drinking Water. Toxins, 2020. 12(5): p. 284. 6. Falcone-Dias, M.F., et al., Occurrence of Cyanobacteria and microcystins in hydroelectric reservoirs used for fish farming. 2020. 18(6): p. 983-994. 6. Falcone-Dias, M.F., et al., Occurrence of Cyanobacteria and microcystins in hydroelectric reservoirs used for f et al., Occurrence of Cyanobacteria and microcystins in hydroelectric reservoirs used for fish farming. 2020. 18(6 7. Mchau, G.J., Cyanotoxins occurrence in drinking waters and risk of exposure to human in Ukerewe district Mw toxins occurrence in drinking waters and risk of exposure to human in Ukerewe district Mwanza, Tanzania. 2020 8. Moreira, C., et al., Cyanotoxins occurrence in Portugal: A new report on their recent multiplication. Toxins, 2020. 12(3): p. 154. ., Cyanotoxins occurrence in Portugal: A new report on their recent multiplication. Toxins, 2020. 12(3): p. 154. 8. Moreira, C., et al., Cyanotoxins occurrence in Portugal: A new report on their recent multiplication. Toxins, 202 9. References Wan, X., et al., Occurrence and risk assessment of microcystin and its relationship with environmental factors in lakes of the eastern plain ecoregion,
China. Environmental Science and Pollution Research, 2020. 27(36): p. 45095-45107. 10. Mohan, R., T. Sathish, and K.B. Padmakumar, Occurrence of potentially toxic cyanobacteriaMicrocystis aeruginosain aquatic ecosystems of central
Kerala (south India). Annales De Limnologie-International Journal of Limnology, 2020. 56. 11. Hamlin, Q.F., et al., Quantifying Landscape Nutrient Inputs With Spatially Explicit Nutrient Source Estimate Maps. Journal of Geophysical Research-
Biogeosciences, 2020. 125(2). J.-S. Ki, Impact of Environmental Factors on the Regulation of Cyanotoxin Production. Toxins, 2014. 6(7). 12. Boopathi, T. and J.-S. Ki, Impact of Environmental Factors on the Regulation of Cyanotoxin Production. To 13. Buratti, F.M., et al., Cyanotoxins: producing organisms, occurrence, toxicity, mechanism of action and human health toxicological risk evaluation. Archives of Toxicology, 2017. 91(3): p. 1049-1130. 14. Campos, A. and V. Vasconcelos, Molecular mechanisms of microcystin toxicity in animal cells. International journal of molecular sciences, 2010. 11(1)
p. 268-287. 15. Mantzouki, E., et al., Temperature Effects Explain Continental Scale Distribution of Cyanobacterial Toxins. Toxins, 2018. 10(4). 15. Mantzouki, E., et al., Temperature Effects Explain Continental Scale Distribution of Cyanobacterial Tox 16. Janssen, E.M.L., Cyanobacterial peptides beyond microcystins – A review on co-occurrence, toxicity, and challenges for risk assessment. Water
Research, 2019. 151: p. 488-499. 17. Baghdady, Y.Z. and K.A. Schug, Online comprehensive high pH reversed phase low pH reversed phase approach for two-dimensional separations of
intact proteins in top-down proteomics. Analytical chemistry, 2019. 91(17): p. 11085-11091. 18. Zhao, Y.Y., et al., Long-term environmental exposure to microcystins increases the risk of nonalcoholic fatty liver disease in humans: A combined fisher-
based investigation and murine model study. Environment International, 2020. 138. 9. McCord, J., et al., pH dependent octanol-water partitioning coefficients of microcystin congeners. Journal of W 20. Thuret-Benoist, H., V. Pallier, and G. Feuillade-Cathalifaud, Quantification of microcystins in natural waters by HPLC-UV after a pre-concentration step: 20. Thuret-Benoist, H., V. Pallier, and G. Feuillade-Cathalifaud, Quantification of microcystins in natural waters by HPLC-UV after a pre-concentration step:
validation of the analytical performances and study of the interferences. Environmental toxicology and pharmacology, 2019. 72: p. 103223. 21. Wood, R., Acute animal and human poisonings from cyanotoxin exposure—A review of the literature. Envi 21. Wood, R., Acute animal and human poisonings from cyanotoxin exposure—A review of the literature. Environment international, 2016. 91: p. 276-282. 22. Funding Not applicable Page 7/20 Availability of data and material Availability of data and material All the experimental data was included in the manuscript. References 562: p. 860-868. 55. Hua, S., et al., The monthly variation tendency of microcystin-LR levels in the Huangpu River (China) by applications of ELISA and HPLC. 2022: p. 1-9. 55. Hua, S., et al., The monthly variation tendency of microcystin-LR levels in the Huangpu River (China) by applications of ELISA and HPLC. 2022: p. 1-9. 55. Hua, S., et al., The monthly variation tendency of microcystin-LR levels in the Huangpu River (China) by applications of ELISA and HPLC. 2022: p. 1-9. References Svirčev, Z., et al., Toxicology of microcystins with reference to cases of human intoxications and epidemiological investigations of exposures to
cyanobacteria and cyanotoxins. Archives of toxicology, 2017. 91(2): p. 621-650. 23. Badar, M., et al., effects of microcystins toxins contaminated drinking water on hepatic problems in animals (cows and buffalos) and toxins removal
chemical method. Buffalo bulletin, 2017. 36(1): p. 43-55. 23. Badar, M., et al., effects of microcystins toxins contaminated drinking water on hepatic problems in animals (cows and buffalos) and toxins removal
chemical method. Buffalo bulletin, 2017. 36(1): p. 43-55. 24. Roy-Lachapelle, A., et al., Detection of Cyanotoxins in Algae Dietary Supplements. Toxins, 2017. 9(3). 25. Zegura, B., An Overview of the Mechanisms of Microcystin-LR Genotoxicity and Potential Carcinogenicity. Mini-Reviews in Medicinal Chemistry, 2016. 16(13): p. 1042-1062. 25. Zegura, B., An Overview of the Mechanisms of Microcystin-LR Genotoxicity and Potential Carcinogenicity. Mini-Reviews in Medicinal Chemistry, 2016. 16(13): p. 1042-1062. 26. Shamsollahi, H.R., et al., Measurement of microcystin-LR in water samples using improved HPLC method. Global journal of health science, 2015. 7(2):
p. 66. 26. Shamsollahi, H.R., et al., Measurement of microcystin-LR in water samples using improved HPLC method. Global journal of health science, 2015. 7(2):
p. 66. 27. Graham, J.L., et al., Cyanotoxin occurrence in large rivers of the United States. Inland Waters, 2020. 10(1): 27. Graham, J.L., et al., Cyanotoxin occurrence in large rivers of the United States. Inland Waters, 2020. 10(1): p. 109-117. 27. Graham, J.L., et al., Cyanotoxin occurrence in large rivers of the United States. Inland Waters, 2020. 10(1): p. 109-117. Page 8/20 Page 8/20 28. Henrie, T., S. Plummer, and J.A. Roberson, Occurrence and state approaches for addressing cyanotoxins in US drinking water. Journal‐American Water
Works Association, 2017. 109(2): p. 40-47. 29. Kumar, P., et al., Recent developments in the methods of quantitative analysis of microcystins. Journal of Biochemical and Molecular Toxicology, 2020. 34(12): p. e22582. 30. Porojan, C., et al., Survey of microcystins in Singapore’s reservoirs using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Marine and
Freshwater Research, 2020. 71(5): p. 659-672. 31. Passos, L.S., et al., Cyanotoxins and water quality parameters as risk assessment indicators for aquatic life in reservoirs. Ecotoxicology and
Environmental Safety, 2022. 241: p. 113828. 32. Niu, Z., et al., Ecological risk assessment of microcystin-LR in the upstream section of the Haihe River based on a species sensitivity distribution model. Chemosphere, 2018. 193: p. 403-411. 33. References Wan, X., et al., Occurrence and risk assessment of microcystin and its relationship with environmental factors in lakes of the eastern plain ecoregion,
China. Environmental Science and Pollution Research, 2020. 27(36): p. 45095-45107. cyanobacterial toxins in the mouse: I microcystin-LR. Human & experimental toxicology, 1999. 18(3): p. 162-167 ., et al., The toxicity of cyanobacterial toxins in the mouse: I microcystin-LR. Human & experimental toxicology, 1 34. Fawell, J., et al., The toxicity of cyanobacterial toxins in the mouse: I microcystin-LR. Human & experimental toxicology, 1999. 18(3): p. 162-167. 35. Munoz, M., et al., Overview of toxic cyanobacteria and cyanotoxins in Ibero-American freshwaters: Challenges for risk management and opportunities
for removal by advanced technologies. Science of the Total Environment, 2021. 761. 36. Hiskia, A., et al., Determination of cyanotoxins by high-performance liquid chromatography with photodiode array. Handbook of cyanobacterial
monitoring and cyanotoxin analysis, 2017. 205. 37. Nyoni, H., et al., Development of a silicone-membrane passive sampler for monitoring cylindrospermopsin and microcystin LR-YR-RR in natural waters. 2017. 100: p. 189-200. nalysis of microcystins in cyanobacterial blooms from freshwater bodies in England. Toxins, 2018. 10(1): p. 39. 38. Turner, A.D., et al., Analysis of microcystins in cyanobacterial blooms from freshwater bodies in England. 39. Ding, Y.-W., et al., Core-shell magnetic zeolite imidazolate framework-8 as adsorbent for magnetic solid phase extraction of brucine and strychnine from
human urine. 2021. 1173: p. 122702. 40. Picardo, M., et al., Recent advances in the detection of natural toxins in freshwater environments. 2019. 1 40. Picardo, M., et al., Recent advances in the detection of natural toxins in freshwater environments. 2019. 112: p. 75-86. 41. Schmidt, J.R., S.W. Wilhelm, and G.L.J.T. Boyer, The fate of microcystins in the environment and challenge ,
,
,
y ,
y
g
g
(
) p
42. García-Mora, A.M., et al., Catalytic wet peroxide oxidation to remove natural organic matter from real surface waters at urban and rural drinking water
treatment plants. 2021. 42: p. 102136. 2. García-Mora, A.M., et al., Catalytic wet peroxide oxidation to remove natural organic matter from real surface
treatment plants. 2021. 42: p. 102136. 43. Majors, R.E.J.A.T., Mississauga, Canada, Sample preparation fundamentals for chromatography. 2013. 43. Majors, R.E.J.A.T., Mississauga, Canada, Sample preparation fundamentals for chromatography. 2013. 44. Wang, P., et al., Development and field evaluation of the organic-diffusive gradients in thin-films (o-DGT) passive water sampler for microcystins. 2022. 287: p. 132079. 4. References Wang, P., et al., Development and field evaluation of the organic-diffusive gradients in thin-films (o-DGT) pass
287: p. 132079. 45. Massey, I.Y., et al., A mini-review on detection methods of microcystins. Toxins, 2020. 12(10): p. 641. 45. Massey, I.Y., et al., A mini-review on detection methods of microcystins. Toxins, 2020. 12(10): p. 641. 46. Capriotti, A.L., et al., Recent applications of magnetic solid-phase extraction for sample preparation. C 46. Capriotti, A.L., et al., Recent applications of magnetic solid phase extraction for sample preparation. Chromatographia, 2019. 82(8): p. 1251 1274. 47. Lawton, L.A., et al., Laboratory analysis of cyanobacterial toxins and bioassays. 2021: p. 745. 47. Lawton, L.A., et al., Laboratory analysis of cyanobacterial toxins and bioassays. 2021: p. 745. 47. Lawton, L.A., et al., Laboratory analysis of cyanobacterial toxins and bioassays. 2021: p. 745. cterial microcystins and human health: First steps towards a congener dependent risk assessment of microcyst 48. Altaner, S., Cyanobacterial microcystins and human health: First steps towards a congener dependent risk 49. Kebede, T.G., et al., The miniaturization of liquid-phase extraction techniques, in Emerging Freshwater Pollutants. 2022, Elsevier. p. 63-93. 49. Kebede, T.G., et al., The miniaturization of liquid-phase extraction techniques, in Emerging Freshwater Pollutants. 2022, Elsevier. p. 63-93. 9. Kebede, T.G., et al., The miniaturization of liquid-phase extraction techniques, in Emerging Freshwater Polluta 0. Shrivastava, A. and R.K.J.T.R. Sharma, Biosensors for the detection of mycotoxins. 2022. 41(2): p. 618-638. 51. Tuzen, M., et al., Synthesis of polystyrene-polyricinoleic acid copolymer containing silver nano particles for dispersive solid phase microextraction of
molybdenum in water and food samples. 2022. 369: p. 130973. 51. Tuzen, M., et al., Synthesis of polystyrene-polyricinoleic acid copolymer containing silver nano particles for dispersive solid phase microextraction of
molybdenum in water and food samples. 2022. 369: p. 130973. 51. Tuzen, M., et al., Synthesis of polystyrene-polyricinoleic acid copolymer containing silver nano particles for dispersive solid phase microextraction of
molybdenum in water and food samples. 2022. 369: p. 130973. 52. Yilmaz, S., et al., Cyanotoxin genotoxicity: a review. 2022. 41(2): p. 699-712. 52. Yilmaz, S., et al., Cyanotoxin genotoxicity: a review. 2022. 41(2): p. 699-712. 53. Stoyneva-Gärtner, M.P., et al., Pilot application of drone observations and pigment marker detection by HPLC in studies of cyanobacterial harmful algal
blooms in Bulgarian inland waters. 2019. 71(5): p. 606-616. 54. Gurbuz, F., et al., Occurrence of microcystins in water, bloom, sediment and fish from a public water supply. 2016. Tables Factor
Response
Std
Run
A: Flow rate
B: pH
C: Column temp
R1
R2
R3
17
1
0.7
5
30
6.538
4.978
1.591
18
2
0.7
5
30
6.531
4.977
1.6
11
3
0.8
4.8
35
5.498
3.986
1.51
15
4
0.8
5.2
35
5.591
4.313
1.791
5
5
0.6
5.2
25
7.598
5.219
1.751
9
6
0.6
4.8
35
7.629
4.886
1.518
3
7
0.8
4.8
25
5.432
5.126
1.66
19
8
0.7
5
30
6.535
4.974
1.595
13
9
0.6
5.2
35
5.591
4.446
1.659
4
10
0.8
4.8
25
5.62
4.198
1.599
2
11
0.6
4.8
25
7.44
5.396
1.558
8
12
0.8
5.2
25
5.595
4.459
1.526
14
13
0.6
5.2
35
7.419
5.146
1.746
12
14
0.8
4.8
35
5.638
4.612
1.541
6
15
0.6
5.2
25
7.549
4.471
1.762
16
16
0.8
5.2
35
5.585
4.448
1.569
10
17
0.6
4.8
35
7.569
5.369
1.592
7
18
0.8
5.2
25
5.578
4.698
1.772
1
19
0.6
4.8
25
7.359
5.12
1.659
R1= Retention time of MC-RR Intraday
MC-RR
MC-YR
MC-LR
Concentration µg L-1
60
380
250
60
380
250
60
380
250
%RSD
3.90
2.80
1.20
1.04
1.92
1.69
0.49
1.46
1.99
Interday
MC-RR
MC-YR
MC-LR
Concentration µg L-1
60
380
250
60
380
250
60
380
250
%RSD
8.85
8.90
8.42
3.69
3.41
0.85
1.87
5.31
5.59 Table 3. DoE’s analysis with responses factors. Table 3. DoE’s analysis with responses factors. Tables Table 1. Analytical standard used to establish MCs LOD and linearity ranged from 3 µg L-1 - 2500 µg L-1
Curve characters
MC-RR
MC-YR
MC-LR
R2
0.9890
0.9880
0.9895
LOD µg L-1
60
10
10
Linear range µg L-1
60 – 2500
10 – 2500
10 – 2500
Standard Curve equation
y = 26809x + 86.88
y = 23203x + 2872
y = 24966x + 3189.3 Table 1. Analytical standard used to establish MCs LOD and linearity ranged from 3 µg L-1 - 2500 µg L-1 Page 9/20 Table 2. Method repeatability Intraday/Interday precision validation at the lower, middle, and higher concentrations. able 2. Method repeatability Intraday/Interday precision validation at the lower, middle, and higher concentration Table 2. Method repeatability Intraday/Interday precision validation at the lower, middle, and higher concentrations. Intraday
MC-RR
MC-YR
MC-LR
Concentration µg L-1
60
380
250
60
380
250
60
380
250
%RSD
3.90
2.80
1.20
1.04
1.92
1.69
0.49
1.46
1.99
Interday
MC-RR
MC-YR
MC-LR
Concentration µg L-1
60
380
250
60
380
250
60
380
250
%RSD
8.85
8.90
8.42
3.69
3.41
0.85
1.87
5.31
5.59 Table 2. Method repeatability Intraday/Interday precision validation at the lower, middle, and higher concentrations. Intraday
MC-RR
MC-YR
MC-LR
Concentration µg L-1
60
380
250
60
380
250
60
380
250
%RSD
3.90
2.80
1.20
1.04
1.92
1.69
0.49
1.46
1.99
Interday
MC-RR
MC-YR
MC-LR
Concentration µg L-1
60
380
250
60
380
250
60
380
250
%RSD
8.85
8.90
8.42
3.69
3.41
0.85
1.87
5.31
5.59
Table 3. DoE’s analysis with responses factors. Tables Factor
Response
Std
Run
A: Flow rate
B: pH
C: Column temp
R1
R2
R3
17
1
0.7
5
30
6.538
4.978
1.591
18
2
0.7
5
30
6.531
4.977
1.6
11
3
0.8
4.8
35
5.498
3.986
1.51
15
4
0.8
5.2
35
5.591
4.313
1.791
5
5
0.6
5.2
25
7.598
5.219
1.751
9
6
0.6
4.8
35
7.629
4.886
1.518
3
7
0.8
4.8
25
5.432
5.126
1.66
19
8
0.7
5
30
6.535
4.974
1.595
13
9
0.6
5.2
35
5.591
4.446
1.659
4
10
0.8
4.8
25
5.62
4.198
1.599
2
11
0.6
4.8
25
7.44
5.396
1.558
8
12
0.8
5.2
25
5.595
4.459
1.526
14
13
0.6
5.2
35
7.419
5.146
1.746
12
14
0.8
4.8
35
5.638
4.612
1.541
6
15
0.6
5.2
25
7.549
4.471
1.762
16
16
0.8
5.2
35
5.585
4.448
1.569
10
17
0.6
4.8
35
7.569
5.369
1.592
7
18
0.8
5.2
25
5.578
4.698
1.772
1
19
0.6
4.8
25
7.359
5.12
1.659 R1= Retention time of MC-RR R1= Retention time of MC-RR
R2= Resolution between MC-RR and MC-YR
R3= Resolution between MC-YR and MC-LR
Table 4. ANOVA statistics analysis R2= Resolution between MC-RR and MC-YR Page 10/20 Page 10/20 Page 10/20 R
Source
Sum of Squares
df
Mean Square
F-value
p-value
R1
Model
11.59
1
11.59
55.94
< 0.0001
significant
A-Flow
11.59
1
11.59
55.94
< 0.0001
Curvature
0.0343
1
0.0343
0.1655
0.6896
Residual
3.31
16
0.2072
Lack of Fit
1.61
6
0.2683
1.57
0.2508
not significant
Pure Error
1.7
10
0.1705
Cor Total
14.94
18
R2
Model
1.11
1
1.11
9.8
0.0065
significant
A-Flow
1.11
1
1.11
9.8
0.0065
Curvature
0.1372
1
0.1372
1.21
0.2873
Residual
1.81
16
0.1132
Lack of Fit
0.4675
6
0.0779
0.5799
0.7393
not significant
Pure Error
1.34
10
0.1344
Cor Total
3.06
18
R3
Model
0.0551
1
0.0551
9.28
0.0077
significant
B-pH
0.0551
1
0.0551
9.28
0.0077
Curvature
0.0047
1
0.0047
0.7859
0.3885
Residual
0.095
16
0.0059
Lack of Fit
0.026
6
0.0043
0.6293
0.7049
not significant
Pure Error
0.069
10
0.0069
Cor Total
0.1548
18 Table 5. Matrix matched analysis at four different surface waters to determine the method LOD and LOQ. Tables Parameters
Tap
River
Wastewater
Milli-Q
Microcystins
RR
YR
LR
RR
YR
LR
RR
YR
LR
RR
YR
LR
Concentration (µg L-1)
1
1
2.5
1
LOD (µg L-1)
0.09
0.01
0.49
0.52
0.24
1.95
0.28
0.36
4.32
0.02
0.03
0.19
LOQ (µg L-1)
0.29
0.03
1.64
1.73
0.79
6.50
0.92
0.25
14.40
0.08
0.09
0.65
Precision (%)
4.72
0.79
7.47
20.36
14.33
14.51
1.24
1.09
3.44
1.56
0.16
0.38 Table 5. Matrix matched analysis at four different surface waters to determine the method LOD and LOQ. hed analysis at four different surface waters to determine the method LOD and LOQ. Table 5. Matrix matched analysis at four different surface waters to determine the method LOD and LOQ. Table 5. Matrix matched analysis at four different surface waters to determine the method LOD Table 6. Application samples from Umgeni River, sport centre tap water, laboratory tap water and UKZN Wetville campus ponds in KZN for MCs
determination. Table 6. Application samples from Umgeni River, sport centre tap water, laboratory tap water and UKZN Wetville campus ponds in KZN for MCs
determination. Page 11/20 Page 11/20 Sampling Point
MC-RR µg L-1
MC-YR µg L-1
MC-LR µg L-1
Blue lagoon dam
Blue lagoon outlet
Umgeni river inlet
Umgeni estuary
Sport centre tap
Laboratory tap water
Campus pond1
Campus pond2
ND
ND
ND
ND
ND
ND
ND
2.160
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND Table 7. Application samples from Darvill Wastewater Treatment Plant for MCs determination on three consecutive weeks
Sampling Point
MC-RR µg L-1
MC-YR µg L-1
MC-LR µg L-1
Return Activated Sludge
1st campaign samples
ND
ND
ND
Digested Thickened WAS
ND
2.015 ± 0.106
ND
Settled sewage
D
ND
ND
Raw sludge
D
ND
ND
Thickened WAS
D
ND
ND
Raw sewage
ND
ND
ND
Final effluent
ND
ND
ND
2nd campaign samples
Sampling points
MC-RR µg L-1
MC-YR µg L-1
MC-LR µg L-1
Effluent secondary
D
ND
ND
Maturation River
D
ND
ND
Balancing tank
6.470
ND
ND
Inlet
4.403
4.938
ND
3rd campaign samples
Sampling points
MC-RR µg L-1
MC-YR µg L-1
MC-LR µg L-1
Effluent secondary
ND
ND
ND
Maturation River
ND
ND
ND
Balancing tank
9.384
ND
ND
Inlet
30.521
ND
ND ication samples from Darvill Wastewater Treatment Plant for MCs determination on three consecutive weeks Table 7. Tables Application samples from Darvill Wastewater Treatment Plant for MCs determination on three consec Page 12/20 Page 12/20 Page 12/20 Table 8. Comparison of the analytical method used in literature with the current developed method Page 12/20 Instrument
Extraction
method
Mobile
phase
Column
Analyte
Method
detection
limit
Method
quantification
limit
R2
Nature of
water
Environmental
concentration
of
microcystins
Reference
HPLC/PDA
SPE
0.05% TFA
in
Acetonitrile
and 0.1%
TFA in
UPW)
(Shimadzu,
5 μm, C18,
250x4.6
mm
MC-RR,
MC-YR,
MC-LR
0.02 µg
L-1
N/A
N/A
Reservoir
in Brazil
MCs total (ND
– 1.59 µg L-1)
Falconer-
Dias
et.al[6]
HPLC/PDA
SPE with
empore
extraction
disks
0.1% TFA in
Acetonitrile
and 0.1%
TFA in UPW
Upelcosil
ABZþPlus
column
(150x 4.6
mm, 5 mm;
Supelco,)
MC-RR,
MC-YR,
MC-LR
0.08 µg
L-1
0.15 µg L-1
N/A
Inland
waters in
Bulgarian
ND
Stoyneva-
Gartener
et.al [53]
HPLC/PDA
0.1% TFA in
Acetonitrile
and 0.1%
TFA in UPW
Waters
C18
Symmetry
(3.9 mm
internal
diameter ×
150 mm, 5
μm particle
size
MC-YR
MC-LR
N/A
N/A
0.9981
Lake
District in
Turkey
2.9 – 13.5 µg
L-1
N/D
Gurbuz
et.al [54]
HPLC-
UV/PDA
SPE
(0.05% TFA
in
Acetonitrile)
and (0.05%
TFA in
UPW)
C18
analytical
column
(XBridge
C18 – 3.5
µm – 4.6
x100mm,
Waters)
MC-RR,
MC-YR,
MC-LR
0.10 µg
L-1
N/A
0.990
Pigeard
pond in
France
MCs total (ND
– 34.4 µg L-1
Thurest-
Benoist
et.al [20]
HPLC/PDA
SPE
(0.025%
TFA in
Acetonitrile)
and
(0.025%
TFA in
UPW)
Hypersil
Gold C18
(Guard
column 2.1
x 10 mm, 3
µm particle
size)
MC-
RR,
MC-
YR,
MC-LR
0.0003
µg mL-1
0.48
0.16
0.0007 µg
mL-1
1.50
0.58
0.9890
0.9880
0.9895
Darvill
wastewater
in KZN
MCs total (ND
– 30.521 ng L-
1)
Current
method Table 9: MCs ecological risk assessment in real water samples
Sampling point
Campus pond1
Digested thickened
WAS
2nd campaign
(balancing tank)
2nd campaign-
inlet
3rd campaign
(balancing tank)
3rd campaign
(Inlet)
Analyte
MC-RR
µg/L
MC-
YR
µg/L
MC-RR
µg/L
MC-YR
µg/L
MC-RR
µg/L
MC-
YR
µg/L
MC-RR
µg/L
MC-YR
µg/L
MC-RR
µg/L
MC-
YR
µg/L
MC-RR
µg/L
MC-
YR
µg/L
MEC
2.160 ±
0.003
-
-
2.015 ±
0.106
6.470 ±
0.149
-
4.403 ±
0.261
4.938 ±
0.181
9.384 ±
0.087
-
30.521±
0.357
-
RCR
0.0540
-
-
0.0503
0.161
-
0.110
0.123
0.234
-
0.764
-
Ecological risk
assessment
Low risk
-
-
Low risk
Medium
risk
-
Medium
risk
Medium
risk
Medium
risk
-
Medium
risk
- Table 9: MCs ecological risk assessment in real water samples Page 13/20 Page 13/20 Figure 1
Microcystins – LR general structure
Figure 2
Microcystins chromatogram obtained by injecting 10 µL of 2500 µg L-1 microcystins standard of targeted analytes Figure 1
Microcystins – LR general structure Figure 1 Figure 1 Microcystins – LR general structure Figure 2
Microcystins chromatogram obtained by injecting 10 µL of 2500 µg L-1 microcystins standard of targeted analytes Figure 2 Microcystins chromatogram obtained by injecting 10 µL of 2500 µg L-1 microcystins standard of targeted analytes Page 14/20 Page 14/20 Figure 3
MC-YR chromatogram obtained when injecting 10 µL of 2500 µg L-1 standard solution in HPLC. Page 12/20 Figure 4
MCs separation spectrum (a) when methanol was used there was no separation, (b) purity curve above the analytes, (c) when acetonitrile was used, 3 Figure 3 MC-YR chromatogram obtained when injecting 10 µL of 2500 µg L-1 standard solution in HPLC. MC-YR chromatogram obtained when injecting 10 µL of 2500 µg L-1 standard solution in HPLC. Figure 4
MCs separation spectrum (a) when methanol was used there was no separation, (b) purity curve above the analytes, (c) when acetonitrile was used, 3
analytes were identified, (d) purity curve below analytes Figure 4
MCs separation spectrum (a) when methanol was used there was no separation, (b) purity curve above the analytes, (c) when acetonitrile was used, 3
analytes were identified, (d) purity curve below analytes Figure 4 Figure 4 MCs separation spectrum (a) when methanol was used there was no separation, (b) purity curve above the analytes, (c) when acetonitrile was used, 3
analytes were identified, (d) purity curve below analytes MCs separation spectrum (a) when methanol was used there was no separation, (b) purity curve above the analytes, (c) when acetonitrile was used, 3
analytes were identified, (d) purity curve below analytes Page 15/20 Page 15/20 Figure 5
Chromatograms of microcystins (a) separation chromatogram (b) where there was no separation. Figure 6
Chromatograms of microcystins at different injection volume (a) 20 µL and (b) 2 µL obtained by analysing 2500 µg L-1 MCs standard. Figure 5
Chromatograms of microcystins (a) separation chromatogram (b) where there was no separation. Figure 5 Chromatograms of microcystins (a) separation chromatogram (b) where there was no separation. Figure 6
Chromatograms of microcystins at different injection volume (a) 20 µL and (b) 2 µL obtained by analysing 2500 µg L-1 MCs standard. Figure 6 Chromatograms of microcystins at different injection volume (a) 20 µL and (b) 2 µL obtained by analysing 2500 µg L-1 MCs standard. hromatograms of microcystins at different injection volume (a) 20 µL and (b) 2 µL obtained by analysing 2500 µ Page 16/20 Page 16/20 Figure 7
Chromatograms of microcystins obtained by injecting 10 µL of 2500 µg L-1 for separation at different flow rate (a) MCs at 0.1 mL min-1, (b) MCs at 0.5 mL
min-1 and (c) MCs at 0.7 mL min-1 gure 7 Figure 7 Chromatograms of microcystins obtained by injecting 10 µL of 2500 µg L-1 for separation at different flow rate (a) MCs at 0.1 mL min-1, (b) MCs at 0.5 mL
min-1 and (c) MCs at 0.7 mL min-1 Chromatograms of microcystins obtained by injecting 10 µL of 2500 µg L-1 for separation at different flow rate (a) MCs at 0.1 mL min-1, (b) MCs at 0.5 mL
min-1 and (c) MCs at 0 7 mL min-1 Page 17/20
Figure 8
Microcystins instrument stability line graph obtained by injecting 375 µg L-1 MCs standard. Page 17/20
Figure 8
Microcystins instrument stability line graph obtained by injecting 375 µg L-1 MCs standard. Figure 8 Figure 9
Calibration curves obtained for (a) Microcystins (MC-RR); (b) MC-YR; (c) MC-LR on HPLC/PDA. Figure 10 Figure 9
Calibration curves obtained for (a) Microcystins (MC-RR); (b) MC-YR; (c) MC-LR on HPLC/PDA. Figure 9
Calibration curves obtained for (a) Microcystins (MC-RR); (b) MC-YR; (c) MC-LR on HPLC/PDA. Figure 9 Calibration curves obtained for (a) Microcystins (MC-RR); (b) MC-YR; (c) MC-LR on HPLC/PDA. Calibration curves obtained for (a) Microcystins (MC-RR); (b) MC-YR; (c) MC-LR on HPLC/PDA. Figure 10 Figure 10 Page 18/20 pH optimisation of microcystins at pH 4, 5, and 6 to obtain MCs recoveries. pH optimisation of microcystins at pH 4, 5, and 6 to obtain MCs recoveries. pH optimisation of microcystins at pH 4, 5, and 6 to obtain MCs recoveries. Figure 11
The contour and Pareto charts for all three responses. Page 19/20
Figure 11
The contour and Pareto charts for all three responses. Figure 11 The contour and Pareto charts for all three responses. The contour and Pareto charts for all three responses. Page 19/20 Page 19/20 Figure 12
Three responses Half normal plot, 3D surfaces, and 3D cube. Figure 12 Three responses Half normal plot, 3D surfaces, and 3D cube. Three responses Half normal plot, 3D surfaces, and 3D cube. GraphicalAbstract.png Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 20/20 Page 20/20
|
https://openalex.org/W2945815696
|
https://europepmc.org/articles/pmc6536532?pdf=render
|
English
| null |
Ultra-small microorganisms in the polyextreme conditions of the Dallol volcano, Northern Afar, Ethiopia
|
Scientific reports
| 2,019
|
cc-by
| 5,280
|
Ultra-small microorganisms in
the polyextreme conditions of
the Dallol volcano, Northern Afar,
Ethiopia Received: 10 April 2018
Accepted: 15 May 2019
Published: xx xx xxxx Received: 10 April 2018
Accepted: 15 May 2019
Published: xx xx xxxx The Dallol geothermal area in the northern part of the Danakil Depression (up to 124–155 meter below
sea level) is deemed one of the most extreme environments on Earth. The area is notable for being part
of the Afar Depression, an incipient seafloor-spreading center located at the triple junction, between
Nubian, Somali and Arabian plates, and for hosting environments at the very edge of natural physical-
chemical extremities. The northern part of the Danakil Depression is dominated by the Assale salt plain
(an accumulation of marine evaporite deposits) and hosts the Dallol volcano. Here, the interaction
between the evaporitic deposit and the volcanisms have created the unique Dallol hot springs, which are
highly acidic (pH ~ 0) and saline (saturation) with maximum temperatures ranging between 90 and 109 °C. Here we report for the first time evidence of life existing with these hot springs using a combination of
morphological and molecular analyses. Ultra-small structures are shown to be entombed within mineral
deposits, which are identified as members of the Order Nanohaloarchaea. The results from this study
suggest the microorganisms can survive, and potential live, within this extreme environment, which has
implications for understanding the limits of habitability on Earth and on (early) Mars. The study of environmental limits of life provides useful information for assessing the limits of habitability both
on Earth and elsewhere in the Solar System1,2. Understanding, and defining these limits, using extreme terrestrial
environments and Earth analogues sites3,4 is therefore a crucial step in selecting sites for future life detection
missions. The Dallol geothermal area (14°14′21″N; 40°17′55″E) is located in the northern part of the Danakil
Depression5. This is a narrow lowland salt plain (up to 124 m below sea level) running inland, quasi-parallel to
the coast of the Red Sea (Fig. 1a), which formed when part of the Red Sea was isolated during the Pleistocene.h g
g
The area is located at the northern segment of the Afar triple junction and is characterized by an attenuated
continental crust, which is less than 15 km thick with shallow (3–5 km deep) magma chambers beneath its axial
zones6,7. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 10 April 2018
Accepted: 15 May 2019
Published: xx xx xxxx Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 Ultra-small microorganisms in
the polyextreme conditions of
the Dallol volcano, Northern Afar,
Ethiopia The Dallol dome-shaped deposits formed as a result of the interaction between the evaporitic deposits
and the shallow magma intrusions7,8.h g
The hydrothermalism of the Dallol area was possibly generated by a phreatic explosion in 1926. Since then
volcanic episodes, seismicity and hydrothermal activity have been reported in the area5. As a consequence of geo-
thermal activity7,9 several minerals, for example, pyrolusite, chlorargyrite, wurtzite and halite, precipitate forming
colourful chimneys (Fig. 1b). It has been postulated that microbial activity does not play a major role in mineral
precipitation and geochemical cycling within Dallol10.h At the surface, the water temperature at the source is above 100 °C and highly acidic (pH ~ 0). The resultant hot
pools vary in color depending on the high metal concentration- (e.g., iron 35.6 g/L, copper 93 mg/l) (Table 1). A 1Centro de Astrobiología (INTA-CSIC) Crtera. Ajalvir km 4 Torrejón de Ardoz, Madrid, 28850, Spain. 2Dipartimento
di Scienze Biologiche, Geologiche e Ambientali (BiGeA), Università di Bologna, Bologna, Italy. 3Department of
Geology, University of Johannesburg, Johannesburg, South Africa. 4Centro de Biología Molecular “Severo Ochoa”
Cantoblanco, Madrid, Spain. 5IRSPS, Universitá d’Annunzio, Pescara, Italy. 6Ibn Battuta Centre, Université Cadi
Ayyad, Marrakech, Morocco. 7School of Environment, Earth and Ecosystems Sciences, The Open University, Milton
Keynes, UK. 8Department of Earth Sciences, Mekelle University, Mekelle, Tigre, Ethiopia. Correspondence and
requests for materials should be addressed to F.G. (email: gomezgf@cab.inta-csic.es) Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 1 www.nature.com/scientificreports/ Figure 1. Study area. (a) Location map of the Dallol geothermal area (red dot) in the Danakil Depression (map
data © 2018 Google). (b) Panoramic view of the sampling sites (D9: central small chimney and D10: water from
the blue pool at the bottom of the chimney). Figure 1. Study area. (a) Location map of the Dallol geothermal area (red dot) in the Danakil Depression (m
data © 2018 Google). (b) Panoramic view of the sampling sites (D9: central small chimney and D10: water fr
the blue pool at the bottom of the chimney). Figure 1. Study area. (a) Location map of the Dallol geothermal area (red dot) in the Danakil Depression (map
data © 2018 Google). (b) Panoramic view of the sampling sites (D9: central small chimney and D10: water from
the blue pool at the bottom of the chimney). Ultra-small microorganisms in
the polyextreme conditions of
the Dallol volcano, Northern Afar,
Ethiopia Sample
D9
D10
pH
0.25
2.42
T
86
47
Cond
188
262
Eh
411
412
H2%
0.02
0.13
CO2%
0
0.15
Na
119333
89323
Mg
3408
5998
Al
383
533
P
10.1
14.9
K
8248
16594
Ca
3810
5927
Mn
538
950
Fe
19159
35652
Cu
40.6
93
Zn
39.6
72
Rb
11.9
20.9
Sr
71.1
116.7
Pb
1.4
0.2
Table 1. Physico-chemical parameters for two sampling sites, D9 and D10 (concentrations in mg/l, average of
three measurements). The units are stated as followed T: °C, Eh: mV; conductivity: mS/cm2. Sample
D9
D10
pH
0.25
2.42
T
86
47
Cond
188
262
Eh
411
412
H2%
0.02
0.13
CO2%
0
0.15
Na
119333
89323
Mg
3408
5998
Al
383
533
P
10.1
14.9
K
8248
16594
Ca
3810
5927
Mn
538
950
Fe
19159
35652
Cu
40.6
93
Zn
39.6
72
Rb
11.9
20.9
Sr
71.1
116.7
Pb
1.4
0.2
Table 1. Physico-chemical parameters for two sampling sites, D9 and D10 (concentrations in mg/l, average of
three measurements). The units are stated as followed T: °C, Eh: mV; conductivity: mS/cm2. Table 1. Physico-chemical parameters for two sampling sites, D9 and D10 (concentrations in mg/l, average of
hree measurements). The units are stated as followed T: °C, Eh: mV; conductivity: mS/cm2. Table 1. Physico-chemical parameters for two sampling sites, D9 and D10 (concentrations in mg/l, average
three measurements). The units are stated as followed T: °C, Eh: mV; conductivity: mS/cm2. combination of these extreme chemical and physical parameters (e.g. temperature, pH, salinity and heavy metals)
has resulted in a unique multi-extreme environment.i combination of these extreme chemical and physical parameters (e.g. temperature, pH, salinity and heavy metals)
has resulted in a unique multi-extreme environment.i First reported thermoacidophilic prokaryote was isolated from coal refuse piles11 and the first eukaryote from
upper oxygenic zones of acidic geothermally heated waters in solfataras12. In 1995 two species of a moderately
thermophilic archaea living around pH 0 were reported13.i p
g
p
p
Here, we describe for the first time morphological and molecular evidence of thermohaloacidophilic nanomi-
croorganism existing in this novel multi-extreme environment. Ultra-small microorganisms in
the polyextreme conditions of
the Dallol volcano, Northern Afar,
Ethiopia h
h
d
l
f
ll l6 7
d
l
l
f
d Due to the unique geochemistry and volcanic activity of Dallol6,7 it is an ideal analogue site for studying
Martian hydrothermal environments, such as Nili Patera caldera9, where hydrothermal sinter deposits are found
in direct association with volcanic activity14, and Gusev crater15, the landing site of the Spirit MER rover. y
g
p
Despite the Dallol hot springs being deemed one of the most extreme environments on Earth the possibility
of microbial life existing within the springs has not been studied. www.nature.com/scientificreports/ 4d–f, members of the Nanohaloarchaea group were detected with the Narc1214 FISH
probe in the salt precipitates at the hydrothermal fluid source from D9, which corroborates the phylogenetic data. l
The presence of ultra-small microorganisms was further investigated using Transmission Electron Microscopy
(TEM) (Fig. 5), Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM)
(Fig. 6). Using these techniques, it was possible to identify ultra-small cellular morphologies (between 50 nm and
500 nm), which supports the molecular data (Figs 5 and 6). The Energy-dispersive X-ray (EDX) elemental micro-
analysis suggested that the cellular morphologies were unambiguously biological given their high carbon content
(Fig. 5A–F). Whilst, SEM-EDX showed the presence of silica precipitates (Fig. 6G–M) surrounding and encrusting the
ultra-small cellular morphologies (Fig. 5A–D), suggesting a hydrothermal origin with a high Fe content (Fig. 5E–F).h Figure 6 shows ultra-small microorganisms entombed within precipitated silica minerals (Fig. 6D–L). The cellular
morphologies were associated with the needle-shaped crystals (Fig. 6M), which suggests a relationship between the
microorganisms and the biomineralization process as has been previously reported in other acidic environments17.hh The ultra-small microorganisms were entombed within the precipitated mineral structures (Fig. 6(D–F)). The
mineral precipitation process gave rise to thin mineral layers (Fig. 6(D)). Figure 6(E,F) show an enlarged view
of the thin mineral layers. Figure 6(G) shows ultra-small microorganisms, which were not covered by minerals,
whilst Fig. 6(H,I) show ultra-small microorganisms partially covered with mineral precipitates. More biominer-
alized ultra-small microorganisms are visible in Fig. 6(J,K). Figure 6(M) shows STEM images with close views
of ultra-small microorganisms surrounded by mineral needles and Fig. 6(F) shows mineral spheres resulting
from the mineralization process. Needles with spike forms are observed in Fig. 6(J–L). Finally, Fig. 6(N,O) show
ultra-small microorganisms surrounded by minerals in a close view using STEM. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. X-Ray Diffraction analyses showing sample D9 mineral composition. (a) chlorargyrite (C) and
wurtzite (W); (b) halite (H), and (c) pyrolusite (P) and halite (H). Figure 2. X-Ray Diffraction analyses showing sample D9 mineral composition. (a) chlorargyrite (C) and
wurtzite (W); (b) halite (H), and (c) pyrolusite (P) and halite (H). DNA was extracted from the salt precipitates at the hydrothermal fluid source (D9), 16S rDNA was amplified
using Archaea primers (Fig. 4c) and phylogenetic analysis was performed. The OTU named Dallol 9 showed close
similarity (95.8%) with the Nanohaloarchaea environmental clone ARDARCSS13 (GenBank accession number
EU869371) (Fig. 3) which closely matched to uncultured sequences from Nanohaloarchaeales Order.h DNA was extracted from the salt precipitates at the hydrothermal fluid source (D9), 16S rDNA was amplified
using Archaea primers (Fig. 4c) and phylogenetic analysis was performed. The OTU named Dallol 9 showed close
similarity (95.8%) with the Nanohaloarchaea environmental clone ARDARCSS13 (GenBank accession number
EU869371) (Fig. 3) which closely matched to uncultured sequences from Nanohaloarchaeales Order.h g
y
q
The clone ARDARCSS13 and Dallol 9 sequences are close to Candidatus Holaredivivus sp. G17 (Fig. 3) that
has been described16 in a Bras of Port saltern pond (Alicante, Spain) as a photoheterotrophic microbe that present
rhodopsin and photolyase. Candidatus Holaredivivus sp. G17 is capable to degrade polysaccharides, likewise
presents typical genes of archaea. In relation to survival strategies in hypersaline environments, Haloredivivus sp. G17 is a salt-in strategist as the isoelectric point of his proteins shows16.i To confirm the presence of members of the Nanohaloarchaea group in the samples Fluorescence In Situ
Hybridization (FISH) was carried out. Samples fixed in situ for FISH were sequentially filtered through 0.45, 0.22
and 0.1 µm filters to select for small (<0.1 µm) microorganisms. Inspection of the DNA stained material retained
in the 0.2 µm filters with SybrGold suggested the presence of ultra-small microorganisms forming compact dense
colonies, including mucilaginous films (Fig. 4a). Ultrasonication was further used to disintegrate the colonies
prior to sequentially filtration. The ultra-small microorganisms passed through the 0.22 µm filter, but were again
retained on the 0.1 µm filters (Fig. 4b). Figure 4c shows 16S rDNA amplification using universal archaea primers. i
g
g
pi
g
p
As it is shown in Fig. Results and Discussion In this system many protuberances can be observed (Figs 1b and 6A–C), which are generated by the precipi-
tation of minerals from the superheated underground solutions. The chimney selected for this study was Site
D9 (Figs 1b and 6A) and composed of chlorargyrite (Fig. 2a), wurtzite (Fig. 2a), halite (Fig. 2b) and pyrolusite
(Fig. 2c). Table 1 reports the physico-chemical parameters of Site D9 and the surrounding pool (Site D10) as
shown in Fig. 6A. The pH and temperature between the two sites varied, for example at Site D9, the source of the
hydrothermal fluid, the fluid was pH 0.25 and 86 °C compared to 2.42 and 47 °C at site D10. Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 2 Concluding Remarks g
We demonstrated the presence of living ultra-small microorganisms in a multi-extreme environment with
adverse conditions for life: extreme low pH (0.25), temperature (90 °C), redox potential, salinity and heavy
metals content. Molecular studies, electron microscopies observation and phylogenetic analyses of amplified
rDNA sequences showed the presence of ultra-small microorganisms related to the Order Nanohaloarchaea. Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 3 www.nature.com/scientificreports/ Figure 3. Neighbor-joining 16S rRNA phylogenetic tree of DPANN archaea. Open circles indicate bootstrap
values between 70–89. Solid circles indicate bootstrap values bootstrap values higher than 90. Figure 3. Neighbor-joining 16S rRNA phylogenetic tree of DPANN archaea. Open circles indicate bootstrap
values between 70–89. Solid circles indicate bootstrap values bootstrap values higher than 90. Figure 3. Neighbor-joining 16S rRNA phylogenetic tree of DPANN archaea. Open circles indicate bootstrap
values between 70–89. Solid circles indicate bootstrap values bootstrap values higher than 90. The ultra-small bacteria observed in the samples were morphologically spherical and entombed in the mineral
layers that form the small chimneys in Dallol under high acidity and high temperature conditions. The saturation
of salts and minerals in the superheated water, which results in precipitation and the formation of the chimneys
may be influenced by entombed microorganisms. We described the presence of ultra-small microorganisms in a
natural environment which is an Earth analogue of some regions of Mars such as Nili Patera Caldera9,10. The pres-
ence of life in the Dallol hot springs expands our understanding of the limits of habitability on Earth and beyond. However, future work is needed to understand how these nanobacteria survive in such an extreme environment
and whether they play a role in geochemical cycling. Materials and Methodsh Dehydration was completed with a mixture of ethanol/propylene oxide (1:1)
for 10 min and pure propylene oxide 3 × 10 min. Infiltration of the resin was accomplished with propylene oxide/
Epon (1:1) for 45 min and pure LR White resin (London Resin Company limited, England), overnight at room
temperature. Polymerization of infiltrated samples was done at 60 °C for 2 days. Ultrathin sections of the samples
were stained with uranyl acetate and lead citrate by standard procedures. y
y
p
Four types of electronic microscopy techniques were used for this study: Scanning Electron Microscope
(SEM) (JEOL-5600 coupled to an EDX, INCA) with an Energy Dispersive X-Ray Analyzer (EDX) and SEM-FI
(Philips XL30-FEG); Scanning Electron Microscopy-Field Emission Gun (SEM-FEG) (Philips XL30-FEG);
Transmission Electronic Microscope (TEM) (JEM-1010); TEM/STEM electronic microscope (JEOL 2100 K) with
FEG. Samples were mounted on conductive graphite stubs and sputter and gold-coated in a Quorum, Q150T-S
apparatus to ensure electrical conductivity and prevent charging under electron beams. pp
y
p
g
g
For SEM analysis, samples were also analyzed with EDX to obtain semiquantitative chemical data. A STEM
unit was coupled to the microscope with acquisition of contrast images z (HAADF). The qualitative element
composition of samples was determined using an INCA-X-SIGHT with a Si-Li detector (Oxford, England) with a
detection limit of 10% for the main element. The operating energy was 200 kv. For TEM analyses, the instrument
was operated at 200 kv (Catalysis Institute and PETROQUIMICA-CSIC), equipped with an energy dispersive
X-RAY microanalysis instrument INCA (Oxford, England). The TEM/STEM was operated at 200 kv with an
EDX, which was coupled to a STEM unit, with acquisition of contrast images z (HAADF). The same grids were
used for both TEM and TEM-STEM. Fluorescence microscopy. Samples were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for
2 h and stored at 4 °C until further processing. Fixed samples were dispersed by 3 cycles of sonication of 30 sec,
with a 30 sec break in between with 1 pulse per second (intensity of 20%). The samples were filtered sequentially
through a 0.4 μm pore size polycarbonate filters, a 0.2 μm pore size filter and a 0.1 μm pore size filter (Millipore,
Germany). Filters were pretreatment as previously described18. FISH was performed as described by Glöckner,
et al.19 using Cy3 single-labeled Narc1214 probe20 (Biomers, Ulm, Germany). Materials and Methodsh Sampling. The sampling campaign was carried out in January 2017 and samples were collected from a fuma-
role’s wall (14°14′21″N; 40°17′55″E) located at the main Dallol hydrothermal outcrop in the Danakil depression
and a blue pool surrounding the small fumarole. Samples were collected aseptically, using sterilized spatulas and
plastic aseptic materials in 12 mL sterile glass vials. The vials were completely filled to prevent head space. To
ensure that the samples did not oxidize during transportation and storage, the vials were sealed with a septum tap,
covered with parafilm tape and kept under anaerobic conditions. Samples were transported at room temperature. “In Situ” physico-chemical parameters measurement. Physico-chemical parameters (T: °C, Eh: mV;
conductivity: mS/cm2) were measured “in situ” using a multi-parametric probe, YSI 556 MPS. Gas composition
by gas chromatography and elemental composition using ICP-MS. Electron microscopy. Samples were fixed in 4% paraformaldehyde and 2% glutaraldehyde, in 0.1 M phos-
phate buffer (pH 7.2), for 2 h at room temperature. Fixed samples were washed three times in the same buffer
and post-fixed with 1% OsO4 in water for 60 min at room temperature in the dark. After three washes in distillate
water, samples were incubated with 2% aqueous uranil acetate for 1 h at room temperature, washed again, and Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Living microorganisms at the Dallol hydrothermal outcrop. (a) SybrGold DNA staining of
microorganisms retained in 0.2 µm filter before ultrasonication. (b) SybrGold DNA stain of microorganisms
retained in 0.1 µm filter after ultrasonication. (c) Electrophoresis of archaeal amplified DNA from sample
D9. (d–f) FISH images of Nanohaloarchaea microorganisms. In green, SybrGold DNA staining (d). In red,
Narc1214 FISH probe signal (e). (f) d and e merge. Scale bars, 5 μm. Figure 4. Living microorganisms at the Dallol hydrothermal outcrop. (a) SybrGold DNA staining of
microorganisms retained in 0.2 µm filter before ultrasonication. (b) SybrGold DNA stain of microorganisms
retained in 0.1 µm filter after ultrasonication. (c) Electrophoresis of archaeal amplified DNA from sample
D9. (d–f) FISH images of Nanohaloarchaea microorganisms. In green, SybrGold DNA staining (d). In red,
Narc1214 FISH probe signal (e). (f) d and e merge. Scale bars, 5 μm. dehydrated in increasing concentrations of ethanol 30, 50, and 70% 20 min each, 90% 2 × 20 min, and 100%
2 × 30 min at room temperature. Materials and Methodsh Stringencies were regulated adjust-
ing formamide and NaCl concentration in hybridization and washing buffer at 30% (vol/vol) and 0.112 M respec-
tively. Filters were counterstaining by incubation with SYBR® Gold (Molecular Probes, Eugene, OR, USA) 1X
diluted in milliQ water for 15 min, before being mounted on glass a slide using Vectashield (Vector Laboratories,
Burlingame, CA, USA): Citifluor (Citifluor, London, United Kingdom) (1:4). Samples were imaged using a con-
focal laser scanning microscope LSM710 (Carl Zeiss, Jena, Germany) equipped with diodo (405 nm), argon
(458/488/514 nm) and helium and neon (543 and 633 nm) lasers. Fiji software21 was used to process images. Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 5 www.nature.com/scientificreports/ Figure 5. Transmission Electron Microscopy (TEM) images of sample D9 showing ultra-small biological
morphologies. Arrows indicate where the EDX analyses were performed. Spectra show the high carbon content
associated with high Si and Fe content in these spots. Figure 5. Transmission Electron Microscopy (TEM) images of sample D9 showing ultra-small biological
morphologies. Arrows indicate where the EDX analyses were performed. Spectra show the high carbon content
associated with high Si and Fe content in these spots. DNA extraction and amplification of ribosomal genes. Samples were filtered through polycarbonate
membranes (0.45, 0.2 and 0.1 µm diameter; Millipore, USA). Total genomic DNA was extracted from those mem-
branes using the DNeasy PowerSoil Kit (Qiagen, Germany) as described in the manufacture’s instructions, and con-
centrated using a SpeedVac Concentrator. The quantity of the extracted DNA was analysed by fluorimetry using a
Qubit 2.0 fluorometer (Thermo Fhiser Scientific, USA). Total genomic DNA were stored at −20 °C for sequencing. Ribosomal genes were amplified using a primers set specific to amplify V2-V3 region of 16S rRNA in Archaea
domain (Arch1F 5′-CGGRAAACTGGGGATAAT-3′ and Arch1R 5′-TRTTACCGCGGCGGCTGBCA-3′). PCR
reactions were performed as described22,23. Gel electrophoreses (1% agarose Conda, Spain in 0.5X TBE buffer,
90 mV during 30 min and staining with GreenSafe Premium NZYTech) were carried out to check the size and
quality of PCR products. Ribosomal genes library preparation and sequencing. Library preparation and 2 × 300 pair-end
sequencing by Illumina MiSeq were made by Genomic Unit in Parque Científico de Madrid Foundation/FPCM
(Madrid Spain) Total genomic DNAs was quantified by Picogreen. Materials and Methodsh Then, an input of 16 pg of DNA and 27 cycles were used in a
first PCR with Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, USA) in the presence of 100 nM
primers for 16S amplification (5′-ACACTGACGACATGGTTCTACACCTACGGGNGGCWGCAG-3′ and 5′-
TACGGTAGCAGAGACTTGGTCTGACTACHVGGGTATCTAATCC-3′, these primers amplify the V3-V4 region of
16S), 200 nM primers for Archaea amplification (5′-ACACTGACGACATGGTTCTACACGGRAAACTGGGGATAAT
-3′ and 5′-TACGGTAGCAGAGACTTGGTCTTRTTACCGCGGCGGCTGBCA-3′),ti After the first PCR, a second PCR of 15 cycles was perfomed with Q5® Hot Start High-Fidelity DNA Polymerase
(New England Biolabs, USA) in the presence of 400 nM of primers (5′-AATGATACGGCGACCA
CCGAGATCTACACTGACGACATGGTTCTACA-3′ and 5′-CAAGCAGAAGACGGCATACGAGAT-[10
nucleotides barcode]-TACGGTAGCAGAGACTTGGTCT-3′) of the Access Array Barcode Library for Illumina
Sequencers (Fluidigm, USA).hii q
g
The amplicons were validated and quantified by a Bioanalyzer. An equimolecular pool was purified by gen
extraction and titrated by quantitative PCR using the “Kapa-SYBR FAST qPCR kit for LightCycler480” with a
reference standard for quantification. The pooled amplicons were denatured and added to the flowcell at a density
of 9 pM. Clusters formed, which were sequenced using a “MiSeq Reagent Kit v3”, in a 2 × 300 pair-end sequencing
run on an Illumina MiSeq sequencer. Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 6 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports
tificreports/
Figure 6. (A) General view of the sampling site, (B) the small chimneys (temperature of water 90 °C. (C) D9
sample from a small chimney in (A). (D–L) SEM and (M–O) Scanning TEM images of sample D9 showing the
morphologies of ultra-small microorganisms entombed in the mineral layers. Figure 6. (A) General view of the sampling site, (B) the small chimneys (temperature of water 90 °C. (C) D9
sample from a small chimney in (A). (D–L) SEM and (M–O) Scanning TEM images of sample D9 showing the
morphologies of ultra-small microorganisms entombed in the mineral layers. Detection of DPANN OTUs by microbiome analyse. Quality of reads was evaluated by means of
FastQC software. PANDAseq Assembler was using for assembling forward and reverse reads and convert
in a fasta file24. Sequencing data were processed using Qiime software package version 1.9.025. High qual-
ity contigs were clustered into OTUs based on 94% sequence similarity with UCLUST. The first sequence
for each OTU as the representative OTU, which were aligned using PYNAST26. The taxonomic identity of
each phylotype was determined using the SILVA_132_QIIME database (https://www.arb-silva.de/down-
load/archive/qiime/)27. g
Europlanet 2020 RI received funding from the European Union’s Horizon 2020 research and innovation program
under Grant Agreement No 654208. g
Europlanet 2020 RI received funding from the European Union’s Horizon 2020 research and innovation program
under Grant Agreement No 654208. References Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-
based diversity studies. Nucleic acids research. 41(1), 1–11 (2013). y
24. Bartram, A. K., Lynch, M. D., Stearns, J. C., Moreno-Hagelsieb, G. & Neufeld, J. D. Generation of multimillion-sequence 16S rRNA
gene libraries from complex microbial communities by assembling paired-end Illumina reads. Applied and environmental
microbiology. 77(11), 3846–3852 (2011). gy
25. Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nature methods. 7(5), 335
(2010).l (
)
26. Caporaso, J. G. et al. PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics. 26(2), 266
(2010).h 27. Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic acids
research. 41(D1), D590–D596 (2012).t 28. Ludwig, W. et al. ARB: a software environment for sequence data. Nucleic acids research 32(4), 1363–1371 (2004). 28. Ludwig, W. et al. ARB: a software environment for sequence Author Contributions Felipe Gómez led the work, did the sampling and the experimentation and wrote the article, including
Figures 1–3. Barbara Cavalazzi contributed to design the field campaign, to the description of the geological
setting and to the corrections of the article. Nuria Rodríguez contributed in the sampling, experimental
and electromicroscopy of the work. She did the Figures 4 and 5 and Table 1. Ricardo Amils advised on the
microbiological part of the work and contributed to the correction of the manuscript. Gian Gabriele Ori
contributed to design the field campaign and advised on the geological part of the work. Karen Olsson-Francis
contributed to the microbiological work and to the correction of the article. Cristina Escudero contributed to
the hybridization and staining experiments. Jose M. Martínez contributed with the metagenomic study of the
work. Hagos Miruts contributed to the sampling campaign and advised on the contributed to the geological
interpretation of the geological Dallol model. References F., Ehlmann, B. L., Milliken, R. E. & Murchie, S. L. Silica deposits in the Nili Pa
volcanic complex on Mars. Nature Geoscience. 3, 838–841, https://doi.org/10.1038/ngeo990 (2010). 4. Skok, J. R., Mustard, J. F., Ehlmann, B. L., Milliken, R. E. & Murchie, S. L. Silica deposits in the Nili Patera caldera on the Syrtis Majo
volcanic complex on Mars. Nature Geoscience. 3, 838–841, https://doi.org/10.1038/ngeo990 (2010).f p
,
,
p
g
g
(
)
15. Ruff, S. W. & Farmer, J. D. Silica deposits on Mars with features resembling hot spring biosignatures at El Tatio in Chile. Nature
communications. 7, 13554, https://doi.org/10.1038/ncomms13554 (2016).i 15. Ruff, S. W. & Farmer, J. D. Silica deposits on Mars with features resembling hot spring biosignatures at El Tatio in Chi
i
ti
7 13554 htt
//d i
/10 1038/
13554 (2016) 15. Ruff, S. W. & Farmer, J. D. Silica deposits on Mars with features resembling hot spring biosignatu
communications. 7, 13554, https://doi.org/10.1038/ncomms13554 (2016).i 15. Ruff, S. W. & Farmer, J. D. Silica deposits on Mars with features resembling hot spring biosignatures at El Tatio in Chile. Nature
communications. 7, 13554, https://doi.org/10.1038/ncomms13554 (2016).i p
g
16. Ghai, R. et al. New abundant microbial groups in aquatic hypersaline environments. Scientific reports 1, 135 (2011). g
y
fi
p
17. Oggerin, M., Tornos, F., Rodriguez, N., Pascual, L. & Amils, R. Fungal Iron biomineralization in Río Tinto. Minerals 6, 37, https://
doi.org/10.3390/min6020037 (2016).li g
8. Ishii, K., Mussmann, M., Macbregor, B. J. & Amann, R. An improved fluorescence in situ hybridization protocol for the identification
of bacteria and archaea in marine sediments. FEMS Microbiol Ecol. 50, 203–13 (2004).i 19. Glöckner, F. O. et al. An In Situ Hybridization Protocol for Detection and Identification of Planktonic Bacteria. Systematic and
Applied Microbiology 19, 403–406, https://doi.org/10.1016/S0723-2020(96)80069-5 (1996). 20. Narasingarao, P. et al. De novo metagenomic assembly reveals abundant novel major lineage of Archaea in hypersaline microbial
communities. The ISME journal 6(1), 81 (2012). h
j
1. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 9(7), 676–82, https://doi.org/10.1038
nmeth.2019 (2012).l 22. Cruaud, P. et al. Influence of DNA extraction method, 16S rRNA targeted hypervariable regions, and sample origin on microbial
diversity detected by 454 pyrosequencing in marine chemosynthetic ecosystems. Applied and Environmental Microbiology. 80(15),
4626–4639 (2014). 23. Klindworth, A. et al. Materials and Methodsh Since Ultra small microorganisms are the target of this study, their representative
sequences were filtered from biom table in order to analysis only these taxa using filter_taxa_from_otu_
table.py and filter_fasta.py scripts in Qiime. Finally, the ARB software package28 was used to reconstruct the
phylogenetic tree. The neighbour-joining with Felsenstein correction included in the ARB package was used
for phylogenetic inference. The robustness of the reconstructed trees was evaluated by bootstrap analysis of
1000 resampled datasets. Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 7 www.nature.com/scientificreports/ References 1. Horneck, G. et al. AstRoMap European Astrobiology Roadmap. Astrobiology. 16(3), 201–243, https://doi.org/10.1089/ast.2015.1441
(2016). 1. Horneck, G. et al. AstRoMap European Astrobiology Roadmap. Astrobiology. 16(3), 201–243, https://doi.org/10.1089/ast.2015.1441
(2016). 2. Stetter, K. O. Extremophiles and their adaptation to hot environments. FEBS Letters. 452, 1–2, https://doi.org/10.1016/S0014
5793(99)00663-8 (2003).h 3. Fairén, A. G. et al. Astrobiology through the Ages of Mars: The Study of Terrestrial Analogues to Understand the Habitability o
Mars. Astrobiology. 10(8), 821–843, https://doi.org/10.1089/ast.2009.0440 (2010). gy
p
g
4. Rothschild, L. J. Earth analogs for Martian life. Microbes in evaporites, a new model system for life on Mars. Icarus. 88-1, 246–260
https://doi.org/10.1016/0019-1035(90)90188-F (1990). p
g
(
)
(
)
5. Miruts, H., Bheemalingeswara, K. & Jemal, A. A preliminary geological and generalized stratigraphy of western margin of northerm
Afar depression, Dallol area, northern Ethiopia. Momona Ethiopian Journal of Science. 8(1), 1–22 (2016). p
p
p
f
6. Pagli, C. et al. Shallow axial magmachamber at the slow spreading Erta Ale Ridge. Nat Geosci 5, 284–288 (2012). 7. Nobile, A. et al. Dyke-fault interaction during the 2004 Dallol intrusion at the northern edge of the Erta AleRidge (Afar, Ethiopia)
Geophys Res Lett 39, L19305 (2012).h p y
8. Cavalazzi, B. et al. The Dallol Geothermal Area, Northern Afar (Ethiopia)—An Exceptional Planetary Field Analog on Earth
Astrobiology Published Online: 17 Jan, https://doi.org/10.1089/ast.2018.1926 (2019).h gy
p
g
(
)
9. Fawdon, P. et al. The geological history of Nili Patera, Mars. JGR. 120(5), 951–977, https://doi.org/10.1002/2015JE004795 (201 gy
p
g
9. Fawdon, P. et al. The geological history of Nili Patera, Mars. JGR. 120(5), 951–977, http h
10. Kotopoulou, E. et al. A Polyextreme hydrothermal system controlled by iron: the case of Dallol at the Afar triangle. ACS Earth Space
Chem. 3(1), 90–99, https://doi.org/10.1021/acsearthspacechem.8b00141 (2019).f p
g
p
11. Darland, G., Brock, T. D., Samsonoff, W. & Conti, S. F. A thermophilic acidophilic Mycoplasma isolated from a coal refuse
Science 170, 1416–1418 (1970).h (
)
12. Brock, T. D. Thermophilic microorganisms and life at high temperatures. Springer-Verlag, New York (1978). h
13. Schleper, C., Pühler, G., Kühlmorgen, B. & Zillig, W. Life at extremely low pH. Nature 375, 741–742 (1995). 14. Skok, J. R., Mustard, J. F., Ehlmann, B. L., Milliken, R. E. & Murchie, S. L. Silica deposits in the Nili Patera caldera on the Syrtis Major
volcanic complex on Mars. Nature Geoscience. 3, 838–841, https://doi.org/10.1038/ngeo990 (2010). 14. Skok, J. R., Mustard, J. Additional Informationh Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. 8 Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 9
|
https://openalex.org/W2743397739
|
https://eprint.ncl.ac.uk/fulltext.aspx?url=267240/E7231A79-D20B-48DE-801D-461FECDED75F.pdf&pub_id=267240
|
English
| null |
Salivary diagnostic markers in males and females during rest and exercise
|
Journal of the International Society of Sports Nutrition
| 2,017
|
cc-by
| 7,334
|
Abstract Background: Saliva is a useful diagnostic tool for analysis in sports, exercise and nutrition research, as collection is
easy and non-invasive and it contains a large number of analytes affected by a range of physiological and
pathological stressors and conditions. This study examined key salivary electrolytes and stress and immune markers
in males and females at rest and during exercise. Methods: Unstimulated whole saliva from 20 healthy, recreationally active participants (8 males and 12 females)
was analysed for flow rate, osmolality, sodium (Na+), potassium (K+), chloride (Cl−), secretory immunoglobulin A
(SIgA), α-amylase activity and cortisol during both rest and moderate intensity (70% peak power) cycling exercise in
a randomised crossover design. Each trial lasted 60 min and sampling was carried out at 15 and 45 min after the
start of the trial. Saliva was collected using the gold-standard drool method; participants were required to provide
at least 1 mL sample over 2 or 3-min period. Results: Females showed a greater response to steady-state exercise stress than males, with significant increases in
osmolality (P < 0.001), α-amylase activity (P = 0.001) and secretion rate (P = 0.023) and SIgA secretion rate (P = 0.023),
with trends for an increase in K+ (P = 0.053) and decrease in Cl−(P = 0.067). There were no differences between rest
and exercise for any salivary analytes in males. In addition, females showed a trend for higher levels of cortisol than
males at both rest (P = 0.099) and exercise (P = 0.070), as well as a higher heart rate (P < 0.001) and greater ratings of
perceived exertion (P < 0.001) during the exercise trial. The coordination of the two stress response pathways
(α-amylase vs cortisol) was positive in males (r = 0.799; P = 0.017) yet negative in females (r = −0.475; P = 0.036). Conclusions: Males and females show a markedly different response to steady-state exercise stress as measured in Conclusions: Males and females show a markedly different response to steady-state exercise stress as measured in
unstimulated whole saliva. Keywords: Hydration, Stress response, Immune markers, Electrolytes, Sex Keywords: Hydration, Stress response, Immune markers, Electrolytes, Sex Hydration, electrolyte status, stress and immune re-
sponses are key markers for exercise performance and
health status [2, 3, 10]. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. 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: A.Ali@massey.ac.nz
2Centre for Metabolic Health Research, Massey University, Palmerston North,
New Zealand
5School of Sport, Exercise and Nutrition Massey University, Auckland, New Zealand
Full list of author information is available at the end of the article Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition
(2017) 14:27 Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition
(2017) 14:27 DOI 10.1186/s12970-017-0185-8 Open Access Open Access Kay Rutherfurd-Markwick1,2, Carlene Starck2,3, Deborah K. Dulson4 and Ajmol Ali2,5* Kay Rutherfurd-Markwick1,2, Carlene Starck2,3, Deborah K. Dulson4 and Ajmol Ali2,5* Abstract A 2–3% dehydration-associated
body mass loss has been linked with a reduction in heat
regulation, cardiovascular function and exercise per-
formance [11] and results in significant changes in
salivary composition [1]. Water is the predominant fluid
constituent of saliva, thus hypo-hydration is expected to
decrease salivary flow-rate, increase osmolality, and may
alter the concentrations of key electrolytes, hormones
and proteins [11–13]. Background Saliva is gaining momentum as a relevant fluid for
clinical and forensic diagnosis, as well as for analysis in
sports, exercise and nutrition research, as collection is
easy and non-invasive and it contains a large number of
analytes affected by a range of physiological and patho-
logical stressors and conditions [1–5]. In addition, saliva
is less complex than serum, including lower protein
content, thus requiring substantially less preparation for
analysis [2, 3]. Furthermore, saliva may be used to exam-
ine the role of sex hormones in stress and disease [6–9]. Exercise modulates both the innate and acquired arms
of the immune system [7] and activates the two major
neuroendocrine stress response arms, the hypothalamic-
pituitary-adrenal
(HPA)
axis
and
the
sympathetic- Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 2 of 8 participants were required to be free of injury, chronic
disease and infection in the 4 weeks prior to the study. adreno-medullary (SAM) axis (sympathetic nervous sys-
tem). While SAM activation is an immediate response to
exercise, the HPA axis shows a delayed response [6, 14]. Saliva carries two primary markers of HPA and SAM ac-
tivation, cortisol and α-amylase, respectively [15]. Alpha-
amylase is a reliable indicator of the established blood
markers for SAM, epinephrine and norepinephrine, as
well as playing a role in mucosal immunity [16]. How-
ever, salivary secretory immunoglobulin A (SIgA) is the
most widely recognised marker of mucosal immunity
[17, 18] and, there appears to be a relationship between
decreases in SIgA and increased risk of upper respiratory
tract infection (URTI) [10, 19]. Immune and stress re-
sponses work together to combat exercise stress [6], with
both the HPA and SAM axes modulating the function of
the immune system. Preliminary procedures A preliminary session was undertaken to familiarise par-
ticipants with the experimental protocol. Upon arrival to
the laboratory, participants were shown the correct tech-
nique for saliva specimen collection by the passive drool
method for the collection of unstimulated whole saliva
(UWS). Each participant then performed an incremental
exercise test on a cycle ergometer (Ergomedic 874E,
Monark Exercise AB, Vansbro Sweden) starting at 60 W,
with intensity increasing by 30 W·min−1 until volitional
fatigue. Following a brief rest of 5–10 min, participants
cycled at a resistance corresponding to 70% of their
previously determined peak power for 10 min, then re-
ported their perceived exertion and level of confidence
regarding completing 60 min of continuous exercise at
this intensity. y
While blood sampling has historically been used to
measure hydration, electrolyte status and markers of
stress and immunity, blood sampling procedures may
not be practical for the setting, they can be expensive
and the invasive approach may not be appealing for all
participants [4]. The analysis of other bodily fluids such
as saliva holds promise in these situations; however, our
understanding of the actions and interactions of the key
salivary diagnostic markers in response to stress is in-
complete. Most studies have presented information
about a selection of markers only and differences in
methodology between studies have led to equivocal in-
formation [4]. Information in the literature is limited
mostly to reviews, in which correlations have been made
between studies using different types of participants and
varying protocols [2, 3, 10]. Moreover, while it appears
that there are sex-related differences in the response of
salivary markers to exercise stress [16, 20] most of the
research focuses on men or a mixed cohort [3, 10];
hence data pertaining to women in isolation, or compar-
ing the male and female response, is scarce [3, 15]. Main trials In a randomised cross-over design, participants per-
formed either an exercising or resting protocol; the
alternative protocol was performed on their subsequent
visit (3–7 days later). The exercising protocol involved
60 min of steady-state cycling at 70% peak power,
whereas for the resting trial participants sat quietly for
60 min. Participants were asked to refrain from consuming caf-
feine and alcohol and avoid exercise in the 24-h period
prior to the trial. They were also asked to replicate the
same food and beverage intake prior to each trial and
report to the laboratory 3 h post-prandial. Four hours
prior to their arrival to the laboratory, participants were
reminded (via text message) to consume the 7 mL·kg−1
BM quantity of water provided by the researcher in the
preliminary session. Upon arrival to the laboratory, a
midstream urine sample was obtained for immediate de-
termination of hydration status by urine specific gravity
(USG) using a handheld refractometer (Sur-Ne, Atago
Co Ltd., Japan); all participants’ USG levels were below
1.020 and therefore were well hydrated prior to exercise. Body mass was measured before and immediately after
each trial period. Our aim was to conduct a thorough analysis of a wide
range of salivary analytes in males and females both at
rest and in response to exercise, in order to provide a
valuable reference dataset for future studies. Methods
Participants In total, 20 recreationally active participants completed
the study (males n = 8; females n = 12: mean age
27.4 ± 5.9 years). Males (height 1.77 ± 0.04 m; weight
81.1 ± 6.5 kg) were significantly taller and heavier than
females (height 1.66 ± 0.06 m; weight 62.8 ± 8.4 kg;
P < 0.001). All procedures had prior approval by the
local institutional ethics committee. Following comple-
tion of a health screening questionnaire, written in-
formed consent was obtained from all participants. In
order to be considered for inclusion in this study Saliva was collected via the UWS drool method at two
time points (15 min and 45 min) during each protocol. Both trials were conducted at the same time of day
(15:00–18:00 h) to overcome any circadian influences. Heart rate (HR; T31 Polar heart rate monitor, Kempele,
Finland) was measured continuously and ratings of
perceived exertion (RPE) were monitored at 10-min
intervals during exercise. Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 3 of 8 Page 3 of 8 Page 3 of 8 Hydration parameters
h
h There was no change in UWS flow rate between rest
and exercise for males (P = 0.248) or females (P = 0.801;
Fig. 1a). However, males produced a higher flow rate
than females during exercise (P = 0.007) and there was a
trend for increase in flow rate at rest (P = 0.056). Within
the exercise trial itself, females showed an increase in
flow rate from 15 to 45 min (P = 0.031; Table 1); this
was not observed in males (P = 0.730). However, females
(P = 0.010) also produced a significant increase in flow
rate within the rest trial (Table 1). Saliva osmolality was measured using a freezing point
depression osmometer according to the manufacturer’s
instructions (Osmomat 030, Gonotec, Berlin, Germany). Salivary electrolyte levels were measured using an Easy-
Lyte analyser according to the manufacturer’s instruc-
tions (Medica Corporation, Bedford, MA, USA). Salivary
secretory IgA concentration was determined by ELISA
as described elsewhere [22]. Salivary cortisol concentra-
tion was determined by radioimmunoassay according to
the
manufacturer’s
instructions
(IBL
International
GMBH, Tecan, Hamburg, Germany, IBMG1206). Saliv-
ary α-amylase activity was determined using the Infinity
Amylase Liquid stable reagent (Thermoscientific, Wor-
thing, UK) according to the manufacturer’s instructions. 1 There was a rise in saliva osmolality for females
(P = 0.01) during exercise compared to rest, but no
change for males (P = 0.838) and no difference between Fig. 1 Mean data for (a). Flow rate (g·min−1) and (b). osmolality
(mOsmol·kg−1) and associated errors between rest and exercise for
males and females. A significant difference between rest and
exercise is indicated with an asterisk (**, P < 0.001; *, P < 0.05) and
between males and females with letters (a, P < 0.001; b, P < 0.05) g
g
The secretion rates of SIgA (μg·min−1) and α-amylase
(U·min−1) were calculated by multiplying the saliva flow
rate (mL·min−1) by the IgA concentration (mg·L−1) and
α-amylase activity (U·mL−1), respectively. Statistical analysis Independent t-tests were used to compare data between
males and females. Paired t-tests were used to compare
15 min vs. 45 min at rest and 15 min vs. 45 min during
exercise. Paired t-tests were used to compare rest vs. exercise data for males and females, separately (mean of
rest vs mean of exercise). Pearson’s correlation was used
to
examine
the
relationships
between
independent
variables. The results are presented as mean values ±
standard deviation. Statistical significance was accepted
at P < 0.05. Saliva collection and analysis (P < 0.001) but there were no sex differences for HR at
rest (P = 0.136). RPE increased with duration of exercise
(P = 0.006) and was higher at 60 min compared to
10 min (P < 0.05). Females reported higher average RPE
than males during exercise (P < 0.001). There was no
difference in body mass loss between males and females
during exercise although females lost more fluid than
males in both absolute mass (P = 0.014) and as a
percentage of body mass (P = 0.001). (P < 0.001) but there were no sex differences for HR at
rest (P = 0.136). RPE increased with duration of exercise
(P = 0.006) and was higher at 60 min compared to
10 min (P < 0.05). Females reported higher average RPE
than males during exercise (P < 0.001). There was no
difference in body mass loss between males and females
during exercise although females lost more fluid than
males in both absolute mass (P = 0.014) and as a
percentage of body mass (P = 0.001). Saliva was collected into a disposable pre-weighed
60 mL plastic container. Participants were instructed to
sit leaning forwards with their head tilted downwards
and swallow before any sampling took place. During
sampling participants were asked to perform minimal
orofacial movement and to allow the saliva to dribble
into the tube. At least 1 mL was collected over a 2-min
period per participant. If insufficient sample was ob-
tained after this time, a further minute of collection was
performed. Saliva
was
weighed
(Sartorius
LE3235,
Germany) and flow rate was calculated on the assump-
tion that saliva density was 1 g·mL−1 [21]. Saliva speci-
mens were then stored at −80 °C until analysis. Exercise trial Although there were no differences in USG between rest
and exercise trials in males (P = 0.178) or females
(P = 0.972), pre-exercise, females exhibited lower USG
than males (P = 0.018). HR was higher during exercise
than rest (P < 0.001), increased during exercise for both
sexes (P < 0.001), but remained constant at rest. During
exercise, females had a higher average HR than males Fig. 1 Mean data for (a). Flow rate (g·min−1) and (b). osmolality
(mOsmol·kg−1) and associated errors between rest and exercise for
males and females. A significant difference between rest and
exercise is indicated with an asterisk (**, P < 0.001; *, P < 0.05) and
between males and females with letters (a, P < 0.001; b, P < 0.05) Fig. 1 Mean data for (a). Flow rate (g·min−1) and (b). osmolality
(mOsmol·kg−1) and associated errors between rest and exercise for
males and females. A significant difference between rest and
exercise is indicated with an asterisk (**, P < 0.001; *, P < 0.05) and
between males and females with letters (a, P < 0.001; b, P < 0.05) Rutherfurd-Markwick et al. Exercise trial Journal of the International Society of Sports Nutrition (2017) 14:27 Page 4 of 8 Page 4 of 8 Table 1 Levels of salivary analytes at two time points during rest and exercise trials in males and females, degree of change
between the time points by percentage (Δ) and the significance of this change (P)
Rest
Exercise
T1
T2
Δ (%)
P
T1
T2
Δ (%)
P
Flow rate (g · min-1)
Males
0.64 ± 0.47
0.72 ± 0.57
8.9 ± 26.6
0.176
0.81 ± 0.38
0.77 ± 0.52
-1.7 ± 39.5
0.730
Females
0.34 0.13
0.40 ± 0.12
21.3 ± 31.0
0.010
↑
0.32 ± 0.19
0.38 ± 0.24
16.7 ± 24.4
0.031
↑
Osmolality (mOsmol · kg-1)
Males
65.4 ± 15.8
65.9 ± 18.7
0.31 ± 9.1
0.850
62.1 ± 18.5
71.3 ± 34.8
11.9 ± 27.1
0.287
Females
57.3 ± 15.1
56.0 ± 11.8
-0.20 ± 15.1
0.650
66.3 ± 16.6
68.8 ± 11.2
6.1 ± 14.0
0.434
Na (mmol · L-1)
Males
4.5 ± 2.2
5.1 ± 1.8
28.3 ± 48.9
0.451
5.1 ± 2.5
9.3 ± 12.0
37.7 ± 69.8
0.285
Females
6.4 ± 3.2
5.0 ± 1.3
-11.0 ± 31.0
0.136
5.3 ± 2.4
6.6 ± 1.8
35.9 ± 61.6
0.199
K (mmol · L-1)
Males
19.7 ± 4.1
21.3 ± 6.2
10.6 ± 40.5
0.528
22.3 ± 4.6
22.8 ± 5.1
2.2 ± 11.0
0.626
Females
21.9 ± 6.0
19.5 ± 4.8
-9.3 ± 15.1
0.060
↓
23.5 ± 5.5
24.3 ± 5.5
3.9 ± 7.5
0.150
Cl (mmol · L-1)
Males
32.6 ± 13.2
35.9 ± 20.4
13.8 ± 64.7
0.634
32.2 ± 13.5
36.6 ± 22.6
10.6 ± 26.2
0.343
Females
49.6 ± 27.2
43.1 ± 23.3
-9.7 ± 26.7
0.193
37.6 ± 19.1
38.7 ± 16.6
5.9 ± 14.3
0.543
SIgA concentration (mg · L-1)
Males
65.7 ± 42.2
66.8 ± 52.0
-3.5 ± 31.1
0.895
56.1 ± 41.2
61.2 ± 49.2
12.0 ± 53.4
0.526
Females
86.4 ± 49.0
60.2 ± 35.8
8.3 ± 130.1
0.120
95.2 ± 103.9
112.9 ± 91.3
46.1 ± 63.4
0.083
↑
SIgA secretion rate (μg · min-1)
Males
33.6 ± 17.5
39.1 ± 37.2
12.8 ± 59.9
0.559
39.6 ± 35.1
31.1 ± 12.9
7.5 ± 62.4
0.486
Females
25.2 ± 17.1
24.1 ± 18.8
34.6 ± 137.1
0.275
29.7 ± 27.8
45.5 ± 34.4
78.8 ± 110.3
0.032
↑
α-amylase activity (U∙mL-1)
Males
34.6 ± 20.4
38.2 ± 22.1
12.3 ± 12.2
0.050
↑
34.8 ± 19.5
44.0 ± 24.6
26.5 ± 19.0
0.034
↑
Females
25.7 ± 23.5
29.5 ± 24.9
24.9 ± 27.4
0.030
↑
41.7 ± 21.9
56.3 ± 22.3
35.2 ± 32.9
<0.001
↑
α-amylase secretion rate (U · min-1)
Males
20.8 ± 18.9
27.6 ± 26.1
26.6 ± 34.4
0.559
28.1 ± 22.2
30.5 ± 22.6
22.9 ± 53.5
0.672
Females
7.3 ± 5.9
11.3 ± 9.6
68.4 ± 51.5
0.275
13.1 ± 9.5
20.6 ± 12.1
68.4 ± 51.5
0.004
↑
Cortisol (nmol · L-1)
Males
4.21 ± 0.73
4.02 ± 0.48
-3.7 ± 8.2
0.210
4.63 ± 0.71
4.55 ± 0.60
-1.0 ± 8.5
0.617
Females
5.95 ± 2.26
5.26 ± 2.16
-11.4 ± 8.3
<0.001
↓
6.00 ± 1.88
5.96 ± 2.08
-0.3 ± 10.8
0.894
UWS was sampled at 15 min (T1) and 45 min (T2) during each protocol. Exercise trial Arrows indicate whether there was an increase (↑) or decrease (↓) in the levels of the
corresponding analyte over time Table 1 Levels of salivary analytes at two time points during rest and exercise trials in males and females, degree of change
between the time points by percentage (Δ) and the significance of this change (P) sexes at rest (P = 0.191) or during exercise (P = 0.926;
Fig. 1b). There was no difference in saliva osmolality
over time within the rest or exercise trials for either sex
(P > 0.05; Table 1). Concentrations of salivary K+ showed a trend for an in-
crease between rest and exercise in females (P = 0.053);
however, there was no change in males (P = 0.107; Fig. 2b). While there was no change in salivary K+ levels
within the rest or exercise trials for either sex (P > 0.05),
there was a trend for a decrease in salivary K+ during
rest in females (P = 0.060; Table 1). Salivary Cl−levels
showed a trend for a decrease in females between rest
and exercise (P = 0.067); however, there was no change
for males (P = 0.971; Fig. 2c). There was no change in
salivary Cl−levels during the rest or exercise trials for ei-
ther sex (P > 0.05; Table 1). Electrolytes mucosal immunity, SIgA
(mg·L−1); (b) the sympathetic stress response, α-amylase activity (U·mL−1)
and c. the adrenal stress response, cortisol (μg·L−1), and associated errors
between rest and exercise for males and females. A significant difference
between rest and exercise is indicated with an asterisk (**, P < 0.001;
*, P < 0.05) and between males and females with letters (b, P < 0.001;
b, P < 0.05) Fig. 2 Mean electrolyte data and associated errors between rest and
exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1)
and c. Cl (mmol·L−1) Fig. 2 Mean electrolyte data and associated errors between rest and
exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1)
and c. Cl (mmol·L−1) Electrolytes There was no change in salivary Na+ levels between rest
and exercise in males or females; nor was there any
change in Na+ levels between males and females in the
rest or exercise trials (P > 0.05; Fig. 2a). There was no
change in saliva Na+ levels during the rest or exercise
trials
for
males
or
females
(P
>
0.05;
Table
1). Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 5 of 8 Mucosal immune and stress markers
Fig. 2 Mean electrolyte data and associated errors between rest and
exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1)
and c. Cl (mmol·L−1)
Fig. 3 Mean data for salivary markers of (a). mucosal immunity, SIgA
(mg·L−1); (b) the sympathetic stress response, α-amylase activity (U·mL−1)
and c. the adrenal stress response, cortisol (μg·L−1), and associated errors
between rest and exercise for males and females. A significant difference
between rest and exercise is indicated with an asterisk (**, P < 0.001;
*, P < 0.05) and between males and females with letters (b, P < 0.001;
b, P < 0.05) Fig. 2 Mean electrolyte data and associated errors between rest and
exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1)
and c. Cl (mmol·L−1) Fig. 3 Mean data for salivary markers of (a). mucosal immunity, SIgA
(mg·L−1); (b) the sympathetic stress response, α-amylase activity (U·mL−1)
and c. the adrenal stress response, cortisol (μg·L−1), and associated errors
between rest and exercise for males and females. A significant difference
between rest and exercise is indicated with an asterisk (**, P < 0.001;
*, P < 0.05) and between males and females with letters (b, P < 0.001;
b, P < 0.05) Fig. 2 Mean electrolyte data and associated errors between rest and
exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1)
and c. Cl (mmol·L−1) Fig. 3 Mean data for salivary markers of (a). mucosal immunity, SIgA
(mg·L−1); (b) the sympathetic stress response, α-amylase activity (U·mL−1)
and c. the adrenal stress response, cortisol (μg·L−1), and associated errors
between rest and exercise for males and females. A significant difference
between rest and exercise is indicated with an asterisk (**, P < 0.001;
*, P < 0.05) and between males and females with letters (b, P < 0.001;
b, P < 0.05) Fig. 3 Mean data for salivary markers of (a). Mucosal immune and stress markers There was no change in SIgA concentration in response
to exercise in males or females (P > 0.05); nor was there a
change between the sexes at rest (P = 0.728) or exercise
(P = 0.235; Fig. 3a). There was no change in SIgA concen-
tration during the rest trial for either sex (P > 0.05; Table
1). There was a trend for an increase in SIgA concentra-
tion during exercise in females (P = 0.083). Alpha-amylase
activity increased in the exercise trial compared to rest for
females (P = 0.001; Fig. 3b) but not males (P = 0.501). There was no change in salivary α-amylase activity be-
tween males and females at rest (P = 0.429) or exercise
(P = 0.345). However, there were significant increases in
salivary α-amylase activity during both the rest and exer-
cise trials for both sexes (P < 0.05; Table 1). increased from 15 min to 45 min during exercise in fe-
males (P < 0.05 for both) with α-amylase secretion rates
increasing
in
the
exercise
trial
compared
to
rest
(P = 0.023). There were no differences between sexes at
rest (P > 0.05) or during exercise (P > 0.05) for SIgA or
α-amylase secretion rate. There was a trend for higher salivary cortisol levels in
females compared to males at both rest (P = 0.099) and
during exercise (P = 0.070). There was no difference in
salivary cortisol levels between rest and exercise for
either sex (P > 0.05). Within the rest trial, a significant
decrease in salivary cortisol was observed for females
(P = 0.003; Table 1) but not males (P = 0.206). Salivary
cortisol levels remained unchanged during the exercise
trial. When expressed as a secretion rate (which takes flow
rate into account) SIgA and α-amylase secretion rates Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 6 of 8 Fig. 4 Correlations between α-amylase activity and cortisol, representing
the sympathetic and adrenal stress responses, respectively, for (a). males;
(b). females for both rest (filled markers) and exercise (empty markers). c
shows a direct comparison of the exercise response at 15 min between
males (squares) and females (circles) Discussion The aim of this study was to examine salivary analytes in
males and females both at rest and in response to exer-
cise. The main finding was that males and females show
a markedly different response to steady-state exercise
stress as measured in unstimulated whole saliva. This
study provides separate novel datasets of salivary re-
sponses to exercise stress in males and females that can
be used as a reference point for future research. A recognised limitation of the gold standard drool
collection method [23] for UWS is the low flow rate rela-
tive to stimulated methods, and females have a lower
UWS flow rate than males due to smaller salivary glands
[24]. Our data showed females had lower flow rates during
both exercise (P = 0.007) and rest (P = 0.056) compared to
males. This difference in salivary flow rate may become
limiting for data analysis, as flow rate has been suggested
to influence the concentrations of some salivary analytes
[1, 10, 24]. Flow rate has been suggested to be affected by
exercise [10], however no difference in salivary flow rate
was observed between rest and exercise for either males
or females in this study. Exercise increased salivary osmolality in females
(Fig 1b); in conjunction with an increase in both α-
amylase activity (Fig 3b) and secretion rate (Table 1),
this supports the consistently reported exercise-driven ac-
tivation of the SAM axis [16, 25]. These results were not
seen in males, indicative of sex-specific differences in the
salivary response to exercise stress. The reason for the in-
crease in α-amylase activity, which was observed during
exercise and rest for both sexes, is unclear but it is
possible that in the absence of exercise stimulation, the
antimicrobial and/or digestive roles of α-amylase may
affect exercise-independent measurements. In addition,
anticipation of sampling, causing premature activation of
the autonomic stress response, may also affect α-amylase
activity levels [6]. Fig. 4 Correlations between α-amylase activity and cortisol, representing
the sympathetic and adrenal stress responses, respectively, for (a). males;
(b). females for both rest (filled markers) and exercise (empty markers). c
shows a direct comparison of the exercise response at 15 min between
males (squares) and females (circles) Fig. 4 Correlations between α-amylase activity and cortisol, representing
the sympathetic and adrenal stress responses, respectively, for (a). males;
(b). females for both rest (filled markers) and exercise (empty markers). Discussion c
shows a direct comparison of the exercise response at 15 min between
males (squares) and females (circles) Unstimulated resting salivary electrolyte levels are
typically in the range of 3–29 mmol·L−1 (Na+), 6.4–
36.6 mmol·L−1 (K+) and 0–27 mmol·L−1 (Cl−) [2]. The
broad ranges reflect the various factors which affect
salivary electrolyte levels such as hydration status and
salivary flow rate; the Na+ and K+ values from this study
fall within normal ranges while the Cl−levels are higher
than the quoted ranges. Although salivary K+ levels in-
creased in females in response to exercise, there were no
accompanying increases in Na+ or Cl−. In fact, there was
a trend for a decrease in Cl−in females. While SAM ac-
tivation may induce electrolyte release, research indi-
cates substantial variation in the effects of exercise on
salivary electrolyte levels which are impacted by exercise
intensity and saliva collection methods [10]. reported a decrease in SIgA in response to exercise [10]. However, SIgA may be affected by training status and
the type of training carried out [10]; although we
recruited recreationally active participants the inter-
individual variation in SIgA measurements was large and
therefore this result must be interpreted with caution. Taken together our results suggest a notable difference
in the physiological response to exercise stress between
males and females, particularly with respect to activation
of the SAM and HPA axes, represented by α-amylase
and cortisol, respectively. While the coordination of the
SAM and HPA pathways indicates a functional stress
response, the activation of one without the other may
represent a dysfunctional response due to the physio-
logical consequences of chronic exposure to fluctuating
or heightened neuroendocrine responses resulting from In this study females showed an increase in SIgA
during the exercise trial, whereas other studies have Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 7 of 8 Page 7 of 8 Page 7 of 8 however, in males the cortisol-SIgA relationship was
positive (r = 0.255; P = 0.592) while in females it was
negative (r = −0.177; P = 0.440). Such weak non-
significant associations limit the conclusions that can be
drawn, but do lend some support to both the SAM ac-
tivity and HPA axis potentially regulating SIgA response. repeated or chronic stress (‘allostatic load’) [26]. Abbreviations
Cl Chl
id
HPA Abbreviations
Cl: Chloride; HPA: Hypothalamic-pituitary-adrenal; HR: Heart rate; K
+: Potassium; Na+: Sodium; RPE: Ratings of perceived exertion;
SAM: Sympathetic-adreno-medullary; SIgA: Secretory immunoglobulin A;
URTI: Upper respiratory tract infection; USG: Urine specific gravity;
UWS: Unstimulated whole saliva The stress pathways also modulate immune responses
[6] with increases in the circulating stress hormones cat-
echolamines and cortisol mediating SIgA responses to
exercise. Whilst there is scarce human research it has
been
suggested
that
cortisol
inhibits
transepithelial
transport of SIgA, while adrenaline appears to enhance
IgA transcytosis [29, 30]. In the current study α-amylase
was positively associated with SIgA in both males
(r = 0.457; P = 0.255) and females (r = 0.214; P = 0.505), Discussion Interest-
ingly, the relationship between α-amylase activity and
cortisol levels produced during exercise gave opposing
trends for males and females (Fig. 4). While both sexes
showed a weak but negative α-amylase versus cortisol
relationship at rest, during exercise this relationship be-
came strongly positive for males (r = 0.799; P = 0.017;
Fig. 4a) yet remained negative for females (r = −0.475;
P = 0.036; Fig. 4b). Considering the dysfunctional stress
response to allostatic load it is unlikely that all females
exhibit dysfunctional stress systems. One explanation is
that the difference between males and females is compli-
cated by the delayed response of cortisol to stress, in
comparison to that of α-amylase [14]. To address this,
we performed correlational analysis at 15 min and
45 min as well as a lagged comparison with α-amylase at
15 min and cortisol at 45 min. The strongest correlation
for both males (r = 0.890; P = 0.003) and females
(r = −0.652; P = 0.021) was observed at 15 min (Fig. 4c),
indicating a result consistent with the overall exercise
stress relationship. Males showed a coordinated increase
in both cortisol and α-amylase, whereas females exhib-
ited a negative relationship, indicating that the stress
axes function independently. This study has some limitations. It is apparent from
this study that there are sex-specific responses to
exercise, likely due to the steroid hormones and thus, to
fully understand these differences, the measurement of
these hormone levels is necessary. In addition, although
the drool method is considered to be the gold standard
approach for diagnosis it has inherent issues, particularly
a low flow rate that differs between males and females. Although menstrual cycle phase has been shown not to
influence neuroendocrine responses [27, 28] future studies
may wish to control for menstruation when using female
participants. Our data show several differences between
males and females but we also report a few non-
significant trends, which may indicate that a larger sample
size may have been required, and so universal conclusions
cannot be made. Conclusions
h While exercise is considered to stimulate both HPA
and SAM stress response systems, the response of each
is dependent on a number of factors including exercise
intensity, duration, training status and the sex hormones
[6]. The spread of the female α-amylase versus cortisol
plot in Fig. 4c indicates three different scenarios: points
in the middle of the plot indicate the two systems work-
ing together at a moderate level; points in the lower
right region show sympathetic activity but no HPA re-
sponse and points in the top left quartile represent HPA
activity without SAM activation. Since exercise intensity
and duration was consistent between the sexes, and
training status is unlikely to be sex-dependent, the most
likely explanation for the contrasting neuroendocrine re-
sponses observed in females involves menstrual status or
menstrual cycle phase, although the latter has been
repeatedly shown not to influence these pathways
[27, 28]. We did not collect hormonal data, and so a
detailed study into the effects of the female hor-
mones on exercise-associated stress pathway coordin-
ation is warranted. Monitoring hydration status, exploring immune re-
sponses to exercise and examining exercise stress are
important considerations for sports and exercise nutri-
tion scientists and practitioners. Saliva sampling is be-
coming increasingly important with regards non-invasive
monitoring of athletes as well as non-athletes. Our data
provides an overview of the electrolyte, immune and
stress response to steady-state submaximal exercise in
both males and females using the current gold standard
drool method to collect unstimulated whole saliva. This
data has revealed some important differences in the re-
sponse of males and females to steady-state exercise
stress, particularly, opposing associations between the
two major neuroendocrine stress axes. While we are un-
able to make specific conclusions about the mechanisms
involved, future studies directly comparing exercise
stress in males and females is warranted. Acknowledgements The authors would like to thank Olivier Morin and John Taulu for help with
data collection, Simon Bennett for technical support and the participants for
providing their time and effort to complete this research. Competing interests Competing interests 20. Li C-Y, Hsu G-S, Suzuki K, Ko M-H, Fang S-H. Salivary Immuno factors,
Cortisol and testosterone responses in athletes of a competitive 5,000 m
race. Chinese J Physiol. 2015;58(4):263–9. 20. Li C-Y, Hsu G-S, Suzuki K, Ko M-H, Fang S-H. Salivary Immuno factors,
Cortisol and testosterone responses in athletes of a competitive 5,000 m
race. Chinese J Physiol. 2015;58(4):263–9. References A review of sex differences in
immune function after aerobic exercise. Exerc Immunol Rev. 2011;17:104–21. 7. Gillum T, Kuennen M, Schneider S, Moseley P. A review of sex differences in
immune function after aerobic exercise. Exerc Immunol Rev. 2011;17:104–21
8. Mastorakos G, Pavlatou M, Diamanti-Kandarakis E, Chrousos GP. Exercise and
the stress system. Hormones (Athens). 2005;4(2):73–89. 8. Mastorakos G, Pavlatou M, Diamanti-Kandarakis E, Chrousos GP. Exercise and
the stress system. Hormones (Athens). 2005;4(2):73–89. • We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
• We provide round the clock customer support
• Convenient online submission
• Thorough peer review
• Inclusion in PubMed and all major indexing services
• Maximum visibility for your research
Submit your manuscript at
www.biomedcentral.com/submit
Submit your next manuscript to BioMed Central
and we will help you at every step: y
All data is presented in the main paper. All data is presented in the main paper. Funding
Th
d This study was funded by the Massey University Research Fund. This study was funded by the Massey University Research Fund. Page 8 of 8 Page 8 of 8 Page 8 of 8 Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. 21. Cole A, Eastoe J. The oral environment. In: Cole A, Eastoe J, editors. Biochemistry and oral biology. 2nd ed. London: Butterworth & Co. Ltd; 1988. p. 478–83. 21. Cole A, Eastoe J. The oral environment. In: Cole A, Eastoe J, editors. Biochemistry and oral biology. 2nd ed. London: Butterworth & Co. Ltd; 1988. p. 478–83. Authors’ contributions 15. Maruyama Y, Kawano A, Okamoto S, Ando T, Ishitobi Y, Tanaka Y, Inoue A,
Imanaga J, Kanehisa M, Higuma H, Ninomiya T, Tsuru J, Hanada H, Akiyoshi
J. Differences in salivary alpha-amylase and Cortisol responsiveness
following exposure to electrical stimulation versus the Trier social stress
tests. PLoS One. 2012;7(7):e39375. AA, KRM and DKD conceived the study and participated in the design of the
study. AA organised data collection. KRM completed biochemical assays. AA
and CS conducted statistical analysis. CS, AA and KRM helped to draft the
manuscript. All authors read and approved the final manuscript. 16. Rohleder N, Nater UM. Determinants of salivary α-amylase in humans and
methodological considerations. Psychoneuroendocrinol. 2009;34(4):469–85. 16. Rohleder N, Nater UM. Determinants of salivary α-amylase in humans and
methodological considerations. Psychoneuroendocrinol. 2009;34(4):469–85. Author details
1 22. Crooks C, Cross M, Wall C, Ali A. Effect of bovine colostrum
supplementation on respiratory tract mucosal defenses in swimmers. Int J
Sport Nutr Exerc Metab. 2010;20(3):224–35. 22. Crooks C, Cross M, Wall C, Ali A. Effect of bovine colostrum
supplementation on respiratory tract mucosal defenses in swimmers. Int J
Sport Nutr Exerc Metab. 2010;20(3):224–35. 1School of Health Sciences, Massey University, Auckland, New Zealand. 2Centre for Metabolic Health Research, Massey University, Palmerston North,
New Zealand. 3Riddet Institute Massey University, Palmerston North, New
Zealand. 4Sports Performance Research Institute New Zealand, Auckland
University of Technology, Auckland, New Zealand. 5School of Sport, Exercise
and Nutrition Massey University, Auckland, New Zealand. 23. Guilhem G, Hanon C, Gendreau N, Bonneau D, Guével A, Chennaoui M. Salivary hormones response to preparation and pre-competitive training of
world-class level athletes. Front Physiol. 2015;6:333. 23. Guilhem G, Hanon C, Gendreau N, Bonneau D, Guével A, Chennaoui M. Salivary hormones response to preparation and pre-competitive training of
world-class level athletes. Front Physiol. 2015;6:333. 24. Inoue H, Ono K, Masuda W, Morimoto Y, Tanaka T, Yokota M, Inenaga K. Gender difference in unstimulated whole saliva flow rate and salivary gland
sizes. Arch Oral Biol. 2006;51(12):1055–60. Received: 11 April 2017 Accepted: 1 August 2017 Received: 11 April 2017 Accepted: 1 August 2017 25. Zouhal H, Jacob C, Delamarche P, Gratas-Delamarche A. Catecholamines
and the effects of exercise, training and gender. Sports Med. 2008;38(5):
401–23. Ethics approval and consent to participate Ethics approval and consent to participate
This study was approved by the Massey University Human Ethics Committee:
Southern A (approval number 11/86). All participants provided written
informed consent prior to taking part in the study. 17. Brandtzaeg P. Humoral immune response patterns of human Mucosae:
induction and relation to bacterial respiratory tract infections. J Infect Dis. 1992;165(Supplement 1):S167–S76. 17. Brandtzaeg P. Humoral immune response patterns of human Mucosae:
induction and relation to bacterial respiratory tract infections. J Infect Dis. 1992;165(Supplement 1):S167–S76. This study was approved by the Massey University Human Ethics Committee:
Southern A (approval number 11/86). All participants provided written informed consent prior to taking part in the study. 18. Papadopoulos E, Muir C, Russell C, Timmons BW, Falk B, Klentrou P. Markers
of biological stress and mucosal immunity during a week leading to
competition in adolescent swimmers. J Immunol Res. 2014:234565. http://
dx.doi.org/10.1155/2014/234565. 18. Papadopoulos E, Muir C, Russell C, Timmons BW, Falk B, Klentrou P. Markers
of biological stress and mucosal immunity during a week leading to
competition in adolescent swimmers. J Immunol Res. 2014:234565. http://
dx.doi.org/10.1155/2014/234565. References 1. Kaufman E, Lamster IB. The diagnostic applications of saliva- a review. Crit
Rev Oral Biol Med. 2002;13(2):197–212. 1. Kaufman E, Lamster IB. The diagnostic applications of saliva- a review. Crit
Rev Oral Biol Med. 2002;13(2):197–212. 1. Kaufman E, Lamster IB. The diagnostic applications of saliva- a review. Crit
Rev Oral Biol Med. 2002;13(2):197–212. 26. Ali N, Pruessner JC. The salivary alpha amylase over cortisol ratio as a marker
to assess dysregulations of the stress systems. Physiol Behav. 2012;106(1):
65–72. 2. Nunes LAS, Mussavira S, Bindhu OS. Clinical and diagnostic utility of saliva
as a non-invasive diagnostic fluid:a systematic review. Biochem Med
(Zagreb). 2015;25(2):177–92. 2. Nunes LAS, Mussavira S, Bindhu OS. Clinical and diagnostic utility of saliva
as a non-invasive diagnostic fluid:a systematic review. Biochem Med
(Zagreb). 2015;25(2):177–92. 27. Boisseau N, Enea C, Diaz V, Dugué B, Corcuff JB, Duclos M. Oral
contraception but not menstrual cycle phase is associated with increased
free cortisol levels and low hypothalamo-pituitary-adrenal axis reactivity. J
Endocrinol Investig. 2013;36(11):955–64. 3. Papacosta E, Nassis GP. Saliva as a tool for monitoring steroid, peptide and
immune markers in sport and exercise science. J Sci Med Sport. 2011;14(5):
424–34. 3. Papacosta E, Nassis GP. Saliva as a tool for monitoring steroid, peptide and
immune markers in sport and exercise science. J Sci Med Sport. 2011;14(5):
424–34. 28. Kraemer RR, Francois M, Castracane VD. Estrogen mediation of hormone
responses to exercise. Metabolism. 2012;61(10):1337–46. 28. Kraemer RR, Francois M, Castracane VD. Estrogen mediation of hormone
responses to exercise. Metabolism. 2012;61(10):1337–46. 4. Yoshizawa JM, Schafer CA, Schafer JJ, Farrell JJ, Paster BJ, Wong DTW. Salivary biomarkers: toward future clinical and diagnostic utilities. Clin
Microbiol Rev. 2013;26(4):781–91. 29. Carpenter GH, Proctor GB, Ebersole LE, Garrett JR. Secretion of IgA by rat
parotid and submandibular cells in response to autonomimetic stimulation
in vitro. Int Immunopharmacol. 2004;4(8):1005–14. 5. Lindsay A, Costello JT. Realising the potential of urine and saliva as
diagnostic tools in sport and exercise medicine. Sports Med. 2017;47:11–31 30. Saxon A, Stevens RH, Ramer SJ, Clements PJ, Yu DTY. Glucocorticoids
administered in vivo inhibit human suppressor T lymphocyte function and
diminish B lymphocyte responsiveness in in vitro immunoglobulin synthesis. J Clin Invest. 1978;61(4):922–30. 6. Fragala M, Kraemer W, Denegar C, Maresh C, Mastro A, Volek J. Neuroendocrine-immune interactions and responses to exercise. Sports
Med. 2011;41(8):621–39. 7. Gillum T, Kuennen M, Schneider S, Moseley P. Consent for publication 19. Gleeson M, Hall ST, McDonald WA, Flanagan AJ, Clancy RL. Salivary IgA
subclasses and infection risk in elite swimmers. Immunol Cell Biol. 1999;
77(4):351–5. 19. Gleeson M, Hall ST, McDonald WA, Flanagan AJ, Clancy RL. Salivary IgA
subclasses and infection risk in elite swimmers. Immunol Cell Biol. 1999;
77(4):351–5. Competing interests
The authors declare that they have no competing interests. Competing interests
The authors declare that they have no competing interests. Availability of data and materials 14. de Vries WR, Bernards NT, de Rooij MH, Koppeschaar HP. Dynamic exercise
discloses different time-related responses in stress hormones. Psychosom
Med. 2000;62(6):866–72. Submit your next manuscript to BioMed Central
and we will help you at every step: 9. Tiidus P. Estrogen and gender effects on muscle damage, inflammation,
and oxidative stress. Can J Appl Physiol. 2000;25(4):274–87. • We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
• We provide round the clock customer support
• Convenient online submission
• Thorough peer review
• Inclusion in PubMed and all major indexing services
• Maximum visibility for your research
Submit your manuscript at
www.biomedcentral.com/submit
p y
y
p 10. Chicharro J, Lucía A, Pérez M, Vaquero A, Ureña R. Saliva composition and
exercise. Sports Med. 1998;26(1):17–27. • We accept pre-submission inquiries 11. Walsh N, Laing S, Oliver S, Montague J, Walters R, Bilzon J. Saliva parameters
as potential indices of hydration status during acute dehydration. Med Sci
Sports Exerc. 2004;36(9):1535–42. 12. Ely B, Cheuvront S, Kenefick R, Spitz MG, Heavens KR, Walsh NP, Sawka MN. Assessment of extracellular dehydration using saliva osmolality. Eur J Appl
Physiol. 2014;114(1):85–92. 13. Munoz CX, Johnson EC, DeMartini JK, Huggins RA, McKenzie AL, Casa DJ,
Maresh CM, Armstrong LE. Assessment of hydration biomarkers including
salivary osmolality during passive and active dehydration. Eur J Clin Nutr. 2013;67(12):1257–63.
|
https://openalex.org/W2078336726
|
https://europepmc.org/articles/pmc3889083?pdf=render
|
English
| null |
Pupil Dilations Reflect Why Rembrandt Biased Female Portraits Leftward and Males Rightward
|
Frontiers in human neuroscience
| 2,014
|
cc-by
| 10,732
|
INTRODUCTION women’s portraits, but only 56% of male portraits have a leftward
bias (McManus and Humphrey, 1973). Portraitures have been shown to exhibit a leftward bias, where the
left check is exposed more often than the right. This occurs more
often in female than male portraits which may be due to a desire
to portray female’s more emotive left-side. Hemispheric lateral-
ization may also play a role by projecting negative emotions to
the left-side of the face and positive emotions to the right-side of
the face. This distinction will be addressed. Given that conscious
judgments of pleasingness are beset with problems, it is impor-
tant to also use an unconscious measure such as pupil size. Hess
(1965, 1972) claimed that pupils enlarge when viewing pleasant
images and constrict when viewing unpleasant images. Yet since
pupil size also covaries with luminance, a novel technique was
employed to measure pupil size in response to portraits and their
mirror image. I found that Hess was incorrect to focus on valence
and should have emphasized arousal instead. This finding was evi-
dent in male but not female portraits, which may be due to the
dominance exhibited in male portraitures. p
y
This gender difference has had several explanations (Lindell,
2013). One promising interpretation suggests that the leftward
bias results from the poser’s preference to portray the left-side
of the face’s emotional qualities (Nicholls et al., 1999). Nicholls
et al., 1999, p. 665) instructed participants to pose for a portrait
to either “put as much real emotion and passion into a portrait
as you can” or “to avoid depicting any emotion at all.” In the first
case, participants were more likely to turn their left-cheek toward
a camera during a picture-taking session, whereas in the second
case participants were more likely to turn their right-cheek toward
the camera. Likewise, multiple studies report that the left-side of
the face is more intense in exhibiting voluntary emotional expres-
sion, especially for women (Sackeim and Gur, 1978; Sackeim et al.,
1978; Borod and Caron, 1980; Borod et al., 1988; Nicholls et al.,
2000). For example,Nicholls et al. (2002a) demonstrated that peo-
ple who are more emotionally expressive are more likely to pose for
a portrait offering the left-cheek; the paper argues that as females
score higher on measures of emotional expressivity, they are more
likely to pose offering the left-cheek. Nicholls et al. HUMAN NEUROSCIENCE HUMAN NEUROSCIENCE REVIEW ARTICLE
published: 13 January 2014
doi: 10.3389/fnhum.2013.00938 REVIEW ARTICLE REVIEW ARTICLE
published: 13 January 2014
doi: 10.3389/fnhum.2013.00938 REVIEW ARTICLE
published: 13 January 2014
doi: 10.3389/fnhum.2013.00938 INTRODUCTION (2002b) then
demonstrated that viewers perceive images of models offering the
left-cheek as more emotionally expressive. Lindell (2013) offers a
review of this literature. These findings are consistent with the idea
that facial expressions are related to cerebral hemispheric lateral-
ity, and that the right brain hemisphere is dominant in processing
and also displaying emotional expressions (to the left-side of the
face) (Bryden and Ley, 1983). Edited by: Edited by:
Sarah Rose Cavanagh, Assumption
College, USA
Reviewed by:
Sarah Rose Cavanagh, Assumption
College, USA
Kristin Prehn, Charité
Universitätsmedizin Berlin, Germany
*Correspondence:
James A. Schirillo, Department of
Psychology, Wake Forest University,
428 Greene Hall, Winston-Salem, NC
27109, USA
e-mail: schirija@wfu.edu *Correspondence:
James A. Schirillo, Department of
Psychology, Wake Forest University,
428 Greene Hall, Winston-Salem, NC
27109, USA
e-mail: schirija@wfu.edu Keywords: hemispheric laterality, pupil size, face perception, emotion, esthetic judgments James A. Schirillo*
Department of Psychology, Wake Forest University, Winston-Salem, NC, USA James A. Schirillo* James A. Schirillo*
Department of Psychology, Wake Forest University, Winston-Salem, NC, USA Department of Psychology, Wake Forest University, Winston-Salem, NC, USA Portrait painters are experts at examining faces and since emotional content may be
expressed differently on each side of the face, consider that Rembrandt biased his male
portraits to show their right-cheek more often and female portraits to show their left-cheek
more often. This raises questions regarding the emotional significance of such biased
positions. I presented rightward and leftward facing male and female portraits. I mea-
sured observers’ pupil size while asking observers to report how (dis)pleasing they found
each image. This was a methodological improvement over the type of research initially
done by Eckhard Hess who claimed that pupils dilate to pleasant images and constrict to
unpleasant images. His work was confounded since his images’ luminances and contrasts
across conditions were inconsistent potentially affecting pupil size. To overcome this limi-
tation I presented rightward or leftward facing male and female portraits by Rembrandt to
observers in either their original or mirror-reversed position. I found that in viewing male
portraits pupil diameter was a function of arousal. That is, larger pupil diameter occurred
for images rated both low and high in pleasantness. This was not the case with female
portraits. I discuss these findings in regard to the perceived dominance of males and how
emotional expressions may be driven by hemispheric laterality. Pupil dilations reflect why Rembrandt biased female
portraits leftward and males rightward James A. Schirillo*
Department of Psychology, Wake Forest University, Winston-Salem, NC, USA PORTRAITURE’S LEFTWARD BIAS So, pos-
itive emotions should be more prevalent on the right-side of the
face (since it is governed by the left cerebral hemisphere) and
negative emotional expressions should be more prevalent on the
left-side of the face (since it is governed by the right cerebral hemi-
sphere) (Rossi and Rosadini, 1967; Gainotti, 1969, 1972; Ahern
and Schwartz, 1979; Schwartz et al., 1979; Sackeim et al., 1982;
Fridlund and Izard, 1983; Natale et al., 1983; Sackeim and Gur,
1983; Davidson, 1984; Silberman and Weingartner, 1986; Schiff
and Lamon, 1989; Mandal et al., 1991; Borod et al., 1997; Jasari
et al., 2000; for a literature review see Powell and Schirillo, 2009). PORTRAITURE’S LEFTWARD BIAS Portraits often expose more of one side of their face than the other
side (McManus and Humphrey, 1973; Grüsser et al., 1988). For
example, in a study of 1,474 Western European portraits created
from the fourteenth to the twentieth century, 891 posers (~60%)
exposed more of their left-cheek, whereas 583 (~40%) exposed
more of their right-check (McManus and Humphrey, 1973). This
left-cheek asymmetry is stronger for women portraits (Gordon,
1974; Grüsser et al., 1988; Conesa et al., 1995). That is, ~68% of January 2014 | Volume 7 | Article 938 | 1 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org Laterality, pupil size, and esthetics Schirillo Given these findings, males may not want to portray their emo-
tive left-side as much as females (or by the behest of the artist). Likewise, artists may prefer to portray women as being more emo-
tive than men, thereby exposing their left-cheek more often. These
notions are supported by Grüsser et al. (1988), who examined
fifteenth to twentieth century portraits and found a left-cheeked
bias, which was always stronger for female than for male portraits. Thus, the current study examines the bias a portrait conveys, not
the viewer’s preference for a right or left-side of an image. This tests
the sitter’s hemispheric asymmetry (contralateral control of facial
musculature) rather than the perceiver will produce differences in
valence or arousal. This makes it peculiar to portray the left-cheek more often. Log-
ically it may follow that the valence hypothesis is less compelling
than the lateralized one. Thus, the field of lateralized portraiture has sought to deter-
mine whether one side of the face is more pleasant than the
other. Schirillo and Fox (2006) (Figure 1) showed observers
all 373 of Rembrandt’s portraits and found that left-cheeked
females were assessed as more approachable than right-cheeked
females portraits while males portraits (for both sides of the
face) were assessed as preferably avoided. Thus, observers were
more likely overall to want to approach female rather than
male Rembrandt portraits. Unfortunately, this study did not use
mirror-reversed images so it could not determine overall effect of
cheek. Alternatively,a valence hypothesis suggests that since each cere-
bral hemisphere controls predominantly the musculature on the
lower two-thirds of the contralateral side of the face (Brodal,1965)
each side of the face portrays different emotive qualities. CONSCIOUS EMOTIONAL JUDGMENTS MEASURED USING AN
UNCONSCIOUS PUPIL SIZE MEASURE Research investigating the interaction of lateralized portraiture
and pleasantness ratings is beset with problems (Rinn, 1984). For
example, the most popular methodology is to obtain an observer’s
subjective impression of the stimuli such as degree of liking (Rus-
sell and George, 1990). This method may cause observers to
use separate, immeasurable, criteria in making judgments (e.g.,
one observer may use an image’s contrast, whereas another may
use facial features, such as eyebrows). This obfuscates linking
esthetic judgments to cerebral laterality. Thus, it makes sense to 1
3
5
-90
0
d
io
v
A
h
c
a
o
r
p
p
A
Orientation (Deg.)
Left-Cheeked Females
1
3
5
-90
0
d
io
v
A
h
c
a
o
r
p
p
A
Orientation (Deg.)
Left-Cheeked Males
1
3
5
0
90
Approach Avoid
Orientation (Deg.)
Right-Cheeked Females
1
3
5
0
90
Approach Avoid
Orientation (Deg.)
Right-Cheeked Males
FIGURE 1 | Scatterplots of the distribution of each of 373 Rembrandt
portrait angles as a function of ratings of approach/avoidance ratings
(collapsed over 73 subjects’ ratings). Upper left (closed squares) – shows
left-cheeked males (−90° to −1° orientation), upper right (open
squares) – shows right-cheeked males (+1° to +90° orientation), lower left
(closed diamonds) – shows left-cheeked females (−90° to −1° orientation),
lower right (open diamonds) – shows right-cheeked females (+1° to +90°
orientation). Linear regressions are plotted for each graph. Here 2 indicates a
rating of “mildly approach,” 3 indicates “neutral,” and 4 indicates “mildly
avoid.”
Frontiers in Human Neuroscience
www.frontiersin.org
January 2014 | Volume 7 | Article 938 | 2 1
3
5
0
90
Approach Avoid
Orientation (Deg.)
Right-Cheeked Females 1
3
5
-90
0
d
io
v
A
h
c
a
o
r
p
p
A
Orientation (Deg.)
Left-Cheeked Females Left-Cheeked Females Right-Cheeked Females Approach Avoi Approach 1
3
5
0
90
Approach Avoid
Orientation (Deg.)
Right-Cheeked Males 1
3
5
-90
0
d
io
v
A
h
c
a
o
r
p
p
A
Orientation (Deg.)
Left-Cheeked Males Left-Cheeked Males Right-Cheeked Males Approach FIGURE 1 | Scatterplots of the distribution of each of 373 Rembrandt
portrait angles as a function of ratings of approach/avoidance ratings
(collapsed over 73 subjects’ ratings). CONSCIOUS EMOTIONAL JUDGMENTS MEASURED USING AN
UNCONSCIOUS PUPIL SIZE MEASURE Upper left (closed squares) – shows
left-cheeked males (−90° to −1° orientation), upper right (open
squares) – shows right-cheeked males (+1° to +90° orientation), lower left (closed diamonds) – shows left-cheeked females (−90° to −1° orientation),
lower right (open diamonds) – shows right-cheeked females (+1° to +90°
orientation). Linear regressions are plotted for each graph. Here 2 indicates a
rating of “mildly approach,” 3 indicates “neutral,” and 4 indicates “mildly
avoid.” Frontiers in Human Neuroscience January 2014 | Volume 7 | Article 938 | 2 www.frontiersin.org www.frontiersin.org Laterality, pupil size, and esthetics Schirillo try to understand portrait laterality to what may be a correlate to
automatic affective reactions. study of “social appealingness” of male and female right and left-
cheeked portraits differs from Schirillo and Fox’s (2006) study of
approach/avoidance of the same? Seemingly, “social appeal” dif-
fers from the desire to approach or avoid. Further work on this
discrepancy is needed. Pupil size is one previously measured unconscious indicator of
affective processing (Hess and Polt,1960; Hess,1965,1972; Janisse,
1974; Loewenfeld, 1999). Hess (1965, 1972), attempted to trans-
form the field of esthetics by claiming that pupils enlarge when
viewing pleasant images and constrict when viewing unpleasant
images (see also Hess and Polt,1960;Hess et al.,1965;Simms,1967;
Fitzgerald, 1968; Goldwater, 1972). However, pupil size also varies
with luminance (Loewenfeld, 1966, 1999; Woodmansee, 1966;
Miller, 1967; Kohn and Clynes, 1969; Goldwater, 1972; Janisse,
1973, 1974; Loftus, 1985; Mannan et al., 1995; Locher, 1996), and
since Hess compared different images with different intensities
and contrast levels (e.g., a snake versus a naked women), his work
was confounded. Given that verbal ratings of pleasingness are a self-report mea-
sure,it is of interest to determine their relationship with an uncon-
scious indicator of pleasingness (i.e., pupil diameter) as suggested
by Hess and others, and how this in turn might be related to the
emotional content of facial musculature. If self-report interpreta-
tions drive assumptions regarding dominance, they may show up
in the portraits’ emotional qualities which are reflected in pupil
size relationships. However, it may be that pupil diameter is more
related to arousal rather than pleasingness. The current study will
help clarify this dependent variable. In an earlier study of Rembrandt portraits (Schirillo, 2000) a
factor analysis revealed that females with their left-cheek exposed
were judged to be much less socially appealing than less commonly
painted right-cheeked females. CONSCIOUS EMOTIONAL JUDGMENTS MEASURED USING AN
UNCONSCIOUS PUPIL SIZE MEASURE Conversely, the more commonly
painted right-cheeked males were judged to be more socially
appealing than either left-cheeked males or females facing either
direction. It was hypothesized that hemispheric asymmetries reg-
ulating emotional facial displays of approach and avoidance influ-
enced the side of the face Rembrandt’s models exposed due to
prevailing social norms. Thus, females would be considered more
appealing than males and left facing males would be considered
the least appealing. A second experiment had different subjects
judge a different collection of 40 portraits by Rembrandt and their
mirror images. Portraits were matched for valence, arousal and
dominance by a second set of 20 subjects. I hypothesized that
mirror-reversed images would produce the same pattern of results
as their original orientation counterparts. I also hypothesized that
hemispheric asymmetries that specify the emotional expression on
each side of the face will account for the obtained results, that is,
original left-cheeked males will be preferred,due to their perceived
dominance while there will be no difference in female portraits. A NOVEL METHODOLOGY Since I used original and mirror-reversed portraits, I was also
able to explore the hemispheric laterality of emotional expression. Rembrandt may have turned his subject’s faces sideways to display
specific emotional content of their facial musculature. If this is the
case, self-reports regarding dominance especially of male subjects
should show up as emotional responses which might be quantified
by pupil size relationships. It was found that Hess was incorrect
to focus on valence. Instead, he should have focused on arousal,
since it was found that arousal, not valence, drives pupil diameter
for male portraits. That is pupils dilated for males that were rated
both most pleasant and most unpleasant (Powell and Schirillo,
2009). Thus, verbal ratings of pleasantness are a self-report mea-
sure which relate to an unconscious indicator of pleasantness (i.e.,
pupil diameter) as suggested by Hess and others (Hess and Polt,
1960; Hess, 1965, 1972; Hess et al., 1965). DOMINANCE, VALENCE, AND AROUSAL Dominance (typically associated with positive, e.g., self-assurance,
arrogance, and feeling bold or triumphant) or negative affective
states (e.g., hostility, irritability, and anger) (Demaree et al., 2005)
can lead to larger pupil size (Darwin, 1872). These arousal dif-
ferences should only be present in viewing male portraits since
they accentuate dominance (Dunbar and Burgoon, 2005). This is
emphasized in images where each side of the face reflects different
emotional expressions a concept first posited by Darwin (1872),
especially as it relates to dominance. Frontiers in Human Neuroscience MATERIALS AND METHODS
SUBJECTS Forty right-handed observers (20 males; ages 18–23) with nor-
mal or corrected-to-normal vision (but no eyeglasses) from the
introductory psychology research pool at Wake Forest Univer-
sity participated in the study. Handedness was determined using
Annett’s Peg-Moving task as right-minus-left latency (peg-moving
speed) (Annett and Kilshaw, 1983). The study was performed
in accordance with the ethical standards of the Declaration of
Helsinki. The use of Rembrandt portraits allowed for the exploration
of esthetic judgments of a famous artist while investigations of
Rembrandt’s work in the context of hemispheric laterality provide
also examining potential differences in facial emotion expression. First, using artwork should elicit stronger esthetic reactions than
photographs of faces. Second,prior preliminary evidence that per-
ceived dominance is greater when viewing his right-cheeked male
portraits (Schirillo, 2000). It is possible that this is the reason for
their prevalence; however due to the correlational nature of the
data it may also be that Rembrandt may have chosen to selec-
tively portray the right-cheeks of more dominant males. Thus, I
attempt to show how hemispheric asymmetries may regulate dis-
plays of facial emotion which are reflected by an observer’s esthetic
judgment of a portrait. It is interesting that Schirillo’s (2000) STIMULI Forty black and white images taken from oil paintings were cho-
sen from a collection of 373 portraits painted by Rembrandt. The specific portraits chosen represent his most rightward and
leftward facing portraits (Schirillo and Fox, 2006). Grayscale
images were used instead of colored images because color can
lead to changes in pupil diameter (Miller, 1967; Kohn and Clynes,
1969). Ten were right-cheeked males (none were self-portraits of
Rembrandt), 10 were left-cheeked males, 10 were right-cheeked January 2014 | Volume 7 | Article 938 | 3 www.frontiersin.org Laterality, pupil size, and esthetics Schirillo Schirillo females, and 10 were left-cheeked females (Lists Painting Names
in Appendix). Next, these images were used to produce 40 mirror-
reversed images using PhotoShop IBM. The portraits were only
of busts. Each portrait was scanned into PhotoShop and was pro-
jected to each observer individually using an IBM CRT computer
monitor using Microsoft PowerPoint. Viewing distance was 24′′
making the image size range from 11.7°(height) × 8.5°(width)
to 11.8° × 12.4°. The observers close distance to the screen lim-
ited their ability to spend considerable time viewing off-screen. To verify this notion, since the head-mounted Applied Science
Laboratories (ASL; series 6000) eye-tracker could also measure
eye position, I determined that observers were only off-screen
~3% of their total viewing time. My data also showed that time
with no record (due to eye closure) was minimal. In addition, 80
blurred images were created (40 from original and 40 from mirror-
reversed images) in PhotoShop using a Gaussian blur function
(See Figure 2). There was no ambient lightning in the experimen-
tal chamber, in that it was a room without windows. Since the
door was closed, the only light available came directly from the
computer screen that showed the images. females, and 10 were left-cheeked females (Lists Painting Names
in Appendix). Next, these images were used to produce 40 mirror-
reversed images using PhotoShop IBM. The portraits were only
of busts. Each portrait was scanned into PhotoShop and was pro-
jected to each observer individually using an IBM CRT computer
monitor using Microsoft PowerPoint. Viewing distance was 24′′
making the image size range from 11.7°(height) × 8.5°(width)
to 11.8° × 12.4°. The observers close distance to the screen lim-
ited their ability to spend considerable time viewing off-screen. STIMULI FIGURE 2 | (A) Blurred original orientation, (B) original orientation of a
left-cheeked female, (C) blurred mirror-reverse, and (D) mirror-reverse
orientation (A woman in fanciful costume). Baltimore, The Walters Art
Gallery; Br. 386). Copyright 1969 Phaidon Press Ltd. Rembrandt, The
Complete Edition of Paintings. A. Bredius, revised by H. Gerson. The eye tracking device was used to determine the pupil size
of the left-eye. The right-eye pupil size was not measured because
pupil size is believed to be conjugate across the two eyes (Loewen-
feld,1999). Pupil diameter was recorded automatically every 17 ms
foreachentire15 strial.Theaveragesizeacrosstheentire15 sview-
ing period was computed minus any time the pupil computation
was off-line (due to blinks, etc.). Given that the ASL eye-tracker
stops recording when the eye closes more than 50% (assumedly
due to blinks or partial eye closure) I have no record of this data. FIGURE 2 | (A) Blurred original orientation, (B) original orientation of a
left-cheeked female, (C) blurred mirror-reverse, and (D) mirror-reverse
orientation (A woman in fanciful costume). Baltimore, The Walters Art
Gallery; Br. 386). Copyright 1969 Phaidon Press Ltd. Rembrandt, The
Complete Edition of Paintings. A. Bredius, revised by H. Gerson. Instead of using linear interpolation to estimate the pupil size
during this off-line period (Steinhauer et al., 2004) I felt it best to
simply eliminate these segments from my dataset since blinks can
alter pupil size (Nakayama, 2006). Given that the ASL eye-tracker
is fixed to the head, head-movements did not alter pupil diameter
recordings or result in loss of tracking. Thus, other than eye-blink
time I did not remove any artifacts from the data. The remaining
data was imported into Excel to do the data cleaning which was
then converted to SPSS to do the statistical analysis. Average pupil
size was calculated for each image across the observation period
which excluded instances where the observer blinked or had par-
tial eye lid closures (since when the eye closes the ASL machine
cannot record any pupil size). Observers used a chinrest to ensure
a fixed 24′′ distance between themselves and the screen to retain a
constant depth of field across the images (Simms, 1967). Figure 2 shows images in their original and mirror-reverse orien-
tation (Figures 2B,D) along with their corresponding preceding
blurred images (Figures 2A,C). These images are significantly
blurred, so that facial pleasantness cannot be extracted from the
blurred images (Bachman, 2007). Frontiers in Human Neuroscience STIMULI Observers viewed 40 images in their original posed orienta-
tion, and in their mirror-reversed orientation (resulting in 80
images in total). Since I only compared pupil size across original
versus mirror-reversed images, my within-subject’s design elimi-
nates potential confounding factors such as age and medication. Right-cheeked mirror-reversed images are portraits that originally
faced rightward but due to reversal appeared to be of original
left-cheeked images. Likewise, left-cheek mirror-reversed images
appeared to be of original right-cheek portraits. Images were ran-
domized, and presented to each observer in random order. Each
of the 80 images was viewed for 15 s and was preceded by a blurred
version of the image for 15 s. Fifteen seconds was decided upon
because of three previous findings. First, Smith and Smith (2001)
found that art viewers examined The Metropolitan Museum of
Art paintings for a median of 17 s. Second, Aboyoun and Dabbs
(1998) showed that pupil size rapidly decreases upon image pre-
sentation, which then recovers to either baseline or above baseline
levels. Consequently, pupil size must be measured for at least sev-
eral seconds to overcome this initial depression. Lastly,Richer et al. (1983) found that pupil size increases begin about 1.5 s before To circumvent Hess’ luminance and contrast confounds, I had
observers view left- and right-cheeked portraits, and their mir-
ror images (e.g., see Figures 2B,D), while they determined the
esthetic pleasantness of each face. Simultaneously, I monitored
their pupil size allowing for a correlate between portrait pleasant-
ness and pupil size. Since original and mirror images have the same
luminance profiles, and I only compared pupil size and pleasant-
ness ratings across matched pairs of faces, Hess’ confounds were
eliminated. Since Woodmansee (1966, p. 133) found “significant pupil-
lary constriction with shifts in gaze from darker to brighter areas
of the picture,” I, like him, presented a blurred image prior to
its clear image to minimize changes in pupil size. For example, January 2014 | Volume 7 | Article 938 | 4 www.frontiersin.org www.frontiersin.org Laterality, pupil size, and esthetics Schirillo stimulus presentation and peak around a second after presen-
tation. As a result, I gave observers more time than needed to
generate an entire response to an image. Observers’ pupil size was
measured during each non-blurred image presentation while they
contemplated how pleasant they found the non-blurred images. PLEASANTNESS RATINGS Before examining individual pleasantness ratings, I obtained each
observer’s average pleasantness ratings for each of the eight por-
trait types (e.g., original right and left, mirror-reverse right and
left,males and females). This resulted in each observer having eight
data points. Then, pleasantness ratings for male and female por-
traits were submitted to a 2 (Portrait Gender: male vs. female) × 2
(Orientation: original vs. mirror-reversed) × 2 (Side of Face: left
vs. right) repeated measures ANOVA. Males and female observers
were included as a between-subjects factor, but showed no effect. j
,
Figure 4 shows the means for each portrait group. There
was a main effect for Side of Face with left-cheeks rated
higher than right-cheeked individuals F(1, 39) = 11.55, p = 0.002,
d = 1.07. Additionally, there were three significant interactions. First, there was a Side of Face by Orientation interaction F(1,
39) = 12.54, p = 0.001, d = 1.12. While left-side portraits (origi-
nal and reversed) were rated higher than right portraits (see Side
of Face main effect), right mirror reversals were rated higher than
right originals (M = 4.23 vs. M = 4.13), while left originals were
rated higher than left mirror reversals (M = 4.75 vs. M = 4.57,
respectively) [t(1, 78) = 2.56, p = 0.01, d = 0.59; t(1, 78) = 2.91,
p = 0.005,d = 0.65].Thatis,leftwardappearingportraits,leftorig-
inals and right reversals whose appearance to the observer seems
to be left faced, are rated higher than right faced originals and
left mirror-reversed (portraits viewed as seemingly right faced). Second, a Side of Face by Portrait Gender interaction was found
F(1, 39) = 15.12, p = 0.001, d = 1.23 with left-side females por-
traits rated higher than right-side females portraits (M = 5.07 vs. M = 4.38), whereas the opposite is true for males portraits though
to a lesser degree (M = 4.23 for left male portraits and M = 4.41
for right male portraits) [t(1, 78) = 3.22, p = 0.002, d = 0.72; t(1,
78) = 2.96, p = 0.004, d = 0.66]. Third, there was an Portrait Gen-
der by Orientation F(1, 39) = 7.42 p = 0.097, d = 0.86, such that
female mirror reversals were rated lower than original female por-
traits (M = 4.38 vs. M = 4.58), whereas the opposite relationship
was found for males (M = 4.35 vs. RESULTS The rationale for presenting a blurred image of a given por-
trait prior to presenting that portrait was to avoid the following
confound prevalent in the pupillometry literature. That is, if a
constant blank gray screen was used as a baseline the subsequent
test-image would produce the following effect (See Figure 2 taken
from Bradley et al., 2008). That is, the most important natural
function of the pupil is to dynamically respond to changes in envi-
ronmental illumination with an initial constriction (i.e., the light
reflex) that is related to stimulus luminosity (Beatty and Lucero-
Wagoner, 2000). Thus, if a constant blank gray screen were used as
a baseline the brighter images would produce a larger constriction
than the dimmer images. This effect takes up to 6 s before reach-
ing a plateau. To circumvent this effect I choose to first present
for 15 s a blurred image of the subsequent test-portrait. This does
two things. First, it makes the large constriction (i.e., light reflex)
occur during the blurred image rather than during the test-image. By the end of the 15 s of viewing the blurred image the pupil has
adjusted to the light level of the image that will subsequently be
presented. Given this very long duration the pupil will no longer
carry-over any information from the previous clear portrait. This
is because the pupil is reflexive and does not contain a memory
loop so by the end of presenting a blurred image there are no resid-
ual effects from the proceeding clear image. However, the blurred
images differ in luminance thus setting a different baseline for the
subsequent portrait. This is actually desired,so that the magnitude
of the effect is not the result of a shift in overall luminance level
(as occurs in Figure 2). Instead, each original and mirror-reversed
blurred image sets the same baseline for their subsequent clear
image. This is important since it is only the results of these two
(original and mirror-reversed test-image) pupil diameters that will
be compared against each other. PLEASANTNESS RATINGS M = 4.29) [t(1, 78) = 2.72,
p = 0.008, d = 0.61; t(1, 78) = 2.51, p = 0.015, d = 0.015]. I manually recorded pleasantness scores for each face by taking
verbal esthetic judgments using a 1–9 numerical scale, with one
meaning most displeasing, five meaning neutral, and nine mean-
ing most pleasing. Pleasingness is just one dimension of esthetics,
but seemed to be appropriate based on a study that used five
evaluative scales (e.g., pleasingness, likeability, preferability, inter-
estingness, and complexity) (Russell and George, 1990). In Russell
and George’s (1990) study,pleasingness was highly correlated with
likeability and preferability, and was the highest in inter-subject
agreement. The difference in verbal rating between the original
and mirror-reverse images was then correlated with the difference
between the average pupil diameters. Frontiers in Human Neuroscience STIMULI Observers were instructed to think about the esthetic pleasingness
of each image for the entire 15 s it was shown and then report their
judgment after the image was removed. This occurred during the
presentation of the subsequent blurry image. session progressed. To determine if observers noticed these mir-
ror duplications, I asked whether they noticed anything unusual
about the images at the end of the session. Sixteen of 40 observers
reported noticing that a number of images were mirror-reversed. Only three observers had formal art training and nine reported
that they had seen less than 25% of the portraits before. Thus,
while many of these images are famous, most observers were not
familiar with them. PUPIL SIZE This variation is termed the light reflex. (Bottom) correlation of the magnitude of the light reflex as a function of
image luminance (Taken from Bradley et al., 2008). FIGURE 4 | Verbal ratings (striped bars) as a function of portrait type
on a 1–9 scale, with 1 indicating most displeasing, 5 indicating neutral,
and 9 indicating most pleasant. Pupil size in mm (solid bars) as a function
of portrait type (R = Right-cheeked, L = Left-cheeked, M = Males,
F = Females, Rev = Mirror-reversed images). Error bars = SEM. FIGURE 5 | Correlation between pupil diameter given the average
luminance for each portrait (n = 40 observers). The figure includes the
portraits with the highest and lowest average luminance. FIGURE 5 | Correlation between pupil diameter given the average
luminance for each portrait (n = 40 observers). The figure includes the
portraits with the highest and lowest average luminance. FIGURE 3 | (Top) Average pupil diameter over time as a function of
luminance of the image viewed. This variation is termed the light reflex. (Bottom) correlation of the magnitude of the light reflex as a function of
image luminance (Taken from Bradley et al., 2008). FIGURE 5 | Correlation between pupil diameter given the average
luminance for each portrait (n = 40 observers). The figure includes the
portraits with the highest and lowest average luminance. females portraits (M = 5.3) [t(1, 78) = 3.07, p = 0.003, d = 0.69]. This analysis also yielded a significant Side of Face × Orientation
interaction F(1, 39) = 14.72, p = 0.001, d = 1.21. Original right-
cheeked portraits (M = 5.42) elicited greater average pupil size
than right-cheeked reversed portraits (M = 5.15) [t(1, 78) = 3.55,
p = 0.001, d = 0.76]. Conversely, average pupil size was largest
for left-cheeked reversed portraits (M = 5.44) than for origi-
nal left-cheeked portraits (M = 5.23) [t(1, 78) = 3.13, p = 0.002,
d = 0.72]. When these findings are examined alongside the Side
of Face × Orientation interaction for verbal ratings, I find that
images with the appearance of being right-cheeked yielded larger
average pupil size and lower verbal ratings. image luminance reinforces my decision to use mirror-reversed
images. PUPIL SIZE Before examining individual pupil size, I obtained each observer’s
average pupil size for each of the eight types of portraits. Mean
pupil sizes for these groups are shown in Figure 3 along-
side the means of the previous three-way ANOVA conducted
for verbal pleasingness. There was a main effect of portrait
gender across the eight types of portraits F(1, 39) = 39.14,
p < 0.0001, d = 1.98 (Figure 4). Average pupil diameter when
viewing male portraits was larger (M = 5.5) than when viewing Following the stimulus presentation, observers were admin-
istered a questionnaire that pertained to their art training and
their familiarity with the portraits. They were not told prior to
the experimental session that they would see original and mirror-
reversed images, but they may have become aware of this as the January 2014 | Volume 7 | Article 938 | 5 Frontiers in Human Neuroscience www.frontiersin.org Laterality, pupil size, and esthetics Schirillo FIGURE 4 | Verbal ratings (striped bars) as a function of portrait type
on a 1–9 scale, with 1 indicating most displeasing, 5 indicating neutral,
and 9 indicating most pleasant. Pupil size in mm (solid bars) as a function
of portrait type (R = Right-cheeked, L = Left-cheeked, M = Males,
F = Females, Rev = Mirror-reversed images). Error bars = SEM. FIGURE 5 | Correlation between pupil diameter given the average
luminance for each portrait (n = 40 observers). The figure includes the
portraits with the highest and lowest average luminance. FIGURE 3 | (Top) Average pupil diameter over time as a function of
luminance of the image viewed. This variation is termed the light reflex. (Bottom) correlation of the magnitude of the light reflex as a function of
image luminance (Taken from Bradley et al., 2008). FIGURE 4 | Verbal ratings (striped bars) as a function of portrait type
on a 1–9 scale, with 1 indicating most displeasing, 5 indicating neutral,
and 9 indicating most pleasant. Pupil size in mm (solid bars) as a function
of portrait type (R = Right-cheeked, L = Left-cheeked, M = Males,
F = Females, Rev = Mirror-reversed images). Error bars = SEM. FIGURE 5 | Correlation between pupil diameter given the average
luminance for each portrait (n = 40 observers). The figure includes the FIGURE 3 | (Top) Average pupil diameter over time as a function of
luminance of the image viewed. Frontiers in Human Neuroscience LINEAR AND QUADRATIC RELATIONSHIPS BETWEEN PLEASANTNESS
RATINGS AND PUPIL DIAMETER Yet, when the mirror-reversed por-
trait was preferred (whether right- or left-cheeked) the pupil was
smaller while viewing the mirror-reversed portrait compared to
the original. Figure 6A shows that there was a significant quadratic effect
for male portraits b = 0.40, t(797) = 3.22, p = 0.002, d = 0.22
when the linear relationship was held constant. Pupil diameter
was largest when there were the greatest differences in verbal
ratings between an original and mirror-reversed male image. That is, when images were extremely liked or disliked, pupil size
increased. This quadratic relationship accounted for significantly
moreof thevariancethanthelinearrelationship,R2 change = 0.32,
p < 0.004, d = 0.22. However, for female portraits, the regression
model failed to find a significant linear or quadratic relation-
ship between pupil diameter and pleasantness F(2, 797) = 0.59,
p = 0.32, d = 0.07 (Figure 6B). Pupil size could be confounded by
the luminance of an image, so difference scores were calculated
to compare across images. If difference scores were not used, and
instead, pupil size for original and mirror-reversed images were
used in the regression, then my findings would be seriously con-
founded because these values do not account for the changes in
luminance/contrast across images and comparisons across images
would be misleading. In sum, there were 800 total cases [i.e., 40
observers × 40 portraits (original-minus mirror-reversed)] used
in the analysis. Next, linear and quadratic functions were entered into a regres-
sion model to evaluate if the relationship is better categorized by a
quadratic function. The following quadratic regression model was
computed separately for male and female portraits: Pupil = b0 + b1
pleasantness
+ b2
pleasantness2 Pupil = b0 + b1
pleasantness
+ b2
pleasantness2 FIGURE 6 | Pupil size difference score (original-minus mirror-reversed)
as a function of the difference in 1–9 verbal rating scores (between an
original-minus mirror-reversed image) for all (A) male portraits,
(B) female portraits. It is important to realize that my images were not necessar-
ily less pleasing in their non-original orientation. Instead, what I
found was that only if there were a large difference in the rating
between original and mirror-reversed images (where either orien-
tation could have been the more pleasing image) there would also
be a large difference in pupil size between those images. LINEAR AND QUADRATIC RELATIONSHIPS BETWEEN PLEASANTNESS
RATINGS AND PUPIL DIAMETER I examined linear and quadratic relationships between pleasant-
ness and pupil size. First, a regression was computed to examine
whether there was a linear relationship between pleasantness and
pupil diameter. This was done by taking each portrait (original
or mirror-reversed) for each observer as an individual case. Then
difference scores between verbal pleasingness were regressed using
the predictor variables of pupil size difference and quadric pupil
size difference. Figure 6A shows that, for males, as the original
verbal ratings became more positive (represented on the x-axis Overall, pupil diameters were well within the normal range,
where, as expected, the luminance of the portrait viewed
dramatically affected pupil size (Figure 5; range = 4.71–5.82 mm). The fact that pupil contraction increased with an increase in January 2014 | Volume 7 | Article 938 | 6 www.frontiersin.org Schirillo Schirillo Laterality, pupil size, and esthetics by verbal rating differences that were greater than zero), the orig-
inal pupil size got smaller. Likewise, as original ratings became
more negative (represented on the x-axis by verbal rating dif-
ferences that were less than zero), the original pupil size got
larger. This negative slope was statistically significant for male
portraits r(39) = −0.41, p < 0.009, d = −0.90 (Figure 6A) but I
failed to find a relationship for female portraits r(39) = −0.22,
p = 0.178, d = −0.90 (Figure 6B). This means that when an orig-
inal male portrait was preferred (whether right- or left-cheeked)
the pupil was smaller while viewing the original portrait compared
to the mirror-reversed image. Yet, when the mirror-reversed por-
trait was preferred (whether right- or left-cheeked) the pupil was
smaller while viewing the mirror-reversed portrait compared to
the original. by verbal rating differences that were greater than zero), the orig-
inal pupil size got smaller. Likewise, as original ratings became
more negative (represented on the x-axis by verbal rating dif-
ferences that were less than zero), the original pupil size got
larger. This negative slope was statistically significant for male
portraits r(39) = −0.41, p < 0.009, d = −0.90 (Figure 6A) but I
failed to find a relationship for female portraits r(39) = −0.22,
p = 0.178, d = −0.90 (Figure 6B). This means that when an orig-
inal male portrait was preferred (whether right- or left-cheeked)
the pupil was smaller while viewing the original portrait compared
to the mirror-reversed image. DISCUSSION They also found an increase in
pupil diameter when observers expected to hear a gunshot. Like-
wise, Polt (1970) found larger pupil size during mental arithmetic
tasks when observers believed they would be shocked for incorrect
answers. This may also account for why females failed to show an
effect of arousal. I suggest that females are considered pleasing (not
aversive) compared to males, thus when viewing female portraits
one’s pupil size is already approaching the floor, and thereby has
less room to show arousal effects. Given that my verbal ratings of pleasingness are a self-report
measure of what may be an emotive expression, it was of interest
to determine their relationship with an unconscious indicator of
pleasingness (i.e., pupil diameter) as suggested by Hess and others,
and how this in turn might be related to the emotional content
of the facial musculature displayed in the images. For example,
self-report assumptions regarding dominance can be present in
the emotional qualities of the portraits, which may be captured
by pupil size relationships. I have explored these variables while
simultaneously eliminating Stern and Strock’s (1987) concern that
one drawback to pupillometry is that changes associated with
such variables are considerably smaller than those associated with
illumination effects. The esthetic verbal pleasantness judgments suggest that the
observers were attending to the actual facial physiognomy of the
posers and that pleasantness was determined to a lesser degree
by the orientation the portrait faced. In agreement with prior
research (Schirillo and Fox, 2006), the left-side of women’s faces
were rated as more esthetically pleasant than their right-side. This
suggests that for women it is important to express more emo-
tive facial qualities than males, in agreement with Nicholls et al. (1999) and the right-hemisphere model of emotion lateralization. Yet how emotion may be lateralized in the cerebral hemispheres
is still under debate (Davidson, 1995; Demaree et al., 2005; Kill-
gore and Yurgelun-Todd, 2007). One recent argument is that an
approach/withdrawal model may provide a more appropriate fit to
the data as opposed to the positive/negative hemispheric difference
model (Demaree et al., 2005). Davidson and others interpretation
of theleft/rightdifferencesinemotionvalenceisthattherightcere-
bral hemisphere regulates withdrawal behaviors whereas the left
cerebral hemisphere regulates approach behaviors (Kinsbourne,
1982;Davidson,1984,1992,1995;Davidsonetal.,1990;Fox,1991). If this is the case, as stated in Schirillo (2000), I speculate that
Rembrandt preferred to paint females left-side because it captured
the attractive quality of being demure. DISCUSSION Male portraits showed both a linear and quadratic relationship
between pupil diameter and esthetic judgments of pleasantness. Male portraits showed both a linear and quadratic relationship
between pupil diameter and esthetic judgments of pleasantness. The linear model indicates that pupil diameter increased when
viewing negative male portraits and decreased when viewing pos-
itive male portraits, whereas a quadratic model shows that pupil
diameter increases to both highly pleasant and unpleasant male
portraits. Thus, it is plausible that researchers who used only a
linear function found that unpleasant images were associated with
larger pupil sizes compared to pleasant images (Woodmansee,
1967; Tinio and Robertson, 1969; Libby et al., 1973). These find-
ings do not support Hess’ (1965) prediction, which suggested that
unpleasant images would have been associated with a smaller pupil
diameter. While it has been shown that pupils get larger to intense (arousing)
stimuli, I only replicated this for male portraits. Schirillo’s (2000)
analysis of Rembrandt’s portraits suggests this may be because
the perceived dominance of male portraits was rated higher than
for female portraits. Dominance may be associated with positive
(e.g., self-assurance, arrogance, and feeling bold or triumphant)
or negative affective states (e.g., hostility, irritability, and anger)
(Demaree et al., 2005). It is possible that while Rembrandt painted
males to exhibit these positive dominant traits (see Humphrey and
McManus, 1973), negative dominance traits may have also been
captured. Thus, the negative linear relationship between pleas-
antness ratings and pupil diameter for males is consistent with
Tinio and Robertson (1969), who found that aggressive Thematic
Apperception Test cards elicited larger pupil size than control
cards. This implies that Rembrandt’s male portraits may actu-
ally be perceived as domineering which is consistent with Libby
et al. (1973) and Woodmansee (1967) who found that unpleas-
ant images were associated with larger pupil sizes compared to
pleasant images. However, a quadratic relationship accounts for significantly
more variance than a linear relationship. In this case, pupil size
increased with large differences among pleasantness ratings for
male faces. This is consistent with previous findings that pupil size
is related to stimulus intensity rather than their specific positive or
negative content (Janisse, 1973, 1974, 1977; Aboyoun and Dabbs,
1998). For example, arousing situations have been shown to pro-
duce larger pupil diameter in non-visual stimuli studies. Nunnally
et al. (1967) found that painfully loud sounds increased muscle
tension causing larger pupil size. LINEAR AND QUADRATIC RELATIONSHIPS BETWEEN PLEASANTNESS
RATINGS AND PUPIL DIAMETER As expected, there were almost as many zero differences
between original and reversed image verbal ratings as there
were difference scores (Figure 7). This inevitable outcome can
potentially affect any of my linear and quadratic relationships. FIGURE 7 | Number of cases for all observers in which there was either
a zero difference in verbal rating or there was a difference of any
magnitude (with the original score being either more or less than the
mirror-reversed image). (R = Right-cheeked, L = Left-cheeked, M = Males,
F = Females). FIGURE 7 | Number of cases for all observers in which there was either
a zero difference in verbal rating or there was a difference of any
magnitude (with the original score being either more or less than the
mirror-reversed image). (R = Right-cheeked, L = Left-cheeked, M = Males,
F = Females). FIGURE 6 | Pupil size difference score (original-minus mirror-reversed)
as a function of the difference in 1–9 verbal rating scores (between an
original-minus mirror-reversed image) for all (A) male portraits,
(B) female portraits. FIGURE 6 | Pupil size difference score (original-minus mirror-reversed)
as a function of the difference in 1–9 verbal rating scores (between an
original-minus mirror-reversed image) for all (A) male portraits,
(B) female portraits. January 2014 | Volume 7 | Article 938 | 7 Frontiers in Human Neuroscience www.frontiersin.org Schirillo Laterality, pupil size, and esthetics DISCUSSION This new methodology eliminates Hess’ (1972) fluctuations in
luminance and contrast across images allowing us to observe how
pupil size varies as a function of the differences in verbal reports of
pleasant and less pleasant original and mirror-reversed portraits. This was done by only comparing measurements between origi-
nal and mirror-reversed images which makes image contrast and
luminance irrelevant. This also means I do not require a baseline
pupil size from the blurred images; since pupil size should not vary
between original and mirror-reversed images because such images
do not vary in image contrast and luminance. If they do differ, this
must be because the emotional content of the images vary, not
their image contrast or luminance. Given that it is impossible to equate apparent contrast or mean
luminance across images, research testing Hess’ hypothesis had
ceased. However, my improved methodology does not need to
equate apparent contrast or mean luminance since this automati-
cally occurs by comparing pupil size only across an original and its
mirror-reversed image. In essence, I created a methodology that
reexamined the relationship between a self-report and emotive
measure (i.e., pleasingness) and an unconscious physiological
measure (i.e., pupil size). I show that Hess (1965, 1972) was
incorrect by focusing on valence. Instead he should have focused
on arousal, since arousal drives the effect for male portraits not
valence. Prehn et al. (2013) demonstrated that oxytocin increased
saliency of all social stimuli regardless whether faces were male
or female. In the placebo group (without oxytocin treatment),
however, they found a strong gender effect (which decreased January 2014 | Volume 7 | Article 938 | 8 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org Laterality, pupil size, and esthetics Schirillo after oxytocin treatment). However, they used Winmorph 3.01
(www.debugmode.com/winmorph) to transform neutral expres-
sions into all of the emotional ones in 5% steps. My study had no
such control. Interestingly, their placebo“happy”male and female
faces did not significantly differ in recorded intensity levels, nor
recorded pupil size. This would have been the condition that I
most like replicated. Like me, they found increased pupil dila-
tions during the processing of male compared with female faces,
which they attribute to reflecting men’s (they only used male sub-
jects) lower interest in male faces. However, I used both sexes
as subjects and found no significant differences between groups. DISCUSSION Yet their oxytocin manipulation is evidence that increased pupil-
lary responses have been observed when stimuli are emotionally
salient. But I controlled for saliency as much as possible by match-
ing for valence and dominance, so I do not believe this was a factor
in my experiment. Brodal, A. (1965). The Cranial Nerves. Oxford: Blackwell. Brodal, A. (1965). The Cranial Nerves. Oxford: Blackwell. Bryden, M. P., and Ley, R. G. (1983). “Right-hemispheric involvement in the per-
ception and expression of emotion in normal humans,” in Neuropsychology of
Human Emotion, eds K. M. Heilman, and P. Satz (New York: Guilford Press),
6–44. Conesa, J., Brunold-Conesa, C., and Miron, M. (1995). Incidence of the half-
left profile pose in single-subject portraits. Percept. Mot. Skills 81, 920–922. doi:10.2466/pms.1995.81.3.920 Darwin, C. (1872). The Expression of the Emotions in Man and Animals. New York:
D. Appleton and Co. Davidson, R. J. (1984). “Affect, cognition, and hemispheric specialization,” in Emo-
tions, Cognition, and Behavior, eds C. E. I. Izard, J. Kagan, and R. B. Zajonc
(Cambridge, MA: Cambridge University Press), 320–365. (Cambridge, MA: Cambridge University Press), 320–365. Davidson, R. J. (1992). Emotion and affective style: hemispheric substrates. Psychol. Sci. 3, 39–43. doi:10.1111/j.1467-9280.1992.tb00254.x Davidson, R. J. (1995). “Cerebral asymmetry, emotion, and affective style,” in Brain
Asymmetry, eds R. J. Davidson, and K. Hughdahl (Cambridge, MA: MIT Press),
361–388. Davidson, R. J., Ekman, P., Saron, C. D., Senuluis, J. A., and Friesen, W. V. (1990). Approach-withdrawal and cerebral asymmetry: emotional expression and brain
physiology I. J. Pers. Soc. Psychol. 58, 330–341. doi:10.1037/0022-3514.58.2.330 Davidson, R. J., Ekman, P., Saron, C. D., Senuluis, J. A., and Friesen, W. V. (1990). Approach-withdrawal and cerebral asymmetry: emotional expression and brain In summary,this study provides a new methodology to research
the association between pupil diameter and esthetic verbal judg-
ments. Based on its findings, Hess’ (1965, 1972) hypothesis that
pupils dilate to pleasant images and constrict to displeasing images
seems incorrect. Instead, at least for male portraits, pupil size is a
function of arousal such that pupil size difference increases when
the difference in verbal reports are both most pleasant and most
displeasing. I consider the possibility that this is related to per-
ceived dominance (Ellis, 2006), in that a linear function showed
that faces rated low in esthetic pleasantness evoked the largest
pupil diameter. REFERENCES Aboyoun, D. C., and Dabbs, J. M. (1998). The Hess pupil dilation findings: sex or
novelty? Soc. Behav. Pers. 26, 415–420. doi:10.2224/sbp.1998.26.4.415 Gainotti, G. (1972). Emotional behavior and hemispheric side of lesion. Cortex 8,
41–55. doi:10.1016/S0010-9452(72)80026-1 Ahern, G. L., and Schwartz, G. E. (1979). Differential lateralization for positive ver-
susnegativeemotion.Neuropsychologia 17,693–698.doi:10.1016/0028-3932(79)
90045-9 Goldwater,B. C. (1972). Psychological significance of pupillary movements. Psychol. Bull. 77, 340–355. doi:10.1037/h0032456 Gordon, I. (1974). “Left and right in art,” in Psychology and the Arts, ed. D. O’Hare
(Brighton: Harevesor), 211–241. Annett, M., and Kilshaw, D. (1983). Right and left-hand skill. 1. Estimating the para-
meters of the distribution of L-R differences in males and females. Br. J. Psychol. 74, 269–283. doi:10.1111/j.2044-8295.1983.tb01862.x Grüsser, O. J., Selke, T., and Zynda, B. (1988). “Cerebral lateralization and some
implications for art, aesthetic perception, and artistic creativity,” in Beauty &
The Brain: Biological Aspects of Aesthetics, eds I. Reutshcler, B. Herzberger, and D. Epstein (Boston: Berkhäuer Verlag), 257–293. Bachman, T. (2007). When beauty breaks down: investigation of the effect of spatial
quantisation on aesthetic evaluation of facial images. Perception 36, 840–849. doi:10.1068/p5509 Hess, E. H. (1965). Attitude and pupil size. Sci. Am. 212, 46–54. doi:10.1038/
scientificamerican0465-46 Beatty, J., and Lucero-Wagoner, B. (2000). “The pupillary system,” in Handbook of
Psychophysiology, eds J. T. Cacioppo, L. G. Tassinary, and G. G. Berntson (Cam-
bridge: Cambridge University Press), 14–162. Hess, E. H. (1972). “Pupillometrics: a method of studying mental, emotional, and
sensory processes,” in Handbook of Psychophysiology, eds N. S. Greenfield and R. A. Sternbach (New York: Holt, Rinehart & Winston), 491–531. Hess, E. H. (1972). “Pupillometrics: a method of studying mental, emotional, and
sensory processes,” in Handbook of Psychophysiology, eds N. S. Greenfield and R. Borod, J. C., and Caron, H. S. (1980). Facedness and emotion related to lateral
dominance,sex and expression type. Neuropsychologia 18,237–241. doi:10.1016/
0028-3932(80)90070-6 nbach (New York: Holt, Rinehart & Winston), 491–531. Hess, E. H., and Polt, J. M. (1960). Pupil size as related to interest value of visual
stimuli. Science 132, 349–350. doi:10.1126/science.132.3423.349 stimuli. Science 132, 349–350. doi:10.1126/science.132.3423.349 Borod, J. C., Haywood, C. S., and Koff, E. (1997). Neuropsychological aspects of
facial asymmetry during emotional expression: a review of the normal adult
literature. Neuropsychol. Rev. 7, 41–60. doi:10.1007/BF02876972 Hess, E. H., Seltzer, A. L., and Shlien, J. M. (1965). Pupil response of hetero- and
homosexual males to pictures of men and women: a pilot study. J. Abnorm. Psychol. ACKNOWLEDGMENTS Fridlund,A. J.,and Izard,C. (1983).“Electromyographic studies of facial expressions
of emotions and patterns of emotion,” in Social Psychophysiology: A Sourcebook, Special thanks to Rebecca White for help with data management
and Lisa Scalzo for editing. eds J. T. Cacioppo and R. E. Petty (New York: Guilford Press), 254–261. Gainotti, G. (1969). “Catastrophic” reactions and manifestations of indifferences
during cerebral disorders. Neuropsychologia 7, 195–204. doi:10.1016/0028-
3932(69)90017-7 DISCUSSION I consider the possibility that this is related to dis-
liking perceived threat (Darwin, 1872), which may have been a
dominance trait that Rembrandt inadvertently depicted. Demaree, H. A., Everhart, D. E., Youngstrom, E. A., and Harrison, D. W. (2005). Brain lateralization of emotional processing: historical roots and a future
incorporating “dominance.” Behav. Cogn. Neurosci. Rev. 4, 3–20. doi:10.1177/
1534582305276837 Dunbar, N. E., and Burgoon, J. K. (2005). Perceptions of power and interac-
tional dominance in interpersonal relationships. J. Soc. Pers. Relat. 22, 207–233. doi:10.1177/0265407505050944 Ellis, L. (2006). Gender differences in smiling: an evolutionary neuroandrogenic
theory. Physiol. Behav. 88, 303–308. doi:10.1016/j.physbeh.2006.03.034 Fitzgerald,H. E. (1968).Autonomic pupillary reflex activity during early infancy and
its relation to social and non-social and non-social visual stimuli. Diss. Abstr. 28,
3896B–3897B. Fox, N. A. (1991). If it’s not left, it’s right: electroencephalograph asymmetry and
the development of emotion. Am. Psychol. 46, 863–872. doi:10.1037/0003-066X. 46.8.863 REFERENCES Hemispheric lateralization of func-
tions related to emotion. Brain Cogn. 5, 322–353. doi:10.1016/0278-2626(86)
90035-7 McManus, C., and Humphrey, N. (1973). Turning the left cheek. Nature 243,
271–272. doi:10.1038/243271a0 Simms, T. M. (1967). Pupillary response of male and female subjects to pupillary
difference in male and female picture stimuli. Percept. Psychophys. 2, 553–555. Miller, R. L. (1967). The Clinical Validation of the Pupillary Response: The Effect of
Chromatic and Achromatic Stimuli Upon Pupil Responsivity. Doctoral Disserta-
tion, Michigan State University, East Lansing. Smith, J. K., and Smith, L. F. (2001). Spending time on art. Empiric. Stud. Arts 19,
229–236. doi:10.2190/5MQM-59JH-X21R-JN5J Nakayama, M. (2006). “Influence of blink on pupillary indices,” in Biomedical Cir-
cuits and Systems Conference (London: IEEE BioCAS 2006), 29–32. Steinhauer, S. R., Siegle, G. J., Condray, R., and Pless, M. (2004). Sympathetic and
parasympathetic innervation of pupillary dilation during sustained processing. Int. J. Psychophysiol. 52, 77–86. doi:10.1016/j.ijpsycho.2003.12.005 Natale, M., Gur, R. E., and Gur, R. C. (1983). Hemispheric asymmetries in pro-
cessing emotional expressions. Neuropsychologia 21,555–565. doi:10.1016/0028-
3932(83)90011-8 Stern, J. A., and Strock, B. D. (1987).“Oculomotor activity and user-system interac-
tion in the workplace,” in Psychophysiology and the Electronic Workplace, eds A. Gale, and B. Christie (Oxford: John Wiley & Sons), 239–254. Nicholls,M. E. R.,Clode,D.,Lindell,A. K.,and Wood,A. G. (2002a). Which cheek to
turn? The effect of gender and emotional expressivity on posing behavior. Brain
Cogn. 48, 480–484. doi:10.1006/brcg.2001.1402 Tinio, F., and Robertson, M. (1969). Examination of two indices of hostility:
fantasy and change in pupil size. Proc. Annu. Convent. Am. Psychol. Assoc. 4,
173–174. Nicholls, M. E. R., Wolfgang, B. J., Clode, D., and Lindell, A. K. (2002b). The effect
of left and right poses on the expression of facial emotion. Neuropsychologia 40,
1662–1665. doi:10.1016/S0028-3932(02)00024-6 Woodmansee, J. J. (1966). “Methodological problems in pupillographic experi-
ments,” in Proceedings of the Annual Convention of the American Psychological
Association, 133–134. Nicholls, M. E. R., Clode, D., Wood, S. J., and Wood, A. G. (1999). Laterality of
expression in portraiture: putting your best cheek forward. Proc. R. Soc. Lond. 226, 1517–1522. doi:10.1098/rspb.1999.0809 Woodmansee, J. J. (1967). The pupil reaction as an index of positive and negative
affect. Paper Presented at the Convention of theAmerican PsychologicalAssociation,
Washington, DC. Washington, DC. Nicholls, M. E. R., Ellis, B. E., Clement, J. G., and Yoshino, M. (2000). Detect-
ing hemifacial asymmetries in emotional expression with three-dimensional
computerized image analysis. Proc. R. Soc. Lond. Biol. Sci. 271, 663–668. REFERENCES 37, 411–420. doi:10.1037/0003-066X.37.4.411 Sackeim, H. A., Greenberg, M. S., Weiman, A. L., and Gurr, R. C. (1982). Hemi-
spheric asymmetry in the expression of positive and negative emotions. Arch. Neurol. 39, 210–218. doi:10.1001/archneur.1982.00510160016003 Kohn, M., and Clynes, M. (1969). Color dynamics of the pupil. Ann. N. Y. Acad. Sci. 156, 931–950. doi:10.1111/j.1749-6632.1969.tb14024.x Libby, W. L., Lacey, B. C., and Lacey, J. I. (1973). Pupillary and cardiac activity
during visual attention. Psychophysiology 10, 270–294. doi:10.1111/j.1469-8986. 1973.tb00526.x Sackeim, H. A., and Gur, R. C. (1978). Lateral asymmetry in intensity of emotional
expression. Neuropsychologia 16, 473–481. doi:10.1016/0028-3932(78)90070-2 Sackeim, H. A., and Gur, R. C. (1983). “Facial asymmetry and the communication
of emotion,” in Social Psychophysiology, eds J. T. Cacioppo, and R. E. Petty (New
York: Guilford), 307–352. Lindell, A. K. (2013). Continuities in emotion lateralization in human and non-
human primates. Front. Hum. Neurosci. 7:464. doi:10.3389/fnhum.2013.00464 Sackeim, H. A., Gur, R. C., and Saucy, M. C. (1978). Emotions are expressed more
intensely on the left side of the face. Science 202, 434–436. doi:10.1126/science. 705335 Locher, P. L. (1996). The contribution of eye-movement research to an understand-
ing of pictorial balance perception: a review of the literature. Empiric. Stud. Arts
14, 143–163. doi:10.2190/D77M-3NU4-DQ88-H1QG Loewenfeld, I. E. (1966). Comment on Hess’ findings. Surv. Ophthalmol. 11,
293–294. Schiff, B. B., and Lamon, M. (1989). Inducing emotion by unilateral contraction of
facial muscles: a new look at hemispheric specialization and the experience of
emotion. Neuropsychologia 27, 923–935. doi:10.1016/0028-3932(89)90068-7 Loewenfeld, I. E. (1999). The Pupil: Anatomy, Physiology, and Clinical Applications. Boston: Butterworth-Heinemann. Schirillo,J.,and Fox,M. (2006). Rembrandt’s portraits: approach or avoid? Leonardo
39, 253–256. doi:10.1162/leon.2006.39.3.253 Loftus, G. R. (1985). Picture perception: effects of luminance on available infor-
mation and information-extraction rate. J. Exp. Psychol. Gen. 114, 342–356. doi:10.1037/0096-3445.114.3.342 Schirillo, J. A. (2000). Hemispheric asymmetries and gender influence Rem-
brandt’s portrait orientations. Neuropsychologia 38, 1593–1606. doi:10.1016/
S0028-3932(00)00063-4 Mandal, M. K., Tandon, S. C., and Asthana, H. S. (1991). Right brain damage
impairs recognition of negative emotions. Cortex 27, 247–253. doi:10.1016/
S0010-9452(13)80129-3 Schwartz, G. E., Ahern, G. L., and Brown, S. (1979). Lateralized facial mus-
cle response to positive and negative emotional stimuli. Psychophysiology 16,
561–571. doi:10.1111/j.1469-8986.1979.tb01521.x Mannan, S., Ruddock, K. H., and Wooding, D. S. (1995). Automatic control of sac-
cadic eye movements made in visual inspection of briefly presented 2-D images. Spat. Vis. 9, 363–386. doi:10.1163/156856895X00052 Silberman, E. K., and Weingartner, H. (1986). REFERENCES 70, 165–168. doi:10.1037/h0021978 Borod, J. C., Kent, J., Koff, E., Martin, C., and Alpert, M. (1988). Facial asymmetry
while posing positive and negative emotions: support for the right hemisphere
hypothesis. Neuropsychologia 26, 759–764. doi:10.1016/0028-3932(88)90013-9 Humphrey, N., and McManus, C. (1973). Status and the left cheek. New Sci. 23,
437–439. Janisse, M. P. (1973). Pupil size and affect: a critical review of the literature since
1960. Can. Psychol. 14, 311–329. doi:10.1037/h0082230 Bradley,M. M.,Miccoli,L.,Escrig,M. A.,and Lang,P. (2008). The pupil as a measure
of emotional arousal and autonomic activation. Psychophysiology 45, 602–607. doi:10.1111/j.1469-8986.2008.00654.x Janisse, M. P. (1974). Pupil size, affect and exposure frequency. Soc. Behav. Pers. 2,
125–146. doi:10.2224/sbp.1974.2.2.125 Frontiers in Human Neuroscience January 2014 | Volume 7 | Article 938 | 9 www.frontiersin.org Laterality, pupil size, and esthetics Schirillo Janisse, M. P. (1977). Pupillometry: The Psychology of the Pupillary Response. New
York, NY: John Wiley & Sons. Rinn,W. E. (1984). The neuropsychology of facial expression: a review of the neuro-
logical and psychological mechanisms for producing facial expressions. Psychol. Bull. 95, 52–77. doi:10.1037/0033-2909.95.1.52 Jasari, A., Tranel, D., and Adolphs, R. (2000). A valence-specific lateral bias for dis-
criminating emotional facial expressions in free field. Cogn. Emot. 14, 341–353. doi:10.1080/026999300378860 Rossi, G. F., and Rosadini, G. (1967). “Experimental analysis of cerebral dominance
in man,” in Brain Mechanisms Underlying Speech and Language, eds C. H. Mil- Rossi, G. F., and Rosadini, G. (1967). “Experimental analysis of cerebral dominance
in man,” in Brain Mechanisms Underlying Speech and Language, eds C. H. Mil-
likan, and F. L. Darley (New York: Grune and Stratton), 167–189. Rossi, G. F., and Rosadini, G. (1967). Experimental analysis of cerebral domina
in man,” in Brain Mechanisms Underlying Speech and Language, eds C. H. M
likan, and F. L. Darley (New York: Grune and Stratton), 167–189. in man,” in Brain Mechanisms Underlying Speech and Language, eds C. H. Mil-
likan, and F. L. Darley (New York: Grune and Stratton), 167–189. y
g p
g
g
likan, and F. L. Darley (New York: Grune and Stratton), 167–189. Killgore, W. D. S., and Yurgelun-Todd, D. A. (2007). The right-hemisphere and
valence hypotheses: could they both be right (and sometimes left)? Soc. Cogn. Affect. Neurosci. 2, 240–250. doi:10.1093/scan/nsm020 Russell, P. A., and George, D. A. (1990). Relationships between aesthetic response
scales applied to paintings. Empiric. Stud. Arts 8, 15–30. doi:10.2190/AU1R-
6UXE-T14R-04WQ Kinsbourne, M. (1982). Hemispheric specialization and the growth of human
understanding. Am. Psychol. REFERENCES doi:10.1098/rspb.2003.2660 Conflict of Interest Statement: The author declares that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest. Nunnally, J. C., Knott, P. D., Duchnowski, A., and Parker, R. (1967). Pupillary
responses as a general measure of activation. Percept. Psychophys. 2, 149–155. Received: 31 July 2013; accepted: 27 December 2013; published online: 13 January 2014. Citation: Schirillo JA (2014) Pupil dilations reflect why Rembrandt biased female
portraits leftward and males rightward. Front. Hum. Neurosci. 7:938. doi:
10.3389/fnhum.2013.00938 Polt, J. M. (1970). Effect of threat of shock on pupillary response in a problem-
solving task. Percept. Mot. Skills 31, 587–593. doi:10.2466/pms.1970.31.2.587 portraits leftward and males rightward. Front. Hum. Neurosci. 7:938. doi:
10.3389/fnhum.2013.00938 Powell, W. R., and Schirillo, J. A. (2009). Asymmetrical facial expressions in por-
traits and hemispheric laterality: a literature review. Laterality 14, 545–572. doi:10.1080/13576500802680336 This article was submitted to the journal Frontiers in Human Neuroscience. j
Copyright © 2014 Schirillo. 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 licen-
sor 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
Copyright © 2014 Schirillo. 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 licen-
sor 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. Prehn, K., Kazzer, P., Lischke, A., Heinrichs, M., Herpertz, S. C., and Domes, G. (2013). Effects of intranasal oxytocin on pupil dilation indicate increased salience
of socioaffective stimuli. Psychophysiology 50, 528–537. doi:10.1111/psyp.12042 Richer, F., Silverman, C., and Beatty, J. (1983). Response selection and initiation
in speeded reactions: a pupillometric analysis. J. Exp. Psychol. Hum. Percept. Perform. 3, 360–370. January 2014 | Volume 7 | Article 938 | 10 Frontiers in Human Neuroscience www.frontiersin.org Laterality, pupil size, and esthetics Schirillo 5. A Young Girl Seated. 1660. London, E. S. Borthwick Norton
Sale, May 15. 1953 (Br. 393). Left facing males 1. Rembrandt’s Sister, 1632. Stockholm, National Museum
(Br. 85) 1. Head of an Old Man. Copenhagen, Statens Museum (Br. 136). 2. Saskia. 1643. Cassel, Gemaldegalerie (Br. 101). 2. A Man in Oriental Costume. 1633. Munich, Alte Pinakothek
(Br. 178). 3. Saskia. 1636. Hartford,
Conn.,
Wadsworth
Atheneum
(Br. 105). 3. A Bearded Man. 1646. Cassel, Germaldegalerie (Br. 230). 4. Hendrickje Stoffels as Flora. 1654. New York, Metropolitain of
Art (Br. 114). 4. Study of an Old Man. 1640. Detroit, Mrs. Standish Backus
(Br. 244). 5. Amalia van Solms. 1632. Paris, Musee Jacquemart-Andre
(Br. 99). 5. Portrait of an Old Man in a Pearl-Trimmed Hat. 1662. Dresden,
Gemaldegalirie (Br. 324). Frontiers in Human Neuroscience January 2014 | Volume 7 | Article 938 | 11 Right facing females 1. Rembrandt’s Mother as a Biblical Prophetess (Hannah?). 1631. Amsterdam, Rijksmuseum (Br. 69). 1. Self-Portrait. 1629. Munich, Alte Pinakothek (Br. 2). 1. Self-Portrait. 1629. Munich, Alte Pinakothek (Br. 2). 2. Self-Portrait. Not dated. The Hague, Mauritshuis (Br. 24). 2. Saskia. 1633. Washington, National Gallery of Art (Widener
Collection) (Br. 96). 3. An Old Man with a Gold Chain. 1630. Los Angeles, Hans Cohn
(Br. 149). 3. Rembrandt’s Sister. 1634. Indianapolis, John Herron Art
Museum (Br. 100). 4. Portrait of a Man Reading. 1645. Williamstown, Mass., The
Sterling and Francine Clark Art Museum (Br. 238). 4. A Woman in Fanciful Costume. 1648. Baltimore, The Walters
Art Gallery (Br. 386). 5. Study of the Head of an Old Man. 1661. New York, John Hay
Whitney (Br. 261). January 2014 | Volume 7 | Article 938 | 11 www.frontiersin.org Frontiers in Human Neuroscience
|
https://openalex.org/W2465647318
|
https://zenodo.org/record/56749/files/Versao_final_publicada.pdf
|
English
| null |
Implementation and Validation of a Self-Consumption Maximization Energy Management Strategy in a Vanadium Redox Flow BIPV Demonstrator
|
Energies
| 2,016
|
cc-by
| 6,636
|
Academic Editor: Xiaoliang Wei Academic Editor: Xiaoliang Wei g
Received: 12 May 2016; Accepted: 20 June 2016; Published: 29 June 2016 g
Received: 12 May 2016; Accepted: 20 June 2016; Published: 29 June 2016 Abstract: This paper presents the results of the implementation of a self-consumption maximization
strategy tested in a real-scale Vanadium Redox Flow Battery (VRFB) (5 kW, 60 kWh) and Building
Integrated Photovoltaics (BIPV) demonstrator (6.74 kWp). The tested energy management strategy
aims to maximize the consumption of energy generated by a BIPV system through the usage of a
battery. Whenever possible, the residual load is either stored in the battery to be used later or is
supplied by the energy stored previously. The strategy was tested over seven days in a real-scale
VRF battery to assess the validity of this battery to implement BIPV-focused energy management
strategies. The results show that it was possible to obtain a self-consumption ratio of 100.0%, and
that 75.6% of the energy consumed was provided by PV power. The VRFB was able to perform the
strategy, although it was noticed that the available power (either to charge or discharge) varied with
the state of charge. Keywords:
vanadium redox flow battery; building integrated photovoltaics (BIPV); energy
management strategy; self-consumption maximization; real-scale battery www.mdpi.com/journal/energies Implementation and Validation of a
Self-Consumption Maximization Energy
Management Strategy in a Vanadium Redox Flow
BIPV Demonstrator Luis Fialho 1,2,*, Tomás Fartaria 1,2, Luis Narvarte 3 and Manuel Collares Pereira 1,2
1
Renewable Energies Chair, Universidade de Évora, 7002-554 Évora, Portugal;
tomasfartaria@uevora.pt (T.F.); collarespereira@uevora.pt (M.C.P.)
2
Instituto de Investigação e Formação Avançada (IIFA), Universidade de Évora, Palácio do Vimioso,
Largo Marquês de Marialva, Apart. 94, 7002-554 Évora, Portugal
3
Instituto de Energía Solar, Universidad Politécnica de Madrid, 28040 Madrid, Spain;
navarte@ies-def.upm.es 1
Renewable Energies Chair, Universidade de Évora, 7002-554 Évora, Portugal;
tomasfartaria@uevora.pt (T.F.); collarespereira@uevora.pt (M.C.P.)
2
Instituto de Investigação e Formação Avançada (IIFA), Universidade de Évora, Palácio do Vimioso,
Largo Marquês de Marialva, Apart. 94, 7002-554 Évora, Portugal
3
Instituto de Energía Solar, Universidad Politécnica de Madrid, 28040 Madrid, Spain;
navarte@ies-def.upm.es *
Correspondence: lafialho@uevora.pt; Tel.: +351-967-886-820 *
Correspondence: lafialho@uevora.pt; Tel.: +351-967-886-820 Academic Editor: Xiaoliang Wei energies energies 2. Real-Scale VRFB Demonstrator The methodology used in this paper is based on the implementation of a real-scale demonstrator,
installed at the University of Évora (Portugal). It consists of a rooftop mounted PV system (BIPV) and
an electrical storage system (Vanadium Redox Flow battery), both connected to a building, which in
turn is connected to the public electric grid (see Figure 1). Figure 1. Demonstrator (photovoltaic (PV) plant and Vanadium Redox Flow battery (VRFB) with
respective converters) simplified schematic. Inverters convert power from PV and battery (DC) to the
grid (AC). Figure 1. Demonstrator (photovoltaic (PV) plant and Vanadium Redox Flow battery (VRFB) with
respective converters) simplified schematic. Inverters convert power from PV and battery (DC) to the
grid (AC). 1. Introduction With the current increase of distributed renewable energies generators (such as rooftop
photovoltaic power) in electrical grids, and more affordable energy storage systems (ESS), it is
becoming much more viable to match individual demand for electricity with on-site photovoltaic (PV)
production [1–3]. In the case of residential installations, the use of ESS for short-term management
of supply—i.e., allowing the shift of PV energy to higher consumption periods (such as the evening
peak or at night)—could increase self-consumed electricity from 30% to 70% [4–6]. To allow this increase, a battery must be integrated in the grid-connected PV system and an ad-hoc
energy management strategy (EMS) must be implemented in a control unit that correctly manages
the electricity flow between the PV generator, the battery, and the grid. In recent years, several
EMSs focused on the maximization of the self-consumption of on-site-produced PV electricity have
been presented [7–10], in which they assert that this strategy is useful and economically viable, even
without feed-in-tariffs. This EMS requires a reliable and efficient battery technology with a low power/energy
ratio, such as the Vanadium Redox Flow Batteries (VRFB). This promising technology has as Energies 2016, 9, 496; doi:10.3390/en9070496 www.mdpi.com/journal/energies 2 of 13 Energies 2016, 9, 496 main advantages the decoupling of power and capacity, a very long lifetime (reported as above
10000 cycles, or 25 years of operation), very low maintenance, and deep discharge capability (almost
100.0%) [11]. Very few batteries of this kind exist in laboratories, especially at such a scale that allows
real-life test conditions, so the investigation of these batteries is mostly done with simulations (and
sometimes in small lab battery cells) [12,13]. In order to test the performance of this kind of EMS, a
real-scale VRB demonstrator was built in the University of Évora, part of the FP7 PVCROPS European
project [14]. This demonstrator consists of a PV generator of 6.74 kWp, a PV inverter of 10 kW,
a battery inverter of 7.4 kW, and a VRFB of 60 kWh. The main objective of this paper is to present the
results of the validation tests of the self-consumption energy management strategy mentioned above
and to show its suitability for building integrated PV applications. Useful conclusions for the future
operation of these kinds of systems are also discussed. 2.1. Equipment The BIPV system is composed of two strings, a 3.24 kWp of monocrystalline PV panels and
3.5 kWp of polycrystalline cells, connected to a three-phase power inverter [15] with two independent
maximum power point tracking (MPPT), connected to the building AC power grid. The energy
storage is provided by a Vanadium Redox Flow Battery with 5 kW nominal power and 12 h
storage [11], providing a total capacity of 60 kWh. The battery is also connected to the triphasic
AC grid by three synchronized inverters (one per phase) [15]. Figure 2 shows the rooftop mounted
PV system while Figure 3 shows the battery and four inverters (PV inverter in gray and the three
battery inverters in green). To operate the demonstrator, a control system was implemented using a PC with Labview
software to send and receive commands, and for overall system communication. This PC also acts
as data logger to receive and store data. This PC receives data with parameter values from all the
equipment (inverters, battery, and other sensors), runs the Labview software with the EMS, and
continuously ensures the operation safety. In addition, each equipment has its own safety measures
and surveillance implemented. 3 of 13 Energies 2016, 9, 496 Energies 2016, 9, 496
3 of 13
Figure 2. Roof mounted PV system. Figure 3. VRFB (right) and the inverters (left). Figure 2. Roof mounted PV system. Figure 2. Roof mounted PV system. Figure 3. VRFB (right) and the inverters (left). Figure 3. VRFB (right) and the inverters (left). The main innovative feature of this demonstrator is the usage of a real-scale Vanadium
Redox Flow Battery. Table 1 shows the main technical specifications of the VRFB installed in
the demonstrator. Table 2 shows the three battery inverters specifications, and Table 3 shows the
photovoltaic inverter manufacturer specifications. In this battery, the electrolyte is stored in two tanks (for positive and negative species). The electrolyte is then pumped through the piping system to a stack of cells where the
reduction–oxidation reaction occurs. The electron exchange takes place in cells (connected in series
to constitute the stack) where a special ion membrane separates the two electrolytes and only permits
H+ and V+ to pass. 2.1. Equipment The use of two tanks and a stack allows the decoupling of the battery power density (determined
by the active area of the cells in the stack) from its energy density—i.e., the total energy that it can
store (given by tank capacity and specific electrolyte energy density). This energy–power decoupling
allows a battery to be tailored for many special cases, such as low power and large capacity
(residential BIPV) or large power and lower capacity to mitigate power fluctuations (wind farms
or PV plants). Another important feature of this battery technology is the absence of self-discharge,
as there is no reaction outside the stack. Table 1. VRFB technical specifications. Specifications
Value
Manufacturer
REDT
Capacity
60 kWh (5 kW for 12 h)
Rated Power
5 kW
Maximum Operating Voltage
62 V
Minimum Operating Voltage
48 V
Electrical connection
Three-phase
Lifetime
+10,000 deep charge/discharge cycles
Full cycle (discharge/charge) Energy Efficiency
70%–80% Table 1. VRFB technical specifications. Table 1. VRFB technical specifications. 4 of 13 Energies 2016, 9, 496 Table 2. Battery inverters technical specifications. Specifications
Value
Manufacturer
Ingeteam
Model
EMS Home 2.4
DC Max Current
50 A
DC Operating Voltage Range
48 V–250 V
Rated AC Power
2.4 kW
Electrical connection
single-phase
Table 3. PV inverter technical specifications. Specifications
Value
Manufacturer
Ingeteam
Model
3Play 12.5TL M
PV Power Range
12.9 kW–16.8 kW
DC Voltage Range MPPT1 and MPPT2
200 V–820 V
Rated Output AC Power
12.5 kW
Electrical connection
three-phase Table 2. Battery inverters technical specifications. Table 3. PV inverter technical specifications. Another distinct characteristic of this battery is the usage of vanadium as the main electrolyte
element. The VRFB exploits the ability of vanadium to exist in four different oxidation states: from
V2+ through V5+, dissolved in sulfuric acid (H2SO4). It is used as the positive electrolyte (V5+/V4+)
and as the negative electrolyte (V2+/V3+). The usage of only one element allows the restoration of the
electrolyte by simply mixing both electrolytes (positive and negative) and then separating them again
by recharging the battery. This process (electrochemical reversibility) makes the electrolyte virtually
infinite and non-degradable. The only source of electrolyte degradation is the occurrence of a side
reaction with elements in air. To avoid this unwanted reaction, the empty space in the electrolyte
tanks is filled with a high purity inert gas (Argon). 2.1. Equipment As these batteries are mostly composed of plastic piping and tanks and as the electrolyte
is isolated from air, its lifetime is longer than other technologies, reported as 10,000 cycles [11]). In addition, as previously referred to in the introduction, another important feature of this technology
is that it allows very deep discharges (close to 100%), so the whole battery capacity can be used. 2.1.1. Determination of the State of Charge The battery voltage is determined by the voltage of
the reference cell multiplied by 40, as the stack is formed by 40 cells in series. Figure 4. State of charge versus battery voltage. The battery voltage is determined by the voltage of
the reference cell multiplied by 40, as the stack is formed by 40 cells in series. Figure 4. State of charge versus battery voltage. The battery voltage is determined by the voltage of
the reference cell multiplied by 40, as the stack is formed by 40 cells in series. 2.1.1. Determination of the State of Charge The VRF battery installed in this demonstrator is equipped with a parallel electrolyte circuit that
carries a small part of the electrolyte flow to just one external cell (called the reference cell) that is
neither subjected to charge nor discharge. As this cell voltage is only affected by the real electrolyte
state, this cell voltage is the actual battery voltage in a rested state (not influenced by charge or
discharge). In addition, the state of charge (SOC) is proportional to this voltage, so by knowing
the reference cell voltage, one can obtain the real SOC. The function relating SOC with reference cell voltage was obtained experimentally by the battery
manufacturer. Figure 4 shows the SOC variation with the battery voltage (determined by the reference
cell voltage multiplied by 40, as the stack is formed by 40 cells in series). In order to obtain the SOC
value during the battery operation, this graphic was fit to Equation (1) so the SOC is computed using
the reference cell voltage measurement. Equation (1) was defined as a piecewise function, computed
as a numerical fit of the data within the operating ranges (from 41.8 V to 65.4 V), as no single function
could be fitted properly. Each function was chosen as the one that yielded the best numerical fit for
their respective ranges (R-Squared ⩾0.99). 5 of 13 Energies 2016, 9, 496 SOC(V) =
0.0031V4 −0.5606V3 + 37.848V2 −1136.7V + 12810,
if 41.8V < V ⩽52.4V
−0.1544V3 + 25.961V2 −1443.4V + 26577,
if 52.4V < V ⩽58.8V
0.0609V3 −11.88V2 + 773.28V −16694,
if 58.8V < V ⩽65.4V
(1)
Figure 4. State of charge versus battery voltage. The battery voltage is determined by the voltage of
the reference cell multiplied by 40, as the stack is formed by 40 cells in series. SOC(V) =
0.0031V4 −0.5606V3 + 37.848V2 −1136.7V + 12810,
if 41.8V < V ⩽52.4V
−0.1544V3 + 25.961V2 −1443.4V + 26577,
if 52.4V < V ⩽58.8V
0.0609V3 −11.88V2 + 773.28V −16694,
if 58.8V < V ⩽65.4V
(1) SOC(V) =
0.0031V4 −0.5606V3 + 37.848V2 −1136.7V + 12810,
if 41.8V < V ⩽52.4V
−0.1544V3 + 25.961V2 −1443.4V + 26577,
if 52.4V < V ⩽58.8V
0.0609V3 −11.88V2 + 773.28V −16694,
if 58.8V < V ⩽65.4V
(1) Figure 4. State of charge versus battery voltage. 2.2. Instrumentation and Data Acquisition In order to properly implement an EMS, several parameters are measured, allowing the
computation of useful variables that enable smooth system operation. Furthermore, to allow a more
complete study of each piece of equipment, several additional measurements (other than critical
variables already provided by the equipment) are necessary. Table 4 summarizes the monitored
variables. For example, regarding the PV system, it is only necessary to know the AC Power given
by the inverter. However, to evaluate the efficiency of the PV system or detect problems, more data is
necessary (global radiation in the plane of the BIPV system, PV cells temperature, and wind speed). These measurements are made using a weather station on the roof consisting of a pyranometer, a
temperature sensor, and an anemometer, as shown in Figure 5. These sensors are read and processed
by a control electronics (arduino Leonardo board), which in turn sends the data via a wireless zigbee
network to another arduino connected to the main PC. Figure 5. Pyranometer and anemometer installed on the roof. Figure 5. Pyranometer and anemometer installed on the roof. The logged variables regarding the battery and inverters system are: DC power, AC power,
battery voltage, current, and state of charge (SOC). Other useful variables are also read to ensure that
the installation is healthy and operating within safe conditions (for example battery temperature,
piping flow and pressure, electric grid parameters, and safe battery voltage range). The data is 6 of 13 Energies 2016, 9, 496 transmitted with Modbus protocol over TCP, via Ethernet. In order to obtain a more precise SOC
value—it being a crucial parameter—an independent digital bench voltmeter was installed to the
reference cell. This independent measurement allows a precision improvement from 0.7% (battery
control measurement) to 0.1% (multimeter measurement) of the reference cell voltage. Furthermore,
a three phase wattmeter (power analyzer) was installed to measure the power exchanged from the
BIPV system and battery to the electric grid. In addition, three wattmeters were installed to obtain
AC power measurements for each phase/ battery inverter. Table
4. Monitored
variables
during
the
demonstrator
operation. BIPV:
Building
Integrated Photovoltaics. BIPV
PV inverter
Battery
Battery Inverter
Grid
Global Radiation
AC Voltage
Ref. Cell Voltage
AC Voltage
AC 3-phase power
PV Cell temperature
AC Current
Temperature
AC Current
DC Voltage
AC Power
DC Voltage
AC Power
DC Current
DC Current
DC Power
DC Power
2.3. Electrical Loads Table
4. 2.3. Electrical Loads The loads used for the tests were simulated using the building to which the demonstrator is
connected, as the consumption of all building loads is much larger than the power and capacity of
the battery and PV system. The loads were simulated as being equally distributed by the three-phase
electric grid. In this way, whenever a load is to be supplied, the power is sent to the building grid
and that same power value is subtracted from the wattmeter measurement. This operation yields the
power that would be exchanged with the grid if the loads were real and directly connected to the
demonstrator. The load profile used is based on real 15 minute interval measurements made during
a year of a real domestic installation [16]. This profile was then scaled according to the demonstrator
specifications in order to provide a load profile level balanced to the system scale in what concerns
battery usage and installed PV power. This method allows the loads to be of a suitable size relative
to the real system specifications. 2.2. Instrumentation and Data Acquisition Monitored
variables
during
the
demonstrator
operation. BIPV:
Building
Integrated Photovoltaics. BIPV:
Building 3. Self-Consumption Maximization Strategy The strategy tested in the demonstrator was the self-consumption maximization strategy. The main objective of this EMS is to maximize the consumption of PV energy produced locally in
the building, hence avoiding injecting energy to the electrical grid. This is achieved by giving priority
to PV power and either storing the residual load (energy produced by PV system minus the energy
consumed by loads) in an ESS or by taking energy stored previously. Thus, the only exceptional case
in which there is an exchange of energy with the grid is when the battery can no longer absorb more
energy or when the PV + battery system cannot provide enough energy to the required loads. According to the regulation of certain countries (such as Spain, Germany, Portugal, South Africa,
Puerto Rico, etc.), the feeding of PV power into the electrical grid is either forbidden or highly
restricted, so it is necessary to take measures to avoid this situation [17–21]. Either the excess power
should be curtailed—i.e., PV production is limited to a sub-optimal value to avoid producing more
power than what is being consumed at the moment—or, ideally, the excess power would be either
stored or used by some deferrable loads (for example, a water heater, washing machine, etc.) [16]. In countries where a feed-in tariff (FIT) is paid for renewable electrical energy, the use of a battery
could help to maximize profit. It could ensure that no PV power is lost due to regulatory restrictions
or technical problems (for example, a sudden power outage) and even delay the sale of energy to a
time when it might be purchased at a higher value (such as during peak hours). In a stand-alone
system or in one where no PV power is allowed to be sold to the electrical grid (or if the selling price Energies 2016, 9, 496 Energies 2016, 9, 496 7 of 13 is too low), the user can store the energy for later usage, lowering the necessity to buy electricity from
the grid. A flowchart of the self-consumption maximization strategy is presented in Figure 6. In the first
step, the control system acquires data from PV production, loads consumption, and the battery state
of charge (SOC). Then, it computes the difference between the loads consumption and PV production,
referred to as residual load (Presidual). 3. Self-Consumption Maximization Strategy If the differential is positive, it means that current PV power
is not enough to supply the loads. Hence, Presidual is provided by the battery (if it has enough
accumulated energy). If the battery cannot provide the total necessary power (due to a low SOC), the
remainder has to be provided by the electric grid. The reverse happens when there is more PV power
being generated than consumed in a given moment. In this case, the remainder should be stored in
the battery. If the battery cannot absorb all of the excess power (for instance, near maximum SOC),
the remaining power must be fed to the grid (considering the case with no active loads management). Figure 6. Flowchart of the self-consumption maximization strategy implemented in the demonstrator. Figure 6. Flowchart of the self-consumption maximization strategy implemented in the demonstrator. This cycle should run at least every second to provide a quick response to any changes in PV
production, consumption, or other situations (for example a blackout or a sudden problem in a piece
of equipment). In this way, the grid is not largely affected by large fluctuations in PV production that
could otherwise be fed to the electric grid with poor quality (high power variations in a short time). With the proper implementation of this EMS, the benefits obtained should be: lower energy
exchange with the public grid, higher self-consumed PV power, and a higher load supply with PV
power (either directly or by the battery). In order to validate this EMS—tested in the real size VRFB
demonstrator—the following merit factors were defined to quantify the obtained improvements:
self-consumption ratio, maximum positive (from the grid), and negative (into the grid) peak grid
power, respectively given by Equations (2) to (4). This cycle should run at least every second to provide a quick response to any changes in PV
production, consumption, or other situations (for example a blackout or a sudden problem in a piece
of equipment). In this way, the grid is not largely affected by large fluctuations in PV production that
could otherwise be fed to the electric grid with poor quality (high power variations in a short time). 4. Validation of the Self-Consumption Maximization Strategy in the VRFB Demonstrator 4. Validation of the Self-Consumption Maximization Strategy in the VRFB Demonstrator 3. Self-Consumption Maximization Strategy With the proper implementation of this EMS, the benefits obtained should be: lower energy
exchange with the public grid, higher self-consumed PV power, and a higher load supply with PV This cycle should run at least every second to provide a quick response to any changes in PV
production, consumption, or other situations (for example a blackout or a sudden problem in a piece
of equipment). In this way, the grid is not largely affected by large fluctuations in PV production that
could otherwise be fed to the electric grid with poor quality (high power variations in a short time). With the proper implementation of this EMS, the benefits obtained should be: lower energy
exchange with the public grid, higher self-consumed PV power, and a higher load supply with PV
power (either directly or by the battery). In order to validate this EMS—tested in the real size VRFB
demonstrator—the following merit factors were defined to quantify the obtained improvements:
self-consumption ratio, maximum positive (from the grid), and negative (into the grid) peak grid
power, respectively given by Equations (2) to (4). With the proper implementation of this EMS, the benefits obtained should be: lower energy
exchange with the public grid, higher self-consumed PV power, and a higher load supply with PV
power (either directly or by the battery). In order to validate this EMS—tested in the real size VRFB
demonstrator—the following merit factors were defined to quantify the obtained improvements:
self-consumption ratio, maximum positive (from the grid), and negative (into the grid) peak grid
power, respectively given by Equations (2) to (4). 3. Self-Consumption Maximization Strategy Energies 2016, 9, 496 Self-consumption ratio =
R ∞
0 (PPV(t) + Pgrid(t)) dt
R ∞
0 Ploads(t) dt
(2)
Maximum positive Pgrid = Max{Pgrid 15min}
(3)
Maximum negative Pgrid = Min{Pgrid 15min}
(4) Self-consumption ratio =
R ∞
0 (PPV(t) + Pgrid(t)) dt
R ∞
0 Ploads(t) dt
(2) (2) R
0 Ploads(t) dt
Maximum positive Pgrid = Max{Pgrid 15min}
(3)
Maximum negative Pgrid = Min{Pgrid 15min}
(4) Maximum positive Pgrid = Max{Pgrid 15min}
(3)
Maximum negative Pgrid = Min{Pgrid 15min}
(4) Maximum positive Pgrid = Max{Pgrid 15min}
(3)
Maximum negative Pgrid = Min{Pgrid 15min}
(4) and Pgrid 15min is the average value for each 15 minute time interval, given by: and Pgrid 15min is the average value for each 15 minute time interval, given by: Pgrid 15min =
1
900
900
∑
t=0
Pgrid(t)
(5) (5) where t is time in seconds. where t is time in seconds. where t is time in seconds. 4. Validation of the Self-Consumption Maximization Strategy in the VRFB Demonstrator 4.1. Testing Campaign A testing campaign was carried out in the time frame of one week. It was considered a sensible
duration for the EMS validation, representing a balance between a short-time test (one day) and
a longer period—for instance, one month. Figure 7 shows fifteen minute averages of the main
parameters (PV power, loads consumption, and power exchanged with the battery) recorded during
the testing of the Self-Consumption Maximization strategy. Figure 8 shows the power exchanged
with the battery and the power exchanged with the grid in the same time period. Figure 7. One week display of the PV, loads, and battery power profiles (PPV, Pload, and
Pbat, respectively). Figure 7. One week display of the PV, loads, and battery power profiles (PPV, Pload, and
Pbat, respectively). Figure 8. Variation of Pbat, Presidual, and state of charge (SOC), one week period. Figure 8. Variation of Pbat, Presidual, and state of charge (SOC), one week period. 9 of 13 Energies 2016, 9, 496 Figure 9 shows the solar radiation on the PV plane (pyranometer) and PV cell temperature in the
same time period. Figure 9 shows the solar radiation on the PV plane (pyranometer) and PV cell temperature in the
same time period. Figure 9. Week profile of Solar Radiation (PV plane) and PV Cell Temperature. Figure 9. Week profile of Solar Radiation (PV plane) and PV Cell Temperature. Figure 9. Week profile of Solar Radiation (PV plane) and PV Cell Temperature. Figure 10. Variation of available discharge (blue) and charge (red) power, relative to the nominal
power with the battery SOC. Figure 9. Week profile of Solar Radiation (PV plane) and PV Cell Temperature. Figure 10. Variation of available discharge (blue) and charge (red) power, relative to the nominal
power with the battery SOC. Figure 10. Variation of available discharge (blue) and charge (red) power, relative to the nominal
power with the battery SOC. From the graphics shown in Figures 7 and 9, the PV generation (blue dotted line in Figure 7)
was mostly regular, with a clean radiation profile with only one day with significant variation (seen
in Figure 9 on 18 May 2015). When the PV power is higher than the consumption, the remaining
power (residual load positive) is stored in the battery (battery power is negative when charging). Usually, later in the day, when PV power is decreasing, the residual load (negative) is supplied by
the battery. 4.1. Testing Campaign However, when comparing Figure 7 with Figure 8, it can be seen that despite the energy stored
in the battery being enough to supply the loads, the power that the battery could deliver was not
enough. This is due to the ratio of power versus SOC not being constant throughout the major range
of SOC. When the battery is partially discharged (lower SOC), the discharge power that it can provide
is lower than the nominal power. The reverse situation occurs with the charge power when the battery
is almost at full capacity (higher SOC), as shown in Figure 10 . Figures 11 and 12 show that, at the beginning, the residual load (blue line) began to be entirely
supplied by the battery (red line). When the battery got below 5% of SOC (purple line), the loads had
to be supplied by the grid, as below this value, the battery voltage gets close to the inverter minimum
voltage range. To avoid this situation, a safety measure was implemented. In Figure 11, one can see
that despite this safety measure, the battery SOC continued to fall, suggesting that the value used
to charge the battery is not enough to overcome the consumption of the inverters and the battery
internal losses. 10 of 13 Energies 2016, 9, 496 10 of 13 Figure 11. First day of the self-consumption maximization test. Figure 11. First day of the self-consumption maximization test. Figure 12. Display of the first day of the self-consumption maximization test. Figure 11. First day of the self-consumption maximization test. Figure 12. Display of the first day of the self-consumption maximization test. Figure 12. Display of the first day of the self-consumption maximization test. When the PV power began to increase, the residual load also began to increase (positively), so
the available power was used to charge the battery. At the end of the day, when PV power decreased,
the previously stored energy was used to supply the loads. At the end of the day (after 21 h), some
power had to come from the grid, as the battery could not entirely provide the necessary power to
the evening load peak. 5. Results To properly assess the validation and to quantify the self-consumption maximization
performance, several merit factors were computed and compared with the case in which the
demonstrator did not have an ESS. Table 5 shows the comparison summary of the merit factors for the demonstrator with and
without storage, evaluating the overall EMS performance for the testing period. Table 6 shows the
merit factors computed for each day of the testing period. In terms of total energy, the installation was able to self-consume 100.0% of the PV generation,
and it was able to supply 75.6% of the total consumption. Hence, it was necessary to get 24.4% of the
energy from the grid. The maximum peak power from the grid was 2.5 kW, without energy injection
into the grid. Considering the demonstrator without storage, only 65.2% of the total energy generated
by the PV system was self-consumed, and the peak power injected to the grid was 3.3 kW, while the
peak power taken from the grid reached 4.2 kW. To decrease the grid contribution, it is possible to increase the installed PV power or decrease
the loads power consumption. In order to have a flexible demonstrator system, allowing the testing Energies 2016, 9, 496 Energies 2016, 9, 496 11 of 13 of different EMS that can require larger storage capacity, the total installed battery capacity ended up
not being completely used in this test. This occurrence hints at the opportunity to develop and implement strategies that can provide
more benefits than an exclusively short-term or long-term EMS. The remaining unused battery
capacity can be used as reserve power in case of an unforeseen blackout, to take advantage
of electricity price fluctuations, to provide power to neighbor installations, or even to charge
electric vehicles. Additionally, a higher available PV power could be stored in the battery for
later use, decreasing the grid necessity. The extra capacity relative to the PV system size also
raises the important question of how to optimize the battery capacity regarding PV generation and
loads consumption. Table 5. Comparison between the full demonstrator and a virtual one without storage. Table 5. Comparison between the full demonstrator and a virtual one without storage. Merit Factor
Storage
No Storage
Self-consumption ratio (%)
100.0
65.2
Maximum positive peak Pgrid (kW)
2.5
4.2
Maximum negative peak Pgrid (kW)
0.0
3.3 Table 6. Comparison between demonstrator results with and without storage. 5. Results Date
Self Consumption Ratio (%)
Maximum Positive
Peak Pgrid (kW)
Maximum Negative
Peak Pgrid (kW)
14/05
storage
100.0
1.2
0.0
no storage
67.5
3.2
3.2
15/05
storage
100.0
2.5
0.0
no storage
61.6
3.1
3.3
16/05
storage
100.0
1.4
0.0
no storage
63.5
4.3
3.2
17/05
storage
100.0
2.1
0.0
no storage
66.1
2.6
3.2
18/05
storage
100.0
2.6
0.0
no storage
66.4
3.2
3.2
19/05
storage
100.0
1.2
0.0
no storage
65.5
3.4
3.4
20/05
storage
100.0
1.2
0.0
no storage
66.9
2.5
3.3 This test yielded several useful results for the future operation of this technology demonstrators •
The Vanadium Redox Flow technology has the ability to decouple power and capacity but the
available power at extreme SOC varies. Figure 10 shows the variation of discharge (blue) and
charge (red) power with the SOC. This fact also reveals the importance of analyzing these systems
as power sources/sinks and not only in terms of global energy. •
The Vanadium Redox Flow technology has the ability to decouple power and capacity but the
available power at extreme SOC varies. Figure 10 shows the variation of discharge (blue) and
charge (red) power with the SOC. This fact also reveals the importance of analyzing these systems
as power sources/sinks and not only in terms of global energy. •
The system formed by the VFRB and inverters has achieved an overall average efficiency of 57.7%
during the testing period. The efficiency is penalized by the battery operating voltage range
being bellow the ideal voltage range of the inverters. The used inverters have a working voltage
ranging from 48 V to 300 V, although their efficiency is higher at the top end of this range [15]. •
The inverters consumption and battery internal losses are not entirely supplied during the safety
procedure. Although the VRF battery is not damaged by an over-discharge, a voltage below the
working range can turn the battery inverters off (the battery inverters are powered by the battery
and require a minimum of 45 V), hence disturbing the normal operation of the installation. Energies 2016, 9, 496 12 of 13 6. Conclusions The test results of the use of a VRFB as the storage system for the testing of a self-consumption
maximization strategy, implemented in a real-size demonstrator, were presented. The results show
that the VRFB is a suitable technology to be combined with BIPV systems. Regarding the EMS testing,
the strategy successfully maximized the consumption of
locally-produced PV energy. Using the battery to store excess PV power (positive residual load),
it was possible to completely avoid the injection of energy into the public electric grid—i.e., 100.0%
self-consumption ratio. In addition, the total energy consumed by the loads was 75.6%, supplied
by PV generation. A closer analysis of the system operation shows that an increase in PV power
could further decrease the necessity of the electrical grid, as a large part of the battery capacity
(approximately 65.0%) was unused. The correct performance of the EMS is also shown by the reduction of power exchanged with the
grid. The peak power from the grid was reduced in 59.0% in comparison with the case without energy
storage. This means that the prosumer—apart from benefiting the whole electrical system—not
only reduces their electric bill due to the reduction of grid energy consumption, but also due to the
reduction of the utility contracted power tariff. This work also showed the relevance of the equipment operational limits (in this case, the
battery inverters DC voltage range) to the EMS, and the non-negligible variation of available
charge/discharge power with the battery SOC. In future developments of energy management
strategies, all these operational limitations should be taken into account to ensure the proper and
safe working mode of the whole system as planned. Acknowledgments: The authors would like to acknowledge the support of this work, developed under the
PVCROPS Project, which has received funding from the European Union’s Seventh Framework Programme for
research, technological development and demonstration under grant agreement n◦308408. This work was also
supported by the Renewable Energies Chair of the University of Évora (PhD scholarship, author Luis Fialho)
and by a PhD scholarship (author Tomás Fartaria) from FCT – Fundação para a Ciência e Tecnologia, Portugal,
Grant number SFRH/BD/84396/2012. The authors also would like to acknowledge the support of the European Commission OPENAIRE FP7
Post-Grant Open Access Pilot for supporting the publication of this work. Conflicts of Interest: The authors declare no conflict of interest. References 2. Feldman, D.; Barbose, G.; Margolis, R.; James, T.; Weaver, S.; Darghouth, D.; Fu, R.; Davidson, C.;
Booth, S.; Wiser, R. Photovoltaic System Pricing Trends: Historical, Recent, and Near-Term Projections. 2014. Available online: http://www.nrel.gov/docs/fy14osti/62558.pdf (accessed on 11 April 2016). 2. Feldman, D.; Barbose, G.; Margolis, R.; James, T.; Weaver, S.; Darghouth, D.; Fu, R.; Davidson, C.;
Booth, S.; Wiser, R. Photovoltaic System Pricing Trends: Historical, Recent, and Near-Term Projections. 2014. Available online: http://www.nrel.gov/docs/fy14osti/62558.pdf (accessed on 11 April 2016). 3. International Energy Agency (IEA). Global EV outlook: Understanding the electric vehicle landscape to
2020. 2013. Available online: http://www.iea.org/publications/globalevoutlook_2013.pdf (accessed on 11
April 2016). 3. International Energy Agency (IEA). Global EV outlook: Understanding the electric vehicle landscape to
2020. 2013. Available online: http://www.iea.org/publications/globalevoutlook_2013.pdf (accessed on 11
April 2016). 3. International Energy Agency (IEA). Global EV outlook: Understanding the electric vehicle landscape to
2020. 2013. Available online: http://www.iea.org/publications/globalevoutlook_2013.pdf (accessed on 11
April 2016). 4. Rickerson, W.; Couture, T.; Barbose, G.L.; Jacobs, D.; Parkinson, G.; Chessin, E.; Belden, A.; Wilson, H.;
Barrett, H. Residential Prosumers-Drivers and Policy Options (Re-Prosumers). 2014. Available online:
http://iea-retd.org/wp-content/uploads/2014/06/RE-PROSUMERS_IEA-RETD_2014.pdf (accessed on
11 April 2016). 5. Solar Power Europe. Global Market Outlook, For Solar Power 2015–2019. Available online: http://www. solarpowereurope.org/insights/global-market-outlook/ (accessed on 11 April 2016). 5. Solar Power Europe. Global Market Outlook, For Solar Power 2015–2019. Available online: http://www. solarpowereurope.org/insights/global-market-outlook/ (accessed on 11 April 2016). 6. Vrettos, E.; Witzig, A.; Kurmann, R.; Koch, S.; Andersson, G. Maximizing local PV utilization using
small-scale batteries and flexible thermal loads. 2013. Available online: http://www.eeh.ee.ethz.ch/
uploads/tx_ethpublications/Vrettos_etal_2013.pdf (accessed on 11 April 2016). 7. Zaheeruddin; Manas, M. Renewable energy management through microgrid central controller design: An
approach to integrate solar, wind and biomass with battery. Energy Rep. 2015, 1, 156–163. Energies 2016, 9, 496 13 of 13 13 of 13 8. Merei, G.; Moshövel, J.; Magnor, D.; Sauer, D.U. Optimization of self-consumption and techno-economic
analysis of PV-battery systems in commercial applications. Appl. Energy 2016, 168, 171–178, 9. Lang, T.; Ammann, D.; Girod, B. Profitability in absence of subsidies: A techno-economic analysis of rooftop
photovoltaic self-consumption in residential and commercial buildings. Renew. Energy. 2016, 87, 77–87. elf-consumption in residential and commercial buildi 10. Pascual, J.; Barricarte, J.; Sanchis, P.; Marroyoa, L. Energy management strategy for a renewable-based
residential microgrid with generation and demand forecasting. Appl. Energy 2015, 158, 12–25. 11. Renewable Energies Dynamics Technology Ltd. PVCROPS case study. Available online: http://www. redtenergy.com/case-studies/energy-storage-80kwh (accessed on 11 April 2016). 12. References Sum, E.; Rychcik, M.; Skyllas-kazacos, M. Investigation of the V(V)/V(IV) system for use in the positive
half-cell of a redox battery. J. Power Sources 1985, 16, 85–95 13. Rychcik, M.; Skyllas-Kazacos, M. Characteristics of a new all-vanadium redox flow battery. J. Power Sources
1988, 22, 59-67 14. PVCROPS. Available online: http://www.pvcrops.eu (accessed on 11 April 2016). 15. Ingeteam. Available online: http://www.ingeteam.com (accessed on 11 April 2016). 16. Pascual, J.; Sanchis, P.; Marroyo, L. Implementation and Control of a Residential Electrothermal Microgrid
Based on Renewable Energies, a Hybrid Storage System and Demand Side Management. Energies 2014, 7,
210–237. 17. Puerto Rico Electric Power Authority (PREPA). Minimum Technical Requirements for Photovoltaic
Generation (PV) Projects. 2012. Available online:
http://www.nrel.gov/docs/fy14osti/57089.pdf
(accessed on 23 June 2016). 18. Red Electrica de España, Instalaciones conectadas a la red de transporte:
requisitos mínimos de
diseño, equipamiento, funcionamiento y seguridad y puesta en servicio. 2005. Available online:
http://www.ree.es/sites/default/files/01_ACTIVIDADES/Documentos/ProcedimientosOperacion/
PO_resol_11feb2005.pdf (accessed on 23 June 2016). 19. Republic of South Africa Grid Code Secretariat (NERSA), Grid connection code for renewable power
plants (RPPs) connected to the electricity transmission system (TS) or the distribution system (DS) in South
Africa. 2012. Available online: http://www.nersa.org.za/Admin/Document/Editor/file/Electricity/
TechnicalStandards/South%20African%20Grid%20Code%20Requirements%20for%20Renewable%
20Power%20Plants%20-%20Vesion%202%206.pdf (accessed on 23 June 2016). p
20. Bundesverband der Energie (BDEW). Technical Guideline Generating Plants Connected to the
Medium-Voltage Network:
Guideline for generating plants connection to and parallel operation
with the medium-voltage network. 2008. Available online:
https://www.bdew.de/internet.nsf/id/
A2A0475F2FAE8F44C12578300047C92F/\protect\T1\textdollarfile/BDEW_RL_EA-am-MS-Netz_engl. pdf (accessed on 23 June 2016). 21. Ministério do Ambiente,
Ordenamento do Território e Energia,
Governo de Portugal,
Decreto
Lei
n.o
153/2014. 2014. Available
online:
http://www.legislacao.org/primeira-serie/
decreto-lei-no-153-2014-306472 (accessed on 23 June 2016). 21. Ministério do Ambiente,
Ordenamento do Território e Energia,
Governo de Portugal,
Decreto
Lei
n.o
153/2014. 2014. Available
online:
http://www.legislacao.org/primeira-serie/
decreto-lei-no-153-2014-306472 (accessed on 23 June 2016). c⃝2016 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/). c⃝2016 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/).
|
https://openalex.org/W4200408458
|
https://www.degruyter.com/document/doi/10.1515/9783110709308-011/pdf
|
English
| null |
Acknowledgments
|
De Gruyter eBooks
| 2,021
|
cc-by
| 1,706
|
Acknowledgments This book is a translation of the revised and expanded version of my PhD disser-
tation, which was approved by the Institute of History at Humboldt-Universität
zu Berlin on 21 January 2015. The German version of the book was published
in 2018 by Vandenhoeck & Ruprecht as Elitenbildung und Dekolonisierung. Die
Évolués in Belgisch Kongo 1944–1960. For the English translation I expanded
and updated sections of the manuscript in light of subsequent publications
and criticism. Only authors know just how much other people contribute to the creation of
books. This volume would never have seen the light of day without the help and
support of many individuals. I would like to expressly thank some of them here. First, I am deeply grateful to Andreas Eckert. As my primary supervisor, he
supported and shaped the composition of this study with commitment, generos-
ity and inspiration. His trusting, motivating and supportive manner was crucial
to the success of this project and to my subsequent progress as a scholar. My
thanks also go to Gabriele Metzler, who took on the second review of my disser-
tation with enthusiastic interest. The suggestions made by both these scholars
were particularly valuable as I reworked my initial study. I also owe a debt of
gratitude to Alexander Nützenadel, who suggested that the German book be pub-
lished in the “Kritische Studien zur Geschichtswissenschaft” series. I am indebt-
ed to the editors for including this work in their programme. Their constructive
suggestions in the reviews and the stimulating support of Dieter Gosewinkel
were of much benefit to the final German-language text, whose publication
was facilitated by the genial and professional stewardship of Daniel Sander at
Vandenhoeck & Ruprecht. The translation of this work was funded by Geisteswissenschaften Interna-
tional – Translation Funding for Work in the Humanities and Social Sciences
from Germany, a joint initiative of the Fritz Thyssen Foundation, the German Fed-
eral Foreign Office, the collecting society VG WORT and the Börsenverein des
Deutschen Buchhandels (German Publishers & Booksellers Association). I’m es-
pecially thankful for the jury’s recognition in awarding my book the Special Prize
for an outstanding work in the humanities.Wilma Kähmer at Vandenhoeck & Ru-
precht was of great help when it came to applying for financial support, while
Anke Simon at the Börsenverein des Deutschen Buchhandels made the funding
process a smooth one. OpenAccess. © 2021 Daniel Tödt, published by De Gruyter.
This work is licensed under the
Creative Commons Attribution 4.0 International License. https://doi.org/10.1515/9783110709308-011 Acknowledgments I am grateful to Joël Glasman and the editors of the Africa
in Global History series for including my text in their ambitious and exciting pub-
lishing programme. The anonymous reviewer provided constructive advice that
helped me put the finishing touches to the manuscript. Rabea Rittgerodt at De 370 Acknowledgments Gruyter supported the translation project with tremendous competence, engage-
ment and an unfailingly positive attitude. The fact that I could recognize my own
language in the English version is down to Alex Skinner, who produced the
translation with great professionalism, sensitivity, conscientiousness and elo-
quence. The dissertation on which the book is based goes back to my participation in
Collaborative Research Centre 640, “Changing Representations of Social Order. Intercultural and Intertemporal Comparisons”, at Humboldt University of Berlin. Its spokesperson Jörg Baberowski, along with Matthias Braun, Xenia Krüger and
all the other project participants created an outstanding research environment. Regular visits to the cafeteria with colleagues were an indispensable source of
support, the shared office time and daily exchanges with Regina Finsterhölzl a
welcome stroke of luck. Michael Pesek and Susann Baller provided important
stimulus for my study at an early stage. The student assistants on the “African
Modernity” sub-project – Sascha Dannenberg, Josephine Friedrich, Lotte Knote
and Anika Stegeman – supported me in a variety of ways. At the Center for Met-
ropolitan Studies at the Technical University of Berlin I was fortunate to be able
to complete the German book manuscript in the pleasant and supportive setting
provided by Dorothee Brantz as well as Elisabeth Asche, Noa Ha and Frederieke
Westermann. When revising the English version, I benefited from the active sup-
port of Felicitas Hentschke at the International Research Center “Work and
Human Lifecycle in Global History” (re: work), who provided a quiet working en-
vironment, as well as Daniela Greca and Zeynep Ecem Pulas, who helped me
with the acquisition of literature. It was my key supporters during my undergraduate and master’s studies,
Stefan Krankenhagen, Rolf Lindner and Silke Strickrodt, who encouraged me
to set forth on the doctoral adventure in the first place. I was privileged to pre-
sent my research project at many workshops, conferences and colloquia, which
provided me with valuable criticism, praise and suggestions. Key decisions along
the way were informed by discussions with Dmitri van den Bersselaar, Frederick
Cooper, Manfred Hettling, Nancy Rose Hunt, Peter Lambertz, Pedro Monaville
and Thomas Mergel. Acknowledgments Joël Glasman supported the project from start to finish
with equal parts enthusiasm and critical input. Through his in-depth reading
at various points in time, Lasse Heerten helped ensure that I paid sufficient at-
tention to global perspectives. Manfred Schmidt and Söhnke Vosgerau read the
entire manuscript carefully and made insightful comments; the latter also proof-
read my doctoral dissertation. Individual chapters were amended in light of com-
ments by Felix Fuhg, Patrick Hege, Lotte Knote, Gesa Trojan, Marlène de Saus-
sure and Hannah Schilling. Mike Sigl updated the index for the English
version of the book. Christoph Kalter and Marc Buggeln were kind enough to Acknowledgments Acknowledgments 371 give me valuable advice on applying for funding from Geisteswissenschaften In-
ternational. The history of the Congo and of Belgium was uncharted territory for me. Many people helped me get my bearings, supported me with useful critique dur-
ing the research process and made my stay in Kinshasa an unforgettable expe-
rience. Some of them, I am delighted to say, have become colleagues and friends. Kwa Kongo Benz, Saint José Camille Inaka, Peter Lambertz, Sapin Makengele,
Cécile Michel, Pedro Monaville, Katrien Pype and Léon Tsambu welcomed me
with open arms and excellent contacts. Sebastien Khonde and the residents
and employees of the Centre d’Information et d’Animation Missionnaire in Limete
made me feel at home and at ease. Filip De Boeck, Susanne Gehrmann, Charles
Didier Gondola, Hermelinde Lanza, Antoine Lumenganeso, Jean-Marie Muta-
mba-Makombo, Jacob Sabakinu, Dominic Thomas and Charles Tshimanga pro-
vided me with valuable leads. The individuals I interviewed gave up a good
deal of time for me and showed great openness and hospitality. When I was try-
ing to find interview partners who already lived in colonial times, Léon Tsambu
took me to the studios of the TV show Sentiments Lipopo, in which more or less
older inhabitants of Kinshasa dance to rumba songs. I hadn’t realized I would be
live on air. I am grateful to the participants in the programme for their kindly
forbearance in view of my clumsy attempts at rumba. My ineptitude may well
have prompted the presenter, Jean de la Croix Mobé, to take pity on me and gen-
erously allow me access to his father’s private archive. Johan Lagae, Amandine
Lauro, Guy Vanthemsche and Jean-Luc Vellut provided me with always instruc-
tive exchanges. Acknowledgments The staff of the archives and libraries I visited were helpful and patient:
Pierre Dandoy and Alain Gérard at the Archives Africaines in Brussels; Lore
Van de Broeck, Sabine Cornelis, Jean Omasombo Tshonda and Anne Welschen
at the Musée Royal de l’Afrique Centrale; Jean-Claude Yoka at the Archives natio-
nales d’outre-mer in Aix-en-Provence; and Tinne Billet of the Société Multimédia
des Auteurs des Arts Visuels (SOFAM). In addition, the staff of the KADOC in
Leuven, the Bontinck Library, the Assanef Archive and the Archives Nationales
de la République Démocratique du Congo helped ensure the success of my re-
search, while Nura Treves facilitated the perusal of several documents. I would like to express my gratitude to the Geschwister Boehringer Ingelheim
Stiftung für Geisteswissenschaften in Ingelheim am Rhein and the Center for Met-
ropolitan Studies at the Technical University of Berlin for helping cover the print-
ing costs of the German edition of the book. My thanks also to the IGK “Work and
Human Lifecycle in Global History” (re: work) for covering the costs of proofread-
ing and the insertion of illustrations. Open Access was kindly made possible by
the Open Access Publication Fund of Humboldt-Universität zu Berlin and by the 372 72
Acknowledgments Translation Prize of the ZEIT-Stiftung Ebelin und Gerd Bucerius awarded by the
German Historical Association (VHD). Finally, I would like to thank my parents, who aroused my curiosity and
wanderlust early on and have always been there for me with their love and
faith in my abilities. I am grateful to my friends, who were of great help during
the hectic period of research culminating in this book. No one is more closely
connected with this project than my wife Farah. Every line in the book bears
the imprint of her encouragement, patience and love. As full time-working pa-
rents with two young children, we are thankful for a great care network, includ-
ing the team at the Kitaquarium nursery and the parents who co-founded and
co-manage it. We are also grateful to Elza Hamoniaux, a fantastic babysitter,
and to my mother whose visits facilitated both travel and writing retreats. It
must be a disappointment to Noam and Elias that this largely picture-free vol-
ume makes for a poor bedtime story. Acknowledgments I wrote the final pages of my dissertation
shortly after Noam was born, and she was by my side in her pram when I defend-
ed my thesis. Elias was born during the production of the translation, and he has
accompanied me ever since as my second lucky charm. I am deeply grateful to
them for reminding me every day that the present and future are more important
than the past. The book is dedicated to Hesdy Olieberg, a close friend. After meeting some
25 years ago on a basketball court on Fuerteventura, he broadened my perspec-
tive on the Black Atlantic and always provided a safe harbour in Amsterdam as I
travelled around the world. He was my role model until his very last breath.
|
https://openalex.org/W4384132315
|
https://www.qeios.com/read/LLRAAG/pdf
|
English
| null |
Review of: "Identifying Psychological Distress Patterns during the COVID-19 Pandemic using an Intersectional Lens"
| null | 2,023
|
cc-by
| 355
|
Qeios, CC-BY 4.0 · Review, July 13, 2023 Qeios ID: LLRAAG · https://doi.org/10.32388/LLRAAG Review of: "Identifying Psychological Distress Patterns
during the COVID-19 Pandemic using an Intersectional
Lens" Panagiotis Pelekasis Potential competing interests: No potential competing interests to declare. Potential competing interests: No potential competing interests to declare. 1) The abstract is not very clear. By reading the results it is not clear which differences were noted. For example, it is
not clear weather males or females had higher distress. 1) The abstract is not very clear. By reading the results it is not clear which differences were noted. For example, it is
not clear weather males or females had higher distress. 2) Psychological distress was not assessed through a single instrument, as it should have, such as the HADS. Instead,
several questions from different instruments were used. This instrument was developed for the study purpose and was not
validated, which consists a major weakness of the study. 2) Psychological distress was not assessed through a single instrument, as it should have, such as the HADS. Instead,
several questions from different instruments were used. This instrument was developed for the study purpose and was not
validated, which consists a major weakness of the study. 3) The study has no other major weaknesses. Yet, it investigates a matter that is currently of low interest, since the
pandemic has ended. It would be a good and interested paper if published earlier. It is obvious by the absence of
references from 2022 and 2023 that the paper was written in the previous years, but for some reason not published. I
believe that currently this paper is unfortunately of low interest. 3) The study has no other major weaknesses. Yet, it investigates a matter that is currently of low interest, since the
pandemic has ended. It would be a good and interested paper if published earlier. It is obvious by the absence of
references from 2022 and 2023 that the paper was written in the previous years, but for some reason not published. I
believe that currently this paper is unfortunately of low interest. Qeios ID: LLRAAG · https://doi.org/10.32388/LLRAAG 1/1
|
https://openalex.org/W4246690928
|
https://www.researchsquare.com/article/rs-154602/latest.pdf
|
English
| null |
How Flushable Wet Wipes Are Causing Sewer Blockages – and An Approach to Prevent That
|
Research Square (Research Square)
| 2,021
|
cc-by
| 150
|
Keywords: 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 Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Scientific Reports on April 12th, 2021. See
the published version at https://doi.org/10.1038/s41598-021-86971-z. Abstract The authors have requested that this preprint be withdrawn due to erroneous posting. The authors have requested that this preprint be withdrawn due to erroneous posting. EDITORIAL NOTE: The full text of this preprint has been withdrawn by the authors while they make corrections to the
work. Therefore, the authors do not wish this work to be cited as a reference. Questions should be
directed to the corresponding author. Page 1/2 Page 1/2 Full Text The authors have withdrawn this preprint from Research Square. Page 2/2
|
https://openalex.org/W2072736278
|
https://www.scielo.br/j/rbso/a/HmwsjFbxy9rWK8RFknchZGL/?lang=pt&format=pdf
|
Portuguese
| null |
Exposição a agentes químicos e a Saúde do Trabalhador
|
Revista Brasileira de Saúde Ocupacional
| 2,007
|
cc-by
| 3,290
|
Exposição a agentes químicos e a Saúde do Trabalhador
Exposure to chemicals and the Workers’ Health Mina Kato1
Eduardo Garcia Garcia2
Victor Wünsch Filho3 Mina Kato1
Eduardo Garcia Garcia2
Victor Wünsch Filho3 1 Editora associada
2 Editor executivo
3 Editor convidado e membro do
Conselho Editorial A relevância do tema deste volume da RBSO é amplamente reconhecida. A
Organização Internacional do Trabalho (OIT) estima em 35 milhões anuais os
casos de doenças relacionadas ao trabalho por exposição a substâncias químicas
com a ocorrência de 439.000 mortes, incluindo, entre outras causas relaciona-
das, 36.000 óbitos por pneumoconioses, 35.500 óbitos por doenças respira-
tórias crônicas, 30.700 óbitos por doenças cardiovasculares e 315.000 óbitos
por câncer (ILO, 2004). A Organização Mundial da Saúde, por sua vez, estima
que esses cânceres provoquem uma perda anual de 1,4 milhão de anos de vida
saudável e que as intoxicações agudas por produtos químicos sejam responsá-
veis por outros 7,5 milhões de anos de vida saudável perdidos pela população
mundial (WHO, 2002). Certamente, há muito por conhecer, discutir e fazer
a respeito da origem, do controle e da prevenção desses problemas, que não
são recentes, embora estejam ganhando amplitude pela inclusão constante de
novas substâncias no mercado e pelo aumento contínuo de seu uso. Neste nú-
mero, são apresentados cinco artigos, um ensaio e duas resenhas relacionados
ao tema. No primeiro dos artigos, intitulado Características da exposição ocupacio-
nal a poeiras em marmorarias da cidade de São Paulo, são reportadas altas
concentrações de poeira originadas pelas ferramentas utilizadas no setor de
acabamento a seco. As concentrações de sílica chegaram a ser muitas vezes
superiores ao valor do limite de exposição ocupacional recomendado, mas foi
apontada a possibilidade de se reduzir significativamente as concentrações
ambientais com a adoção de um processo de acabamento a úmido, uma medi-
da de proteção coletiva dos trabalhadores. Uma das conseqüências possíveis da exposição a poeiras contendo sílica
é uma pneumoconiose – a silicose. As pneumoconioses podem ser causadas
por outros compostos de origem mineral (asbestos, carvão, berílio) ou vegetal
(algodão, sisal). Mesmo conhecidas desde a Antiguidade, ainda acometem os
trabalhadores do século XXI. Em 2006, foram registrados na Previdência So-
cial, através das Comunicações de Acidentes de Trabalho (CATs), 56 casos de
pneumoconiose relacionados a poeiras contendo sílica e 10 pneumoconioses
por outros agentes ou não especificadas (MPAS, 2008). Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 Exposição a agentes químicos e a Saúde do Trabalhador
Exposure to chemicals and the Workers’ Health É importante observar
que os casos registrados na Previdência se referem àqueles trabalhadores que
estão inseridos em meios com conhecimento e acesso ao sistema da Previdên-
cia, não representando a totalidade dos casos incidentes no país. Em seu compêndio sobre as doenças dos trabalhadores (De Morbis Ar-
tificum Diatriba), originalmente publicado em 1700, Ramazzini comentou
sobre a qualidade da poeira a que os mineradores estavam expostos – tão
fina que ultrapassava a bexiga de um boi que estava pendurada na oficina
de um lapidário (RAMAZZINI, 1999). Plínio, “O Velho” (23 a 79 d.C.), um
estudioso romano, já relatava o uso de bexigas de animais como uma tenta-
tiva de se proteger contra inalação de poeiras e fumos de chumbo (OSHA, 6 6 Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 2007; RAMAZZINI, 1999), demonstrando a preocupação em impedir ou reduzir o contato do trabalhador com as
substâncias químicas por meio de proteção individual. Portanto, a noção de proteção pelo uso de equipamentos
de proteção individual (EPIs) não é nova, mas, mesmo atualmente, pode ser difícil de obter-se. É o que ilustra o ensaio A contaminação por agrotóxicos e os Equipamentos de Proteção Individual (EPIs). Os
autores reportam que os EPIs, neste caso a vestimenta de proteção na aplicação de agrotóxicos, além de não prote-
gerem integralmente o trabalhador contra o produto químico, ainda agravaram os riscos, pois se tornaram fontes
de contaminação. Discutem, também, a possibilidade dos EPIs apresentarem lacunas funcionais na concepção,
no projeto, no uso, na manutenção, no armazenamento e no descarte. Assim, esses equipamentos deveriam ser
continuamente avaliados por meio de estudos de desenvolvimento tecnológico para melhoria da sua funcionali-
dade e não ocasionarem, paradoxalmente, danos à saúde humana. Os primeiros agrotóxicos utilizados foram os de sais inorgânicos. Ainda se utilizam no mundo compostos
de arsênio, cobre, chumbo e mercúrio, cujos efeitos tóxicos são conhecidos desde a Antiguidade (EPA, 1986;
DECC, 2008). Plantas e seus extratos, como o das folhas de tabaco, foram também utilizados na agricultura para
combater insetos. Mas os agrotóxicos organossintéticos começaram a ser produzidos e utilizados em larga escala
a partir da Segunda Guerra Mundial, com a descoberta da propriedade inseticida do DDT em 1939, apesar de
sintetizado em 1874. O sucesso inicial do DDT foi observado no controle de vetores de doenças transmissíveis,
como malária e tifo epidêmico. Exposição a agentes químicos e a Saúde do Trabalhador
Exposure to chemicals and the Workers’ Health Surgiram, porém, críticas à utilização do inseticida e preocupação com os seus
impactos ao ambiente a partir da identificação de inúmeros casos de intoxicação aguda e do aumento de seu uso
indiscriminado na agricultura, com conseqüente acúmulo de resíduos no ambiente e na cadeia alimentar, assim
como a constatação da ativação de resistência entre os organismos alvo e o risco de câncer em animais, incluindo
o ser humano (D´AMATO et al., 2002). O livro de Rachel Carson, Primavera Silenciosa, de 1962, tornou pública
a preocupação ecológica com o DDT e condenou a liberação de grandes quantidades da substância e seus congê-
neres no ambiente sem o conhecimento integral de seus efeitos nocivos (LEAR, 2002). Os agrotóxicos também estão focalizados no artigo intitulado Incidência de suicídios e uso de agrotóxicos por
trabalhadores rurais em Luz (MG), Brasil. Em 2005, o Sistema Nacional de Informações Tóxico-Farmacológicas
(SINITOX) registrou 84.456 casos de intoxicações em 28 dos 34 Centros de Informação e Assistência Toxicológica
em atividade no país. Desses casos, 15.804 foram intoxicações atribuídas às tentativas de suicídio, dentre os quais
14% aos agrotóxicos de uso agrícola e 11% aos raticidas, superados apenas pelos casos provocados por medica-
mentos (59%). Dos 6.827 casos de intoxicação atribuídos à circunstância ocupacional, 37% foram causados por
animais peçonhentos, 25% por agrotóxicos de uso agrícola e 13% por produtos químicos industriais. Entre os 477
óbitos registrados, os agrotóxicos foram a causa mais freqüente (33%), seguida por medicamentos (18%), raticidas
(11%), drogas de abuso (11%) e animais peçonhentos (9%). O suicídio respondeu por 54% dos óbitos e a faixa
etária produtiva, de 20 a 59 anos, respondeu por 68% (325 casos) do total dos óbitos (SINITOX, 2005). De acordo com o artigo de Minas Gerais, a fácil disponibilidade dos produtos poderia ser uma das razões
para a escolha em caso de suicídios. A necessidade de aprofundamento da discussão sobre o papel da exposição
a esses produtos na indução ao suicídio também se mostrou relevante no estudo. A dificuldade de se usar uma
vestimenta confortável e protetora, seja por questões financeiras ou ergonômicas ou por inadequação do produto,
além do despreparo dos aplicadores de agrotóxicos no que diz respeito ao uso e ao conhecimento dos riscos são
apontados como os determinantes das intoxicações. Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 Exposição a agentes químicos e a Saúde do Trabalhador
Exposure to chemicals and the Workers’ Health No ambiente de trabalho de laboratórios clínicos, são observados também ou-
tros tipos de fatores de risco à saúde – agentes biológicos, posturas inadequadas e movimentos repetitivos, entre
outros. Para este tipo de atividade de prestação de serviços na área de saúde, delineou-se uma área específica de
atuação dentro da Saúde do Trabalhador: a Biossegurança. A Biossegurança, apesar de ter recebido algumas definições que restringem seu campo de ação, é considera-
da, na Saúde do Trabalhador, parte integrante da Segurança e da Higiene do Trabalho, que se preocupa com os
trabalhadores da área de saúde e afins, em cujos ambientes de trabalho estão presentes não somente os fatores de
riscos biológicos, mas outros que podem diretamente agravar a saúde ou podem ser desencadeadores de aciden-
tes biológicos (VIEIRA & LAPA, 2006). Por longo tempo, o modelo da unicausalidade das doenças dominou o pensamento na área da saúde. Essa
forma de pensar dificultava aceitar as doenças multifatoriais ou multicausais e também doenças cujos sintomas
eram inespecíficos e difíceis de serem mensurados objetivamente. O modelo unicausal era reducionista e funcio-
nal para estudos experimentais, mas impossibilitava explicar a complexidade da realidade. Em contrapartida, o
modelo multicausal é complexo e difícil de representar, necessitando-se para tanto utilizar modelos matemáticos
complexos. Atualmente, mesmo com essa concepção sendo hegemônica e com o presente desenvolvimento tec-
nológico, os aspectos legais da previdência social e trabalhista preservam ainda abordagens reducionistas, como,
por exemplo, na procura em estabelecer o nexo causal e o julgamento da insalubridade do ambiente, mesmo
quando há exposição a múltiplos agentes (FRANCO & KATO, 1997; SANTOS et al., 2004). Os resultados do artigo Dados sociodemográficos e condições de trabalho de pintores expostos a solventes em
uma universidade pública da cidade do Rio de Janeiro fazem parte de um estudo mais amplo que avaliou altera-
ções neuropsicológicas que podem estar relacionadas à exposição a solventes orgânicos. Embora os efeitos agudos
dos solventes orgânicos sobre o sistema nervoso central fossem conhecidos há muito tempo, o estabelecimento da
relação causal entre os efeitos neuropsicológicos em trabalhadores expostos a solventes em longo prazo ocorreu
apenas no final do século passado (RAMOS et al., 2004). O artigo mostra que os pintores entrevistados referi-
ram uma fonte de renda paralela, uma questão importante quando se busca traçar a relação entre o ambiente de
trabalho e os efeitos sobre a saúde. Exposição a agentes químicos e a Saúde do Trabalhador
Exposure to chemicals and the Workers’ Health Uma vez que um acidente de grande porte não pode
ser reproduzido ou testado experimentalmente, os dados do estudo e a análise de risco realizada são elementos
preciosos para fundamentar medidas de emergência e de prevenção de ocorrências futuras. Ainda, permitem o
planejamento de políticas públicas com o objetivo de controlar fatores desencadeadores deste tipo de acidente. O desenvolvimento e a aplicação de modelos matemáticos e de novos softwares para a análise de riscos favo-
recem a idéia de que as avaliações dos fatores de risco devem ser quantitativas – as concentrações de poeiras no
ar ambiente, o nível de contaminação do solo por agrotóxicos, a incidência de radiação, o calor nos ambientes de
trabalho etc. Porém, para realizar uma avaliação quantitativa que represente as condições que se deseja estudar
é necessário realizar inicialmente uma boa avaliação qualitativa. Até mesmo para ponderar a necessidade das
mensurações, a avaliação qualitativa é imprescindível (PUGAS et al., 2001). O manual Avaliação qualitativa de riscos químicos: princípios básicos para o controle das substâncias nocivas
à saúde em fundições, apresentado por resenha neste volume, é um instrumento colocado à disposição dos que
atuam na área da Saúde do Trabalhador em pequenas e médias empresas. O texto aborda as fundições, mas deriva
de um sistema de gerenciamento de risco desenvolvido e padronizado pelo Programa sobre Segurança e Saúde no
Trabalho e no Meio Ambiente (SafeWork) da Organização Internacional do Trabalho, a partir do Health and Safety
Executive (HSE). O programa, implementado desde 1998, no Reino Unido, tem o objetivo de apoiar esforços na
prevenção de exposições a agentes de risco ocupacional por meio de tomada de decisões baseadas em análise
qualitativa, sem necessidade de utilizar sempre avaliações quantitativas (PAPP, EIJKEMANS & VICKERS, 2004). Obviamente haverá situações em que avaliações quantitativas do ambiente serão indispensáveis ou desejáveis,
porém, o custo e a complexidade dessas avaliações podem dificultar a sua realização. O artigo Avaliação dos riscos associados ao uso do xilol em laboratórios de anatomia patológica e citologia con-
tém uma avaliação baseada nas informações dos trabalhadores. Na conclusão, os autores enfatizam a necessidade
de educar o trabalhador com base numa visão prevencionista em relação aos riscos ocupacionais e ambientais
provenientes do uso daquele agente químico. Provavelmente, o desconhecimento dos trabalhadores não se res-
tringe apenas ao agente químico. Exposição a agentes químicos e a Saúde do Trabalhador
Exposure to chemicals and the Workers’ Health Mas certamente as causas vão além, passando pelo modelo
de produção agrícola adotado, pelas estratégias de difusão dessa tecnologia (sem evidenciar os riscos à saúde e ao
ambiente e, ainda, sem considerar o despreparo do usuário e a ausência de recursos para o controle), pela indu-
ção ao uso excessivo por vendedores e propagandas, pelas relações e condições precárias de trabalho, além dos
determinantes socioeconômicos predominantes no meio rural, como a baixa escolaridade, moradias inadequadas
e difícil acesso à atenção à saúde (GARCIA, 2001). Reconhecendo a complexidade da questão da exposição a agentes químicos, na segunda metade do século
passado a área da Saúde do Trabalhador passou a operar com abordagens multi e interdisciplinares para solução
de problemas, preconizando que a substituição, ou a modificação do processo, ou a intervenção na fonte do risco
(equipamentos de proteção coletiva – EPCs) são mais eficazes que os EPIs, que estabelecem barreiras de exposição
sem redução da emissão (SANTOS et al., 2004). A alteração no processo de produção com a introdução da umi-
dificação descrita no artigo Características da exposição ocupacional a poeiras em marmorarias da cidade de São
Paulo é um exemplo. Aliás, Ramazzini, há mais de trezentos anos, já relatava a adoção de EPC em minas subter-
râneas – um sistema de ventilação exaustora – quando descreveu a doença dos mineiros (RAMAZZINI, 1999). No universo da Saúde do Trabalhador, entende-se como fonte de emissão um equipamento ou uma situação
dentro do processo de produção no local onde o trabalhador executa suas atividades, geralmente demarcada pelos
muros das plantas industriais. Para a área ambiental, acrescem-se como fontes geradoras de agentes poluidores os
produtos finais e secundários e os resíduos gerados no processo. Assim, tanto o trabalhador quanto a população
geral sofrem os efeitos do processo de produção num ambiente transformado pela produção no entorno da fonte 7 Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 ou como consumidores dos produtos gerados nesse processo. Portanto, as questões do ambiente saudável estão
intimamente ligadas às da Saúde do Trabalhador. No artigo Acidente químico com dióxido de enxofre em um populoso distrito de uma grande metrópole, os
autores estudaram um acidente ambiental a partir de um modelo matemático de dispersão e propuseram alguns
cenários possíveis de exposição da população do entorno. Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 Exposição a agentes químicos e a Saúde do Trabalhador
Exposure to chemicals and the Workers’ Health O pintor não estaria exposto somente às condições avaliadas, também deve
ter sido exposto em seus outros ambientes de trabalho, que podem ser ainda mais precários por serem trabalhos
temporários e sem normas contratuais que incluam questões de proteção à saúde dos trabalhadores. Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 8 As investigações das causas que levam o trabalhador a assumir uma segunda atividade, transpõe o âmbito dos
fatores de risco imediatamente ligados ao ambiente de trabalho e passa-se para o nível de fatores sociais e eco-
nômicos do grupo social ao qual esses trabalhadores pertencem. Por meio de um modelo multicausal, é possível
estudar estes fatores em seus diferentes níveis de proximidade em relação ao trabalhador. Para reforçar o fato de que a complexidade da Saúde do Trabalhador é inerente à realidade do ser humano
e que, para a compreensão e gestão do risco ocupacional de forma integral, somente a avaliação da exposição a
um determinado agente químico não é suficiente, este volume da RBSO inclui a resenha do livro A desmedida
do capital, da socióloga francesa Danièle Linhart. A coletânea é formada por diversos artigos que versam sobre
as transformações na organização e na gestão do trabalho na empresa moderna, analisando os modelos organi-
zacionais na França nos últimos 40 anos. A autora da resenha remete-nos à realidade brasileira, questionando os
paralelos desta com a experiência francesa analisada. O conjunto dos trabalhos aqui apresentados traz à tona um questionamento fundamental: o que realmente
se alcançou na Saúde do Trabalhador no tocante à exposição a agentes químicos? A comunicação dos riscos e
de medidas de proteção, já levantada como necessidade por Ramazzini, ainda não atinge todos os trabalhadores. Ainda se confere um valor excessivo às medidas individuais de proteção, que não condizem com o seu papel
efetivo na garantia da proteção integral à saúde do trabalhador. Por outro lado, as medidas de proteção coletiva,
algumas já sugeridas e utilizadas há mais de trezentos anos, ainda são recebidas com relutância por parte daque-
les que podem modificar o processo de produção. Os trabalhadores ainda executam suas atividades em condições
precárias, não apenas no que diz respeito à segurança e à higiene do trabalho, mas também no que tange às ga-
rantias de um trabalho digno e decente. Convidamos a todos a refletir sobre esses aspectos com a leitura dos textos aqui publicados. Referências D’AMATO, C.; TORRES, J. P. M.; MALM, O. DDT (dicloro difenil tricloroetano): toxicidade e contaminação
ambiental - uma revisão. Quím. Nova, São Paulo, v. 25, n. 6, p. 995-1002, 2002. DECC. Department of Environment and Climate Change. New South Wales Government. Contaminated
Agricultural Land. Australia, 2008. Available from: http://www.environment.nsw.gov.au/mao/
contaminatedagriland.htm. Access: 06 mar. 2008. EPA. Environmental Protection Agency. Lead arsenate EPA Pesticide Fact Sheet 12/86. EPA, 1986. Available
from: http://pmep.cce.cornell.edu/profiles/insect-mite/fenitrothion-methylpara/lead-arsenate/insect-prof-leadars. html. Access: 06 mar. 2008. FRANCO, T.; KATO, M. Os riscos ambientais e os indivíduos. In: FRANCO T. (org.) Trabalho, riscos industriais e
meio ambiente: rumo ao desenvolvimento sustentável? Salvador: EDUFBA – CRh FFCH UFBA, 1997. p. 215-242. GARCIA, E. G. Segurança e saúde no trabalho rural: a questão dos agrotóxicos. São Paulo: Fundac ILO. International Labor Office. Safe work and safety culture. The ILO report for word day for safety and health
at work 2004. ILO, 2004. Available from: http://www.ilo.org/public/english/protection/safework/worldday/
products04/report04_eng.pdf . Access: 07 abr. 2008. LEAR, L. Introduction. In: CARSON, R. Silent Spring. New York: Mariner Books / Houghton Mifflin Co., 2002
p. X-XIX. MPAS. Ministério da Previdência Social. Informações estatísticas de acidentes do trabalho – Bases de dados do
Anuário Estatístico de Acidentes de Trabalho, edição 2008 – Disponível em: http://creme.dataprev.gov.br/AEAT/
Inicio.htm. Acesso em: 06 mar. 2008. OSHA. Occupational Safety & Health Administration. Office of Training and Education - Industrial Hygiene. Available from: http://www.osha.gov/dcsp/ote/library/industrial_hygiene/industrial_hygiene.html. Access: 06
mar. 2008. PAPP, E. M.; EIJKEMANS, G.; VICKERS C. Reducing worker exposure by using the occupational risk
management toolbox. GOHNET News letter, n. 7, 1-3, Summer, 2004. Available from: http://www.who.int/
occupational_health/publications/newsletter/gohnet7e.pdf. Access: 20 mar. 2008. PUGAS, E. et al. Risco ocupacional: Caracterização básica, avaliação qualitativa e priorização. Rev. bras. Saúde
ocup., São Paulo, v. 26, n. 99/100, p. 9-30, 2001. RAMAZZINI, B. As doenças dos trabalhadores. Trad. Raimundo Estrela. 2.ed. São Paulo: Fundacentro, 1999. 9 Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 RAMOS, A.; JARDIM, S. R.; SILVA FILHO, J. F. Solvent-related chronic toxic encephalopathy as a target in the
worker’s mental health research. An. Acad. Bras. Ciênc., v. 76, n. 4, p.757-769, 2004. SANTOS, A. M. A. et al. Introdução à Higiene Ocupacional. São Paulo: Fundacentro, 2004. SINITOX. Sistema Nacional de Informações Tóxico-Farmacologicas. Ministério da Saúde. Casos registrados
de intoxicação humana e envenenamento. Brasil, 2005. Uma análise. Disponível em: http://www.fiocruz.br/
sinitox/2005/umanalise2005.htm. Acesso em: 06 mar. 2008. VIEIRA, V. M.; LAPA, R. Riscos em laboratório: prevenção e controle. Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 Referências Cadernos de Estudos Avançados, Rio de
Janeiro, v. 3, n. 1, p. 25-43, 2006. WHO. Word Health Organization. The world health report 2002. Reducing risks, promoting health WHO. Word Health Organization. The world health report 2002. Reducing risks, promoting healthy life. WHO,
2002. Available from: http://www.who.int/whr/2002/en/. Access: 07 abr. 2008. 2002. Available from: http://www.who.int/whr/2002/en/. Access: 07 abr. 2008. 10 10
|
https://openalex.org/W4379107617
|
https://egusphere.copernicus.org/preprints/2023/egusphere-2023-742/egusphere-2023-742.pdf
|
English
| null |
Comment on egusphere-2023-742
| null | 2,023
|
cc-by
| 28,122
| ERROR: type should be string, got "https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Correspondence: Maurus Borne (maurus.borne@kit.edu) Correspondence: Maurus Borne (maurus.borne@kit.edu) Abstract. Since its launch by the European Space Agency in 2018, the Aeolus satellite has been using the first Doppler\nwind lidar in space to acquire three-dimensional atmospheric wind profiles around the globe. Especially in the tropics, these\nmeasurements compensate for the currently limited number of other wind observations, making an assessment of the quality of\nAeolus wind products in this region crucial for numerical weather prediction. To evaluate the quality of the Aeolus L2B wind products across the tropical Atlantic Ocean, 20 radiosondes corresponding to Aeolus overpasses were launched from the islands\n5\nof Sal, Saint Croix and Puerto Rico during August-September 2021 as part of the Joint Aeolus Tropical Atlantic Campaign. During this period, Aeolus sampled winds within a complex environment with a variety of cloud types in the vicinity of the\nInter-tropical Convergence Zone and aerosol particles from Saharan dust outbreaks. On average, the validation for Aeolus\nRaleigh-clear revealed a random error of 3.8 – 4.3 ms–1 between 2–16 km and 4.3 – 4.8 ms–1 between 16–20 km, with a 5 systematic error of -0.5±0.2 ms–1. For Mie-cloudy, the random error between 2–16 km is 1.1 – 2.3 ms–1 and the systematic\n10\nerror is -0.9 ±0.3 ms–1. Below clouds or within dust layers, the quality of Rayleigh-clear measurements can be degraded when\nthe useful signal is reduced. In these conditions, we also noticed an underestimation of the L2B estimated error. Gross outliers\nwhich we define with large deviations from the radiosonde but low error estimates account for less than 5% of the data. These\noutliers appear at all altitudes and under all environmental conditions; however, their root-cause remains unknown. Finally, systematic error of -0.5±0.2 ms–1. For Mie-cloudy, the random error between 2–16 km is 1.1 – 2.3 ms–1 and the systematic\n10\nerror is -0.9 ±0.3 ms–1. Below clouds or within dust layers, the quality of Rayleigh-clear measurements can be degraded when\nthe useful signal is reduced. In these conditions, we also noticed an underestimation of the L2B estimated error. Gross outliers\nwhich we define with large deviations from the radiosonde but low error estimates account for less than 5% of the data. These\noutliers appear at all altitudes and under all environmental conditions; however, their root-cause remains unknown. Validation of Aeolus L2B products over the tropical Atlantic using\nradiosondes Maurus Borne 1, Peter Knippertz 1, Martin Weissmann 2, Benjamin Witschas 3, Cyrille Flamant 4,\nRosimar Rios-Berrios 5, and Peter Veals 6 1Institute of Meteorology and Climate Research, Karlsruhe Institute of Technology (KIT), 76131 Karlsruhe, Germany\n2Universität Wien, Institut für Meteorologie und Geophysik, Wien, Austria g\np y\n3Deutsches Zentrum für Luft- und Raumfahrt e.V. (DLR), Institut für Physik der Atmosphäre, 82234 Oberpfaffenhofen,\nGermany\n4 3Deutsches Zentrum für Luft- und Raumfahrt e.V. (DLR), Institut für Physik der Atmosphäre, 82234 Oberpfaffenhofen,\nGermany 4Laboratoire Atmosphères, Milieux, Observations Spatiales (LATMOS), UMR 8190, CNRS, Sorbonne Université and\nUniversité Paris Saclay, Paris, France\n5N i\nl C\nf\nA\nh i R\nh B\nld\nCO USA 4Laboratoire Atmosphères, Milieux, Observations Spatiales (LATMOS), UMR 8190, CNRS, Sorbonne Université and\nUniversité Paris Saclay, Paris, France\n5 5National Center for Atmospheric Research, Boulder, CO, USA 5National Center for Atmospheric Research, Boulder, CO, USA\n6 p\n6Department of Atmospheric Sciences, University of Utah, Salt Lake City, Utah, USA 6Department of Atmospheric Sciences, University of Utah, Salt Lake City, Utah, USA https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 1\nIntroduction In situ measurements derived from aircraft reports, ground stations or radiosondes\nare not globally distributed and lead to a lack of observations in the aforementioned regions. To address these deficiencies, the European Space Agency (ESA) deployed the Atmospheric Dynamics Mission Aeolus in To address these deficiencies, the European Space Agency (ESA) deployed the Atmospheric Dynamics Mission Aeolus in\n2018, the first satellite capable of measuring atmospheric winds around the globe from space with a homogeneous space-time\n30\nwind coverage and altitude-resolved profiles up to 30 km height (Reitebuch, 2012). The instrument carries a direct detection\nDoppler wind lidar called ALADIN (Atmospheric LAser Doppler INstrument) that emits short ultraviolet (UV) pulses at 355\nnm along the Line Of Sight (LOS) of the instrument. The Doppler shift of the backscatter signal is detected by a dual-channel\nreceiver consisting of the following elements: a Fizeau interferometer analysing the Doppler shift of the narrowband particle 2018, the first satellite capable of measuring atmospheric winds around the globe from space with a homogeneous space-time\n30\nwind coverage and altitude-resolved profiles up to 30 km height (Reitebuch, 2012). The instrument carries a direct detection\nDoppler wind lidar called ALADIN (Atmospheric LAser Doppler INstrument) that emits short ultraviolet (UV) pulses at 355\nnm along the Line Of Sight (LOS) of the instrument. The Doppler shift of the backscatter signal is detected by a dual-channel\nreceiver consisting of the following elements: a Fizeau interferometer analysing the Doppler shift of the narrowband particle backscatter signal (cloud droplets and aerosols or ice crystals) using the fringe imaging technique (McKay, 2002), referred to\n35\nas the Mie channel, and a dual Fabry-Pérot interferometer detecting the Doppler-shifted frequency of the Rayleigh-Brillouin\nbackscatter spectrum (air molecules) using the double-edge technique (Flesia and Korb, 1999), called the Rayleigh channel. The processing algorithm also distinguishes between retrievals originating from \"cloudy\" or \"clear\" atmospheric conditions,\nresulting in Rayleigh-clear and Mie-cloudy observation types. The two channels complement each other, as Mie-cloudy winds backscatter signal (cloud droplets and aerosols or ice crystals) using the fringe imaging technique (McKay, 2002), referred to\n35\nas the Mie channel, and a dual Fabry-Pérot interferometer detecting the Doppler-shifted frequency of the Rayleigh-Brillouin\nbackscatter spectrum (air molecules) using the double-edge technique (Flesia and Korb, 1999), called the Rayleigh channel. The processing algorithm also distinguishes between retrievals originating from \"cloudy\" or \"clear\" atmospheric conditions,\nresulting in Rayleigh-clear and Mie-cloudy observation types. Correspondence: Maurus Borne (maurus.borne@kit.edu) Finally, we confirm the presence of an orbital-dependent bias of up to 2.5 ms–1 observed with both radiosondes and European Centre\n15\nfor Medium-Range Weather Forecasts model equivalents. The results of this study contribute to a better characterization of\nthe Aeolus wind product in different atmospheric conditions and provide valuable information for further improvement of the\nwind retrieval algorithm. we confirm the presence of an orbital-dependent bias of up to 2.5 ms–1 observed with both radiosondes and European Centre\n15\nfor Medium-Range Weather Forecasts model equivalents. The results of this study contribute to a better characterization of\nthe Aeolus wind product in different atmospheric conditions and provide valuable information for further improvement of the\nwind retrieval algorithm. 1 1 1\nIntroduction According to the World Meteorological Organisation (WMO), wind is the most critical atmospheric variable lacking in the\n20\ncurrent Global Observing System (GOS) (Baker et al., 2014). Especially in the Southern Hemisphere (SH), over the oceans\nand near equatorial regions, numerical weather prediction (NWP) models require additional wind observations with sufficient\ncoverage in time and space to identify key atmospheric dynamics (Stoffelen et al., 2005; Straume et al., 2020). Before the\nlaunch of Aeolus in 2018, satellite wind observations in these regions were only available for a limited number of tropospheric According to the World Meteorological Organisation (WMO), wind is the most critical atmospheric variable lacking in the\n20\ncurrent Global Observing System (GOS) (Baker et al., 2014). Especially in the Southern Hemisphere (SH), over the oceans\nand near equatorial regions, numerical weather prediction (NWP) models require additional wind observations with sufficient\ncoverage in time and space to identify key atmospheric dynamics (Stoffelen et al., 2005; Straume et al., 2020). Before the\nlaunch of Aeolus in 2018, satellite wind observations in these regions were only available for a limited number of tropospheric layers and were mainly provided by atmospheric motion vectors (AMVs) estimated from tracking cloud and water vapour\n25\nfeatures (Bormann et al., 2003; Folger and Weissmann, 2014), or by scatterometer measurements of surface winds (Naderi\net al., 1991; Portabella and Stoffelen, 2009). In situ measurements derived from aircraft reports, ground stations or radiosondes\nare not globally distributed and lead to a lack of observations in the aforementioned regions. dd\nh\nd fi i\ni\nh\nd\nl\nd h\nh i\ni\ni i\nl\ni layers and were mainly provided by atmospheric motion vectors (AMVs) estimated from tracking cloud and water vapour\n25\nfeatures (Bormann et al., 2003; Folger and Weissmann, 2014), or by scatterometer measurements of surface winds (Naderi\net al., 1991; Portabella and Stoffelen, 2009). In situ measurements derived from aircraft reports, ground stations or radiosondes\nare not globally distributed and lead to a lack of observations in the aforementioned regions. To address these deficiencies the European Space Agency (ESA) deployed the Atmospheric Dynamics Mission Aeolus in layers and were mainly provided by atmospheric motion vectors (AMVs) estimated from tracking cloud and water vapour\n25\nfeatures (Bormann et al., 2003; Folger and Weissmann, 2014), or by scatterometer measurements of surface winds (Naderi\net al., 1991; Portabella and Stoffelen, 2009). 1\nIntroduction The two channels complement each other, as Mie-cloudy winds can compensate for gaps in Rayleigh-clear measurements, especially in cloudy and aerosol-loaded regions. Various NWP\n40\ncentres have demonstrated the added value of assimilating Aeolus winds through significant improvements in model fields\nand model background information, especially in tropical regions, the upper tropical troposphere and the lower stratosphere\n(Rennie et al., 2021; Martin et al., 2022a, b; Garrett et al., 2022). can compensate for gaps in Rayleigh-clear measurements, especially in cloudy and aerosol-loaded regions. Various NWP\n40\ncentres have demonstrated the added value of assimilating Aeolus winds through significant improvements in model fields\nand model background information, especially in tropical regions, the upper tropical troposphere and the lower stratosphere\n(Rennie et al., 2021; Martin et al., 2022a, b; Garrett et al., 2022). For an optimal use of the Aeolus wind observations in NWP models, an assessment of the data quality is essential. To achieve\nthis, several scientific and technical studies are carried out in the framework of Calibration/Validation (Cal/Val) activities\n45\norganised by ESA. For wind validation, several reference products have been used such as ground-based remote sensing\nobservations (Belova et al., 2021; Guo et al., 2021; Iwai et al., 2021; Abril-Gago et al., 2022), in situ measurements (Baars\net al., 2020; Chen et al., 2021; Ratynski et al., 2022), airborne measurements (Lux et al., 2020; Witschas et al., 2020; Bedka\net al., 2021; Witschas et al., 2022) or NWP model equivalents (Martin et al., 2021; Zuo et al., 2022). For an optimal use of the Aeolus wind observations in NWP models, an assessment of the data quality is essential. To achieve\nthis, several scientific and technical studies are carried out in the framework of Calibration/Validation (Cal/Val) activities\n45\norganised by ESA. For wind validation, several reference products have been used such as ground-based remote sensing\nobservations (Belova et al., 2021; Guo et al., 2021; Iwai et al., 2021; Abril-Gago et al., 2022), in situ measurements (Baars\net al., 2020; Chen et al., 2021; Ratynski et al., 2022), airborne measurements (Lux et al., 2020; Witschas et al., 2020; Bedka\net al., 2021; Witschas et al., 2022) or NWP model equivalents (Martin et al., 2021; Zuo et al., 2022). this, several scientific and technical studies are carried out in the framework of Calibration/Validation (Cal/Val) activities\n45\norganised by ESA. The contribution\n70\nof the radiosondes in JATAC is complementary to other instruments as they provide accurate wind measurements throughout\nthe troposphere up to the lower stratosphere, which is not probed by many other instruments and provides an almost unique\ndata set for validating the Aeolus winds at this altitude. This article is structured as follows: Section 2 describes the instruments and data while section 3 details the quality con the warm and moist Caribbean, where heavy rainfall events and tropical cyclones frequently affect the area. The contribution\n70\nof the radiosondes in JATAC is complementary to other instruments as they provide accurate wind measurements throughout\nthe troposphere up to the lower stratosphere, which is not probed by many other instruments and provides an almost unique\ndata set for validating the Aeolus winds at this altitude. This article is structured as follows: Section 2 describes the instruments and data while section 3 details the quality con-\ntrol and co-location criteria used for the validation study. Section 4 deals with the quantification of errors, their dependency\n75\non temporal and spatial distance between the compared observations as well as on the presence of clouds and dust. For this\npurpose, we use the Satellite Application Facility for supporting NoWCasting and very short range forecasting (SAFNWC,\nAlonso Lasheras et al. (2005)) satellite-based meteorological Cloud Type (CT) product and the Copernicus Atmosphere Mon-\nitoring Service (CAMS) dust mixing ratio reanalysis. Furthermore, the section includes a case study illustrating the different\nbehaviour of Rayleigh-clear and Mie-cloudy winds under different environmental conditions Finally, section 5 summarises\n80 trol and co-location criteria used for the validation study. Section 4 deals with the quantification of errors, their dependency\n75\non temporal and spatial distance between the compared observations as well as on the presence of clouds and dust. For this\npurpose, we use the Satellite Application Facility for supporting NoWCasting and very short range forecasting (SAFNWC,\nAlonso Lasheras et al. (2005)) satellite-based meteorological Cloud Type (CT) product and the Copernicus Atmosphere Mon-\nitoring Service (CAMS) dust mixing ratio reanalysis. Furthermore, the section includes a case study illustrating the different behaviour of Rayleigh-clear and Mie-cloudy winds under different environmental conditions. Finally, section 5 summarises\n80\nthe main results and provides recommendations for improving the Aeolus wind retrieval algorithm. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. lies\" (Weiler et al., 2021a), which reduced the systematic and random errors in the Rayleigh channel. One phenomenon that\nstill needs to be explored is the sensitivity of Aeolus wind quality to the presence of aerosols and clouds, potentially affecting lies\" (Weiler et al., 2021a), which reduced the systematic and random errors in the Rayleigh channel. One phenomenon that\nstill needs to be explored is the sensitivity of Aeolus wind quality to the presence of aerosols and clouds, potentially affecting\nkey parameters such as the signal levels or scattering ratio (SR) used to calculate the Horizontal Line of Sight (HLOS) winds\n55\nand the associated error estimate (EE). The tropical Atlantic during the boreal summer, spanning from West Africa to the\nCaribbean, is the ideal place to explore these dependencies, with a wide range of atmospheric aerosols (Saharan dust aerosols,\nsea salt aerosols, biomass combustion aerosols) and convective cloud types associated with the West African Monsoon (WAM) lies\" (Weiler et al., 2021a), which reduced the systematic and random errors in the Rayleigh channel. One phenomenon that\nstill needs to be explored is the sensitivity of Aeolus wind quality to the presence of aerosols and clouds, potentially affecting key parameters such as the signal levels or scattering ratio (SR) used to calculate the Horizontal Line of Sight (HLOS) winds\n55\nand the associated error estimate (EE). The tropical Atlantic during the boreal summer, spanning from West Africa to the\nCaribbean, is the ideal place to explore these dependencies, with a wide range of atmospheric aerosols (Saharan dust aerosols,\nsea salt aerosols, biomass combustion aerosols) and convective cloud types associated with the West African Monsoon (WAM)\ncirculation and the Inter Tropical Convergence Zone (ITCZ). key parameters such as the signal levels or scattering ratio (SR) used to calculate the Horizontal Line of Sight (HLOS) winds\n55\nand the associated error estimate (EE). The tropical Atlantic during the boreal summer, spanning from West Africa to the\nCaribbean, is the ideal place to explore these dependencies, with a wide range of atmospheric aerosols (Saharan dust aerosols,\nsea salt aerosols, biomass combustion aerosols) and convective cloud types associated with the West African Monsoon (WAM)\ncirculation and the Inter Tropical Convergence Zone (ITCZ). 1\nIntroduction For wind validation, several reference products have been used such as ground-based remote sensing\nobservations (Belova et al., 2021; Guo et al., 2021; Iwai et al., 2021; Abril-Gago et al., 2022), in situ measurements (Baars\net al., 2020; Chen et al., 2021; Ratynski et al., 2022), airborne measurements (Lux et al., 2020; Witschas et al., 2020; Bedka\net al., 2021; Witschas et al., 2022) or NWP model equivalents (Martin et al., 2021; Zuo et al., 2022). Several anomalies in the Aeolus data have already been detected and improvements in the processing chain and the in-\n50\nstrument have been made accordingly. These include the implementation of a bias correction in both channels related to the\norbital-dependent temperature variations of ALADIN’s M1 mirror (Weiler et al., 2021b) and the correction of \"pixel anoma- Several anomalies in the Aeolus data have already been detected and improvements in the processing chain and the in-\n50\nstrument have been made accordingly. These include the implementation of a bias correction in both channels related to the\norbital-dependent temperature variations of ALADIN’s M1 mirror (Weiler et al., 2021b) and the correction of \"pixel anoma- Several anomalies in the Aeolus data have already been detected and improvements in the processing chain and the in-\n50\nstrument have been made accordingly. These include the implementation of a bias correction in both channels related to the\norbital-dependent temperature variations of ALADIN’s M1 mirror (Weiler et al., 2021b) and the correction of \"pixel anoma- 2 For this purpose, ESA organized the Joint Aeolus Tropical Atlantic Campaign (JATAC) in the period July to September\n60\n2021, which deployed sophisticated airborne lidar instruments over Cabo Verde (German Aerospace Center (DLR), Labo-\nratoire ATmosphères, Milieux, Observations Spatiales (LATMOS)) and the Virgin Islands (National Aeronautics and Space\nAdministration (NASA)) but also ground-based instruments such as radiosondes (Karlsruhe Institute of Technology (KIT),\nUniversity of Oklahoma, University of Utah) and Doppler lidar systems (Leibniz Institute for Tropospheric Research (TRO- For this purpose, ESA organized the Joint Aeolus Tropical Atlantic Campaign (JATAC) in the period July to September\n60\n2021, which deployed sophisticated airborne lidar instruments over Cabo Verde (German Aerospace Center (DLR), Labo-\nratoire ATmosphères, Milieux, Observations Spatiales (LATMOS)) and the Virgin Islands (National Aeronautics and Space\nAdministration (NASA)) but also ground-based instruments such as radiosondes (Karlsruhe Institute of Technology (KIT),\nUniversity of Oklahoma, University of Utah) and Doppler lidar systems (Leibniz Institute for Tropospheric Research (TRO- POS), National Observatory of Athens (NOA)). In this study, we validate Aeolus wind products using radiosondes launched\n65\nfrom western Puerto Rico, northern St. Croix and Sal airport on Cabo Verde. The semi-arid island of Sal is located over the\ntropical East Atlantic off the West African coast, near the northern boundary of the WAM. Rain events are relatively sporadic\nthere, as most synoptic and mesoscale precipitation systems propagate south of the island. The region is exposed to mineral\ndust plumes emanating from Saharan dust outbreaks. In contrast, the islands of St. Croix and Puerto Rico are located within POS), National Observatory of Athens (NOA)). In this study, we validate Aeolus wind products using radiosondes launched\n65\nfrom western Puerto Rico, northern St. Croix and Sal airport on Cabo Verde. The semi-arid island of Sal is located over the\ntropical East Atlantic off the West African coast, near the northern boundary of the WAM. Rain events are relatively sporadic\nthere, as most synoptic and mesoscale precipitation systems propagate south of the island. The region is exposed to mineral\ndust plumes emanating from Saharan dust outbreaks. In contrast, the islands of St. Croix and Puerto Rico are located within the warm and moist Caribbean, where heavy rainfall events and tropical cyclones frequently affect the area. 2.1\nALADIN and Aeolus wind products Aeolus is the second Earth Explorer Core mission and measures global atmospheric wind profiles from a 320 km high sun-\nsynchronous dusk-dawn orbit. It carries the ALADIN instrument (Schillinger et al. (2003)), which is a direct-detection high-\n85 85 3 3 The L1B product comprises the geolocated and observation data as well as optical information (SNR, useful signal, scattering ratio , etc.). The wind product called L2B contains the final horizontal projection of the LOS wind speed profiles of\n105\nthe Rayleigh and Mie channels, where all necessary calibration and instrument corrections have been performed (Dabas et al.,\n2008). This product is suitable for the assimilation in NWP models and scientific research. The L2B product also provides scene\nclassification based upon the backscatter ratio corresponding to the wind originating from a ’cloudy’ or ’clear’ atmospheric\nregion, resulting in Rayleigh-clear, Rayleigh-cloudy, Mie-clear and Mie-cloudy observation types. Throughout the processing scattering ratio , etc.). The wind product called L2B contains the final horizontal projection of the LOS wind speed profiles of\n105\nthe Rayleigh and Mie channels, where all necessary calibration and instrument corrections have been performed (Dabas et al.,\n2008). This product is suitable for the assimilation in NWP models and scientific research. The L2B product also provides scene\nclassification based upon the backscatter ratio corresponding to the wind originating from a ’cloudy’ or ’clear’ atmospheric\nregion, resulting in Rayleigh-clear, Rayleigh-cloudy, Mie-clear and Mie-cloudy observation types. Throughout the processing\nchain, the L1B and L2B processors are continuously updated into different baseline versions to account for revisions and\n110 chain, the L1B and L2B processors are continuously updated into different baseline versions to account for revisions and\n110\nidentified problems. This leads to different HLOS wind observations and quality in different time periods. In this study, data from the near-real-time version Baseline product 12 (L2bP 3.50) are used. We evaluate all observation\ntypes and corresponding Error Estimates (EEs) of the L2B product except Mie-clear observations as the Mie signal should is\nusually weak in clear sky conditions. Additionally, two L1B products are used, namely the scattering ratio (SR) and the useful chain, the L1B and L2B processors are continuously updated into different baseline versions to account for revisions and\n110\nidentified problems. This leads to different HLOS wind observations and quality in different time periods. In this study, data from the near-real-time version Baseline product 12 (L2bP 3.50) are used. We evaluate all observation\ntypes and corresponding Error Estimates (EEs) of the L2B product except Mie-clear observations as the Mie signal should is\nusually weak in clear sky conditions. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. spectral-resolution wind lidar with a Nd:YAG laser transmitter that operates at an ultraviolet wavelength of 354.8 nm. It points\nat 35 ◦with and angle of ∼10° from the zonal direction. ALADIN consists of a two-channel receiver that allows the instrument to measure wind speed from molecular backscatter\n(Rayleigh channel) and particle backscatter (Mie channel). The Rayleigh channel relies on the double-edge technique (Flesia\nand Korb, 1999) using a sequential Fabry-Perot interferometer, where the Doppler shift of the backscattered molecular spectrum\n90\nis retrieved from the signal intensities that are transmitted through two band-pass filters A and B. The final Rayleigh response\nis computed from a contrast-function between both filter signals. For Mie winds, the computation is based on a fringe-imaging\ntechnique (McKay, 2002), in which the Fizeau interferometer forms a linear interference fringe on the detector from the\nnarrowband particle backscatter signal. The lateral displacement of the interference fringe is then used to calculate the Doppler 90 shift. 95\nTo ensure a sufficient Signal-to-Noise Ratio (SNR), the wind measurements are averaged vertically and horizontally into\nsingle observations. Vertical sampling is performed within 24 vertical elevation bins with a resolution that can vary from\n0.25 km at lower elevations to 2 km at higher elevations. They are defined by the Range Bin Settings (RBS) and can vary\ngeographically and between the respective detection channel (Rayleigh and Mie). Horizontally, the measurements are averaged shift. 95\nTo ensure a sufficient Signal-to-Noise Ratio (SNR), the wind measurements are averaged vertically and horizontally into\nsingle observations. Vertical sampling is performed within 24 vertical elevation bins with a resolution that can vary from\n0.25 km at lower elevations to 2 km at higher elevations. They are defined by the Range Bin Settings (RBS) and can vary\ngeographically and between the respective detection channel (Rayleigh and Mie). Horizontally, the measurements are averaged over 87 km and 10 km integration lengths for Rayleigh and Mie channels, respectively, owing to the lower signal levels of the\n100\nRayleigh measurements. The data products are processed through a multi-stage processing chain, with each level containing different information\n(Reitebuch et al., 2018; Tan et al., 2008). In this study, the Level1B (L1B) and Level2B (L2B) products are of particular\ninterest. Additionally, two L1B products are used, namely the scattering ratio (SR) and the useful signal. The SR represents the ratio between the total (molecular and particulate) and the molecular backscatter coefficients. 115\nIt is strictly equal to or greater than one and describes the contribution of the particles to the backscattered signal. Note that\nthe SRs of the L2B products are not used, as some SRs were manually set to one during the processor baseline to eliminate a\ncross-talk correction, which had detrimental effects on the wind quality. The useful signal represents the returned signal levels\nper observation and comprises corrections for the solar background, the dark current and the detection chain offset (DCO). signal. The SR represents the ratio between the total (molecular and particulate) and the molecular backscatter coefficients. 115\nIt is strictly equal to or greater than one and describes the contribution of the particles to the backscattered signal. Note that\nthe SRs of the L2B products are not used, as some SRs were manually set to one during the processor baseline to eliminate a\ncross-talk correction, which had detrimental effects on the wind quality. The useful signal represents the returned signal levels\nper observation and comprises corrections for the solar background, the dark current and the detection chain offset (DCO). signal. The SR represents the ratio between the total (molecular and particulate) and the molecular backscatter coefficients. 115\nIt is strictly equal to or greater than one and describes the contribution of the particles to the backscattered signal. Note that\nthe SRs of the L2B products are not used, as some SRs were manually set to one during the processor baseline to eliminate a\ncross-talk correction, which had detrimental effects on the wind quality. The useful signal represents the returned signal levels\nper observation and comprises corrections for the solar background, the dark current and the detection chain offset (DCO). We apply an additional range correction and signal normalization that takes into account the different range bin thickness and\n120 4 4 At the end of each assimilation cycle, the feedback files with the Aeolus winds\nand their model equivalents can be retrieved from the Meteorological Archival and Retrieval System (MARS). These reports\ncontain information on the assimilated observations, their model background (short-range forecast) and analysis equivalents as\nwell as various quality control flags.. In this study, background equivalents of Aeolus observations are used as an additional\nreference to validate Aeolus HLOS winds. Note that only Rayleigh-clear and the Mie-cloudy winds are in operational use for\n130\nNWP. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. distances between the instruments and the height bins. Due to the sequential implementation of the Fizeau and the Fabry-Perot\ninterometers, signal from Mie scattering can leak into the Rayleigh channel signal. This optical \"cross-talk\" can cause biases,\nespecially in the case of strong Mie returns, as the Rayleigh-channel assumes pure molecular signal in the processing chain. distances between the instruments and the height bins. Due to the sequential implementation of the Fizeau and the Fabry-Perot\ninterometers, signal from Mie scattering can leak into the Rayleigh channel signal. This optical \"cross-talk\" can cause biases,\nespecially in the case of strong Mie returns, as the Rayleigh-channel assumes pure molecular signal in the processing chain. Along with many other NWP centers, the data were assimilated in the European Centre for Medium-Range Weather Fore-\ncasts (ECMWF) Integrated Forecasting System (IFS) by means of the operational four-dimensional ensemble-variational (4D-\n125\nEnVar) data assimilation scheme (4D-EnVar). At the end of each assimilation cycle, the feedback files with the Aeolus winds\nand their model equivalents can be retrieved from the Meteorological Archival and Retrieval System (MARS). These reports\ncontain information on the assimilated observations, their model background (short-range forecast) and analysis equivalents as\nwell as various quality control flags.. In this study, background equivalents of Aeolus observations are used as an additional\nreference to validate Aeolus HLOS winds. Note that only Rayleigh-clear and the Mie-cloudy winds are in operational use for\n130\nNWP. distances between the instruments and the height bins. Due to the sequential implementation of the Fizeau and the Fabry-Perot\ninterometers, signal from Mie scattering can leak into the Rayleigh channel signal. This optical \"cross-talk\" can cause biases,\nespecially in the case of strong Mie returns, as the Rayleigh-channel assumes pure molecular signal in the processing chain. interometers, signal from Mie scattering can leak into the Rayleigh channel signal. This optical \"cross-talk\" can cause biases,\nespecially in the case of strong Mie returns, as the Rayleigh-channel assumes pure molecular signal in the processing chain. Along with many other NWP centers, the data were assimilated in the European Centre for Medium-Range Weather Fore-\ncasts (ECMWF) Integrated Forecasting System (IFS) by means of the operational four-dimensional ensemble-variational (4D-\n125\nEnVar) data assimilation scheme (4D-EnVar). different research components of JATAC. Between the 7 and 28th of September 2021, a total of 37 radiosondes were launched\nfrom Sal airport in Cape Verde, 9 of them corresponding to Aeolus overflights. The launches were coordinated by the Karlsruhe\n135\nInstitute of Technology (KIT) with local support from the JATAC team. This was accomplished using the DFM-09 (GRAW)\nlight weather radiosondes, which measure air pressure, air temperature, relative humidity, wind speed and wind direction. The\nvertical resolution depends on the ascent speed, which varies with the amount of helium in the balloon, but can generally be\nestimated at about 5 ms–1. Most of the radiosondes launched at Sal were ingested into the Global Telecommunication System\n(GTS).\n140 As for Sal, these\n145\nmeasurements were performed with the radiosonde instrument DFM-09 (GRAW). Lastly, 32 launches were conducted from\nthe University of Puerto Rico at Mayagüez (UPRM) campus between 26 August and 14 September 2021, 7 of which could be\nused for the validation of Aeolus. All launches were performed with iMet-4 radiosondes from the International Met System. As with DFM-09, the iMet-4 radiosondes provide measurements of wind speed, wind direction, temperature, humidity and air pressure. The radiosonde data also underwent a quality control check using the Atmospheric Sounding Processing Envi-\n150\nronment (ASPEN) software (Martin and Suhr, 2021) developed by the Earth Observing Laboratory at the National Center for\nAtmospheric Research (NCAR). A summary of the radiosonde launches and weather events sampled at UPRM was provided\nby Rios-Berrios et al. (2023). The total number of radiosonde profiles corresponding to Aeolus overpasses thus amounts to 20, of which 12 correspond to air pressure. The radiosonde data also underwent a quality control check using the Atmospheric Sounding Processing Envi-\n150\nronment (ASPEN) software (Martin and Suhr, 2021) developed by the Earth Observing Laboratory at the National Center for\nAtmospheric Research (NCAR). A summary of the radiosonde launches and weather events sampled at UPRM was provided\nby Rios-Berrios et al. (2023). The total number of radiosonde profiles corresponding to Aeolus overpasses thus amounts to 20 of which 12 correspond to The total number of radiosonde profiles corresponding to Aeolus overpasses thus amounts to 20, of which 12 correspond to\nascending and 8 to descending orbits of Aeolus. An overview of the launches from the different sites can be found in Table 1,\n155\nalong with other co-location parameters fully discussed in Section 3.1. ascending and 8 to descending orbits of Aeolus. An overview of the launches from the different sites can be found in Table 1,\n155\nalong with other co-location parameters fully discussed in Section 3.1. ascending and 8 to descending orbits of Aeolus. An overview of the launches from the different sites can be found in Table 1,\n155\nalong with other co-location parameters fully discussed in Section 3.1. 2.2\nRadiosondes Table 1. Overview of Aeolus overflights and associated radiosonde profiles. Table 1. Overview of Aeolus overflights and associated radiosonde profiles. Week day\nStart and stop time\nOrbit node\nCo-location radius\nNumber of profiles\nSal\nTuesday\n07:28 – 07:29 UTC\nDescending\n50 km\n3\nThursday\n19:23 – 19:24 UTC\nAscending\n180 km\n3\nFriday\n19:36 – 19:37 UTC\nAscending\n280 km\n3\nSaint Croix\nMonday\n10:17 - 10:18 UTC\nDescending\n90 km\n3\nWednesday\n22:12 – 22:13 UTC\nAscending\n160 km\n3\nThursday\n22:25 – 22:26 UTC\nAscending\n340 km\n1\nPuerto Rico\nTuesday\n10:29 – 10:30 UTC\nDescending\n160 km\n2\nThursday\n22:25 – 22:26 UTC\nAscending\n100 km\n2 Week day\nStart and stop time\nOrbit node\nCo-location radius\nNumber of profiles During the campaign, radiosondes were launched from three different locations over the tropical Atlantic and coordinated by\ndifferent research components of JATAC. Between the 7 and 28th of September 2021, a total of 37 radiosondes were launched from Sal airport in Cape Verde, 9 of them corresponding to Aeolus overflights. The launches were coordinated by the Karlsruhe\n135\nInstitute of Technology (KIT) with local support from the JATAC team. This was accomplished using the DFM-09 (GRAW)\nlight weather radiosondes, which measure air pressure, air temperature, relative humidity, wind speed and wind direction. The\nvertical resolution depends on the ascent speed, which varies with the amount of helium in the balloon, but can generally be\nestimated at about 5 ms–1. Most of the radiosondes launched at Sal were ingested into the Global Telecommunication System (GTS). 140 5 5 2.3\nEUMETSAT SAFNWC Cloud type product The Satellite Application Facility for supporting NoWCasting and very short range forecasting (SAFNWC; Alonso Lasheras\net al., 2005) developed a number of satellite-based meteorological products distributed by the European Organisation for the\nExploitation of Meteorological Satellites (EUMETSAT). Among others, they provide the Cloud Type (CT) product (Derrien\n160\nand Le Gléau, 2005), which is a detailed scenery classification of clouds based on different main classes. The Satellite Application Facility for supporting NoWCasting and very short range forecasting (SAFNWC; Alonso Lasheras\net al., 2005) developed a number of satellite-based meteorological products distributed by the European Organisation for the Exploitation of Meteorological Satellites (EUMETSAT). Among others, they provide the Cloud Type (CT) product (Derrien\n160\nand Le Gléau, 2005), which is a detailed scenery classification of clouds based on different main classes. The baseline data originate from the Spinning Enhanced Visible and Infrared Imager (SEVIRI) operated onboard the second\ngeneration METEOSAT geostationary satellites (MSG). Multispectral thresholding techniques (Saunders and Kriebel, 1988;\nDerrien et al., 1993; Stowe et al., 1999) are subsequently applied in the NWCSAF software to process the SEVIRI/MSG images Exploitation of Meteorological Satellites (EUMETSAT). Among others, they provide the Cloud Type (CT) product (Derrien\n160\nand Le Gléau, 2005), which is a detailed scenery classification of clouds based on different main classes. The baseline data originate from the Spinning Enhanced Visible and Infrared Imager (SEVIRI) operated onboard the second\ngeneration METEOSAT geostationary satellites (MSG). Multispectral thresholding techniques (Saunders and Kriebel, 1988;\nDerrien et al., 1993; Stowe et al., 1999) are subsequently applied in the NWCSAF software to process the SEVIRI/MSG images into the various NWC products. The product is available with a temporal resolution of 15 minutes and a nadir spatial resolution\n165\nof 3 km, compared to 11 km at the edge of the field of view. In this study, CT is used to identify the cloud type and cloud cover along the Aeolus tracks and to assess the quality of\nthe Aeolus wind products relative to the presence of clouds. More specifically, we identify the pixels closest to each track of\nAeolus and determine the average percentage of cloud cover at each altitude based on a cloud classification. According to this classification, a measurement bin is considered as cloudy, if it is situated within or below a cloud. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The radiosondes launched on the Virgin Islands were organised by National Aeronautics and Space Administration (NASA)’s\nConvective Processes Experiment-Aerosols and Winds (CPEX-AW) campaign component of JATAC, with the University of\nUtah conducting the launches on Saint Croix and the University of Oklahoma conducting the launches from Puerto Rico. On Saint Croix, launches were conducted from Carambola between 19 August 2021 and 14 September 2021. Altogether 73\nlaunches were conducted, of which a total of seven radiosondes were used to validate Aeolus in this study. As for Sal, these\n145\nmeasurements were performed with the radiosonde instrument DFM-09 (GRAW). Lastly, 32 launches were conducted from The radiosondes launched on the Virgin Islands were organised by National Aeronautics and Space Administration (NASA)’s\nConvective Processes Experiment-Aerosols and Winds (CPEX-AW) campaign component of JATAC, with the University of\nUtah conducting the launches on Saint Croix and the University of Oklahoma conducting the launches from Puerto Rico. On Saint Croix, launches were conducted from Carambola between 19 August 2021 and 14 September 2021. Altogether 73\nlaunches were conducted, of which a total of seven radiosondes were used to validate Aeolus in this study. As for Sal, these\n145\nmeasurements were performed with the radiosonde instrument DFM-09 (GRAW). Lastly, 32 launches were conducted from\nthe University of Puerto Rico at Mayagüez (UPRM) campus between 26 August and 14 September 2021, 7 of which could be\nused for the validation of Aeolus. All launches were performed with iMet-4 radiosondes from the International Met System. As with DFM-09, the iMet-4 radiosondes provide measurements of wind speed, wind direction, temperature, humidity and ,\ng\np\ng\nlaunches were conducted, of which a total of seven radiosondes were used to validate Aeolus in this study. As for Sal, these\n145\nmeasurements were performed with the radiosonde instrument DFM-09 (GRAW). Lastly, 32 launches were conducted from\nthe University of Puerto Rico at Mayagüez (UPRM) campus between 26 August and 14 September 2021, 7 of which could be\nused for the validation of Aeolus. All launches were performed with iMet-4 radiosondes from the International Met System. As with DFM-09, the iMet-4 radiosondes provide measurements of wind speed, wind direction, temperature, humidity and launches were conducted, of which a total of seven radiosondes were used to validate Aeolus in this study. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 3.1\nCo-location criteria For the comparison of Aeolus against radiosonde profiles, several steps are required to fit the radiosonde wind measurements\nto the Aeolus measurement grid and to co-locate them in time and space. To ensure vertical consistency, the high-resolution radiosonde measurements are vertically averaged within the 24 range bins\n190\nas specified in the Aeolus L2B product. Subsequently, the radiosondes total horizontal wind speed VRS and direction φRS are\nprojected to the Aeolus HLOS (HLOSRS) using the azimuth angle φAEOLUS also specified in the L2B product, in accordance\nto\nHLOSRS = VRS × cos(φAEOLUS – φRS)\n(1) To ensure vertical consistency, the high-resolution radiosonde measurements are vertically averaged within the 24 range bins\n190\nas specified in the Aeolus L2B product. Subsequently, the radiosondes total horizontal wind speed VRS and direction φRS are\nprojected to the Aeolus HLOS (HLOSRS) using the azimuth angle φAEOLUS also specified in the L2B product, in accordance\nto\nHLOSRS = VRS × cos(φAEOLUS – φRS)\n(1) (1) Moreover, we have chosen co-location radii of up to 340 km, as we assume typical variations in zonal wind to be of a larger\n195\nscale. In fact, during boreal summer, African Easterly Waves (AEWs) and tropical disturbances dominate the tropospheric zonal\nwind variability over the tropical Atlantic, which generally have a horizontal wavelength of 2000-5000 km with a periodicity\nof 2-7 days (Belanger et al., 2016). Section 4.3.2 discusses the error dependencies related to co-location aspects in more detail. Moreover, we have chosen co-location radii of up to 340 km, as we assume typical variations in zonal wind to be of a larger\n195\nscale. In fact, during boreal summer, African Easterly Waves (AEWs) and tropical disturbances dominate the tropospheric zonal\nwind variability over the tropical Atlantic, which generally have a horizontal wavelength of 2000-5000 km with a periodicity\nof 2-7 days (Belanger et al., 2016). Section 4.3.2 discusses the error dependencies related to co-location aspects in more detail. Moreover, we have chosen co-location radii of up to 340 km, as we assume typical variations in zonal wind to be of a larger\n195\nscale. In fact, during boreal summer, African Easterly Waves (AEWs) and tropical disturbances dominate the tropospheric zonal\nwind variability over the tropical Atlantic, which generally have a horizontal wavelength of 2000-5000 km with a periodicity\nof 2-7 days (Belanger et al., 2016). Section 4.3.2 discusses the error dependencies related to co-location aspects in more detail. 2.4\nCAMS Dust products The dust-aerosol mixing ratio is thereby averaged along each track and projected onto Rayleigh-clear and\nMie-cloudy measurement bins to obtain an estimate of the dust concentration for each observation. 185 Mie-cloudy measurement bins to obtain an estimate of the dust concentration for each observation. 185 Mie-cloudy measurement bins to obtain an estimate of the dust concentration for each observation. 185 2.3\nEUMETSAT SAFNWC Cloud type product This refers to following\n170\nclasses for altitudes above 16 km (very high clouds), between 7 and 16 km (very high and high cloud types), between 3 and 7\nkm (very high, high, mid-level, low and fractional cloud types) and finally below 3 km (very high, high, mid-level, low, very\nlow and fractional cloud types). classification, a measurement bin is considered as cloudy, if it is situated within or below a cloud. This refers to following\n170\nclasses for altitudes above 16 km (very high clouds), between 7 and 16 km (very high and high cloud types), between 3 and 7\nkm (very high, high, mid-level, low and fractional cloud types) and finally below 3 km (very high, high, mid-level, low, very\nlow and fractional cloud types). classification, a measurement bin is considered as cloudy, if it is situated within or below a cloud. This refers to following\n170\nclasses for altitudes above 16 km (very high clouds), between 7 and 16 km (very high and high cloud types), between 3 and 7\nkm (very high, high, mid-level, low and fractional cloud types) and finally below 3 km (very high, high, mid-level, low, very\nlow and fractional cloud types). 6 6 2.4\nCAMS Dust products The fourth generation of ECMWF Global Atmospheric Composition Reanalysis (EAC4) (Inness et al., 2019) is produced by\n175\nthe Copernicus Atmosphere Monitoring Service (CAMS) with the main objective of global aerosol monitoring. EAC4 relies on\nECMWF’s IFS, which has been extended to predict and assimilate aerosols (Rémy et al., 2019), trace gases (Flemming et al.,\n2015; Huijnen et al., 2019) and greenhouse gases. The IFS meteorological and atmospheric composition models are combined\nwith data assimilation from satellite products using the 4D-Var data assimilation scheme in CY42R1. In particular, CAMS assimilates the Aerosol Optical Depth (AOD) at 550 nm derived from MODIS and the Polar Multi-Sensor Aerosol Optical\n180\nProperties (PMAp). Reanalysis outputs are provided on three-dimensional time-consistent fields interpolated on 25 pressure\nlevels, a horizontal resolution of about 80 km and a sub-daily time resolution of 6 hours. assimilates the Aerosol Optical Depth (AOD) at 550 nm derived from MODIS and the Polar Multi-Sensor Aerosol Optical\n180\nProperties (PMAp). Reanalysis outputs are provided on three-dimensional time-consistent fields interpolated on 25 pressure\nlevels, a horizontal resolution of about 80 km and a sub-daily time resolution of 6 hours. assimilates the Aerosol Optical Depth (AOD) at 550 nm derived from MODIS and the Polar Multi-Sensor Aerosol Optical\n180\nProperties (PMAp). Reanalysis outputs are provided on three-dimensional time-consistent fields interpolated on 25 pressure\nlevels, a horizontal resolution of about 80 km and a sub-daily time resolution of 6 hours. Similar to the SAFNWC CT, the dust-aerosol mixing ratio is used to assess the quality of the Aeolus wind products in\npresence of dust. The dust-aerosol mixing ratio is thereby averaged along each track and projected onto Rayleigh-clear and Properties (PMAp). Reanalysis outputs are provided on three-dimensional time-consistent fields interpolated on 25 pressure\nlevels, a horizontal resolution of about 80 km and a sub-daily time resolution of 6 hours. Similar to the SAFNWC CT, the dust-aerosol mixing ratio is used to assess the quality of the Aeolus wind products in\npresence of dust. The dust-aerosol mixing ratio is thereby averaged along each track and projected onto Rayleigh-clear and\nMie-cloudy measurement bins to obtain an estimate of the dust concentration for each observation. 185 Similar to the SAFNWC CT, the dust-aerosol mixing ratio is used to assess the quality of the Aeolus wind products in\npresence of dust. 3.2\nStatistical metrics Different metrics were used to validate and estimate the systematic and random error of Aeolus wind products. The bin-to-bin\n200\nwind speed difference between Aeolus and radiosonde along the HLOS is defined as Different metrics were used to validate and estimate the systematic and random error of Aeolus wind products. The bin-to-bin\n200\nwind speed difference between Aeolus and radiosonde along the HLOS is defined as\nΔdiffHLOS = (HLOSAEOLUS – HLOSRS)\n(2) Different metrics were used to validate and estimate the systematic and random error of Aeolus wind products. The bin-to-bin\n200\nwind speed difference between Aeolus and radiosonde along the HLOS is defined as\nΔdiffHLOS = (HLOSAEOLUS – HLOSRS)\n(2) wind speed difference between Aeolus and radiosonde along the HLOS is defined as\nΔdiffHLOS = (HLOSAEOLUS – HLOSRS)\n(2) (2) 7 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Thus, the bias μ is defined as the total mean difference μ = 1\nN\nN\nX\ni=1\nΔdiffHLOS (3) with the Mean Absolute Difference (MADI) yielding\n205 MADI = 1\nN\nN\nX\ni=1\n|ΔdiffHLOS| MADI = 1\nN\nN\nX\ni=1\n|ΔdiffHLOS| (4) and N the total number of data points. and N the total number of data points. and N the total number of data points. Additionally, we calculated the standard deviation of the difference STD =\nv\nu\nu\nt 1\ni – 1\nN\nX\ni=1\n(HLOSAEOLUS – HLOSRS)2 (5) and the scaled median absolute deviation (SMAD)\n210 SMAD = 1.4826 × median\n\u0000\f\fΔdiffHLOS – median(ΔdiffHLOS)\n\f\f\u0001 (6) The SMAD is equivalent to the standard deviation for a normal distribution of errors, but is often used in Aeolus validation\nstudies as it is less sensitive to individual outliers with very large differences than the standard deviation. Since the number of data points varies greatly depending on the measurement channel and height, we define the uncertainty\nof the mean bias εμ as\n215 (7) (7) https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. study is comparably homogeneous, we estimate the representativeness error for our comparison to be in the range of 1.5 ms–1\nto 2.5 ms–1. The radiosonde observation error σRS is estimated to be 0.7 ms–1 based on Dirksen et al. (2014). study is comparably homogeneous, we estimate the representativeness error for our comparison to be in the range of 1.5 ms–1\nto 2.5 ms–1. The radiosonde observation error σRS is estimated to be 0.7 ms–1 based on Dirksen et al. (2014). The representativeness and radiosonde observations errors also need to be considered when comparing the differences be-\ntween Aeolus and radiosonde observations with the expected error provided in the Aeolus data product (EEAeolus). To account\nfor this, we add the the radiosonde observation error and an estimated representativeness error of 2 ms–1 to achieve the total\nexpected error for the comparison (EEtot) as follows: The representativeness and radiosonde observations errors also need to be considered when comparing the differences be-\ntween Aeolus and radiosonde observations with the expected error provided in the Aeolus data product (EEAeolus). To account\nfor this, we add the the radiosonde observation error and an estimated representativeness error of 2 ms–1 to achieve the total\nexpected error for the comparison (EEtot) as follows: 230 EEtot =\nq\nEE2\nAeolus + σ2\nRS + σ2rep (9) 3.3\nRepresentativeness The difference between Aeolus and radiosonde observations is the sum of the Aeolus observation error, the radiosonde ob-\nservation error and the error arising from spatial and temporal displacement of the observations and different observation\ngeometries. The latter is usually referred to as representativeness error (Weissmann et al., 2005). As the three error components\n220\ncan be assumed to be uncorrelated, the standard deviation of the Aeolus HLOS winds observation error (σAeolus) can therefore\nbe calculated as The difference between Aeolus and radiosonde observations is the sum of the Aeolus observation error, the radiosonde ob-\nservation error and the error arising from spatial and temporal displacement of the observations and different observation servation error and the error arising from spatial and temporal displacement of the observations and different observation\ngeometries. The latter is usually referred to as representativeness error (Weissmann et al., 2005). As the three error components\n220\ncan be assumed to be uncorrelated, the standard deviation of the Aeolus HLOS winds observation error (σAeolus) can therefore\nbe calculated as geometries. The latter is usually referred to as representativeness error (Weissmann et al., 2005). As the three error components\n220\ncan be assumed to be uncorrelated, the standard deviation of the Aeolus HLOS winds observation error (σAeolus) can therefore\nbe calculated as σAeolus =\nq\nσ2tot – σ2\nRS – σ2rep (8) where σtot is the standard deviation of the total difference between Aeolus and radiosonde observations (STD), σRS is the standard deviation of the radiosonde observation error and σrep is the standard deviation of the representativeness error. Martin\n225\net al. (2021) estimated that the representativeness error for the comparison of Aeolus and radiosonde observations in mit-\nlatitudes is about 2.5 ms–1 based on high-resolution model simulations. As the wind fields in the area of the present validation standard deviation of the radiosonde observation error and σrep is the standard deviation of the representativeness error. Martin\n225\net al. (2021) estimated that the representativeness error for the comparison of Aeolus and radiosonde observations in mit-\nlatitudes is about 2.5 ms–1 based on high-resolution model simulations. As the wind fields in the area of the present validation 8 8 3.4\nQuality control\n235 Quality control (QC) is an important step in the evaluation of Aeolus wind errors. The aim is to check for the validity of the\nmeasurements and discard nonphysical wind results from the analysis process. The QC we apply here is based on the existing\nquality control recommendations (Rennie et al., 2020) from the Aeolus Data Science and Innovation Cluster (DISC), and\nprimarily rely on the HLOS wind error estimate (EE) in the L2B product and the validity flags. The Rayleigh channel EE is based on the uncertainty of the SNR spectrometer response and takes into account error propaga-\n240\ntion arising from the sensitivity of the Fabry-Perot interferometer, Poisson noise in the useful signal and the solar background. Ultimately, the Rayleigh EE is proportional to the inverse squared root of the useful signal on the detector. Future baseline\nversions will include additional noise terms, such as noise related to atmospheric temperature and pressure, or cross-talk con-\ntamination. In contrary, the Mie EE is determined from the accuracy of the fringe peak position using the solution covariance of the Lorentzian fitting algorithm based on four characteristics of the signal shape, i.e the peak position, height, width and\n245\noffset. Following the default QC flags, all Aeolus wind products with a validity flag of 0, EE above 8 ms–1 for Rayleigh and 4\nms–1 for Mie, are omitted. Nevertheless, the used QC might not be enough and the data algorithm may contain gross errors in\nthe wind estimate that have not been flagged as invalid. These errors are usually due to non-Gaussian error sources, such as of the Lorentzian fitting algorithm based on four characteristics of the signal shape, i.e the peak position, height, width and\n245\noffset. Following the default QC flags, all Aeolus wind products with a validity flag of 0, EE above 8 ms–1 for Rayleigh and 4\nms–1 for Mie, are omitted. Nevertheless, the used QC might not be enough and the data algorithm may contain gross errors in\nthe wind estimate that have not been flagged as invalid. These errors are usually due to non-Gaussian error sources, such as instrument/transmission failure, or to a misrepresentation of the measurements in space and time. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4.1\nStatistical comparison of Aeolus with radiosonde observations and model winds In this section, the L2B HLOS winds (L2bP 3.50) from Aeolus are compared statistically with radiosonde observations and\n260\nmodel winds. This includes a comparison with the ECMWF model equivalents (subsection 4.1.1), an overview of systematic\nand random differences with respect to Cal/Val sites and orbital nodes (subsection 4.1.2), and finally the identification of an\norbital- and altitude-dependent bias in the Rayleigh-clear channel (subsection 4.1.3). In this section, the L2B HLOS winds (L2bP 3.50) from Aeolus are compared statistically with radiosonde observations and\n260\nmodel winds. This includes a comparison with the ECMWF model equivalents (subsection 4.1.1), an overview of systematic\nand random differences with respect to Cal/Val sites and orbital nodes (subsection 4.1.2), and finally the identification of an\norbital- and altitude-dependent bias in the Rayleigh-clear channel (subsection 4.1.3). The present study relies on a total of 384 Rayleigh-clear and 59 Mie-cloudy bin pairs, of which ∼60% and ∼53% are\nfrom ascending orbits, respectively, with the majority of observations obtained from the Caribbean launch sites (∼56% for\n265\nRayleigh-clear and ∼64% for Mie-cloudy). Rayleigh-cloudy bin pairs are also available, but only in a very small number (16\ncounts), which makes a statistical analysis difficult. 3.4\nQuality control\n235 Since the two aforementioned\n250\nQC are not sufficient to remove these gross errors, an additional QC parameter is used, namely the modified Z-score (Lux et al.,\n2022b; Witschas et al., 2022; Iglewicz and Hoaglin, 1993). The modified Z-score Zm,i is defined as instrument/transmission failure, or to a misrepresentation of the measurements in space and time. Since the two aforementioned\n250\nQC are not sufficient to remove these gross errors, an additional QC parameter is used, namely the modified Z-score (Lux et al.,\n2022b; Witschas et al., 2022; Iglewicz and Hoaglin, 1993). The modified Z-score Zm,i is defined as (10) and describes the median deviations between each wind speed difference normalized with the SMAD. The modified Z-score\nsignificantly influences small data sets, such as those used in this study. Following literature recommendations (Lux et al.,\n255\n2022b; Witschas et al., 2022; Sandbhor and Chaphalkar, 2019; Tripathy et al., 2013), we discard wind observations with a\nmodified Z-score greater than 3 as a final QC. significantly influences small data sets, such as those used in this study. Following literature recommendations (Lux et al.,\n255\n2022b; Witschas et al., 2022; Sandbhor and Chaphalkar, 2019; Tripathy et al., 2013), we discard wind observations with a\nmodified Z-score greater than 3 as a final QC. 9 The generally good agreement between radiosonde and model equivalent\nshows that the co-location parameters used in this study are reliable, as most of the systematic and random errors seem to be\nspecific to the Aeolus Rayleigh-clear data. This stresses the need to identify the underlying potential error sources of Rayleigh- in the model equivalent is smaller than for Aeolus Mie-cloudy winds, with biases of 0.4 ± 0.3 ms–1 and –0.9 ± 0.3 ms–1,\n280\nrespectively. For Rayleigh-cloudy, the STD is larger at 6.6 ms–1 with a bias of 1.0 ± 1.4 ms–1, but given the small statistical\nsample size, there is a risk of a large margin of error. The generally good agreement between radiosonde and model equivalent\nshows that the co-location parameters used in this study are reliable, as most of the systematic and random errors seem to be\nspecific to the Aeolus Rayleigh-clear data. This stresses the need to identify the underlying potential error sources of Rayleigh- clear observations with respect to the presence of clouds and dust aerosols, which are frequent in the region of interest. It is\n285\nalso worth noting that this good agreement indicates that the model equivalent is a robust reference for validating the Aeolus\nwinds in the tropical Atlantic. clear observations with respect to the presence of clouds and dust aerosols, which are frequent in the region of interest. It is\n285\nalso worth noting that this good agreement indicates that the model equivalent is a robust reference for validating the Aeolus\nwinds in the tropical Atlantic. 4.1.1\nComparative analysis with the ECMWF model equivalents 4.1.1\nComparative analysis with the ECMWF model equivalents Figure 1. (a) Aeolus HLOS Rayleigh-clear (blue), Mie-cloudy (red) and Rayleigh-cloudy (orange) wind products plotted against radiosonde\nmeasurements projected along the HLOS for the 20 radiosonde profiles. The gross errors (crosses) are determined using the modified Z-score\nwith a threshold of 3. (b) Aeolus HLOS model equivalents from the ECMWF feedback files plotted against radiosonde measurements. The\ndashed lines are located at the ±10 ms–1 and ±20 ms–1 wind speed difference between two measurements. Figure 1. (a) Aeolus HLOS Rayleigh-clear (blue), Mie-cloudy (red) and Rayleigh-cloudy (orange) wind products plotted against radiosonde\nmeasurements projected along the HLOS for the 20 radiosonde profiles. The gross errors (crosses) are determined using the modified Z-score\nwith a threshold of 3. (b) Aeolus HLOS model equivalents from the ECMWF feedback files plotted against radiosonde measurements. The\ndashed lines are located at the ±10 ms–1 and ±20 ms–1 wind speed difference between two measurements. 10 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Figure 1 shows a scatter plot of the radiosonde HLOS (HLOSRS) against Aeolus L2B (HLOSAEOLUS) Rayleigh-clear (blue), Figure 1 shows a scatter plot of the radiosonde HLOS (HLOSRS) against Aeolus L2B (HLOSAEOLUS) Rayleigh-clear (blue),\nMie-cloudy (red) and Rayleigh-cloudy (orange) wind products (a) as well as against Aeolus ECMWF model equivalents\n270\n(HLOSECMWF) (b). Since Rayleigh-cloudy wind observations are not assimilated at ECMWF, they are not displayed in Fig. 1b. The × symbol represent the gross errors rejected with a Z-score threshold of 3 (∼3.5%, ∼4.8% and ∼6.7% of the\ntotal Rayleigh-clear, Mie-cloudy and Rayleigh-cloudy data points, respectively). The dashed lines represent the ±10 ms–1\nand ±20 ms–1 difference between two measurements. The Aeolus model equivalent HLOSECMWF for Rayleigh-clear shows a Mie-cloudy (red) and Rayleigh-cloudy (orange) wind products (a) as well as against Aeolus ECMWF model equivalents\n270\n(HLOSECMWF) (b). Since Rayleigh-cloudy wind observations are not assimilated at ECMWF, they are not displayed in Fig. 1b. The × symbol represent the gross errors rejected with a Z-score threshold of 3 (∼3.5%, ∼4.8% and ∼6.7% of the\ntotal Rayleigh-clear, Mie-cloudy and Rayleigh-cloudy data points, respectively). The dashed lines represent the ±10 ms–1\nand ±20 ms–1 difference between two measurements. The Aeolus model equivalent HLOSECMWF for Rayleigh-clear shows a much better agreement with the radiosonde measurements HLOSRS with a STD of 2.1 ms–1 (Fig. 1b) compared to the Aeolus\n275\nHLOSAEOLUS Rayleigh-clear observations, which have a larger spread and a STD of 4.8 ms–1 (Fig. 1a). The systematic\ndifference of the model equivalent is also smaller with a bias of 0.1 ± 0.1 ms–1 compared to –0.5 ± 0.2 ms–1 for the Aeolus\nobservations. In contrast, the Mie-cloudy winds of both Aeolus model equivalents and HLOSAEOLUS behave similarly with\nrespect to the radiosonde measurements, with STD of 2.93 ms–1 and 2.9 ms–1, respectively. Again, the systematic difference in the model equivalent is smaller than for Aeolus Mie-cloudy winds, with biases of 0.4 ± 0.3 ms–1 and –0.9 ± 0.3 ms–1,\n280\nrespectively. For Rayleigh-cloudy, the STD is larger at 6.6 ms–1 with a bias of 1.0 ± 1.4 ms–1, but given the small statistical\nsample size, there is a risk of a large margin of error. 4.1.2\nSystematic and random errors using radiosondes An overview of the bias and random differences of both channels can be found in Table 2. In terms of systematic errors,\nRayleigh-clear shows a relatively small negative bias of –0.5 ± 0.2 ms–1, on average, which is below ESA’s specification of\n290\n0.7 ms–1 (Ingmann and Straume, 2016). This bias is, however, the result of a large heterogeneity with respect to the Cal/Val\nsites and orbital nodes, with compensating biases of –1.5±0.6 ms–1 and 0.6±0.4 ms–1 for the descending and ascending nodes\non Sal, respectively, compared to negative biases of –1.0±0.3 ms–1 (ascending) and –0.6±0.4 ms–1 (descending) in the Virgin\nIsland. As for random differences, Rayleigh-clear has an average STD of 4.8 ms–1, which varies only marginally between the Rayleigh-clear shows a relatively small negative bias of –0.5 ± 0.2 ms–1, on average, which is below ESA’s specification of\n290\n0.7 ms–1 (Ingmann and Straume, 2016). This bias is, however, the result of a large heterogeneity with respect to the Cal/Val\nsites and orbital nodes, with compensating biases of –1.5±0.6 ms–1 and 0.6±0.4 ms–1 for the descending and ascending nodes\non Sal, respectively, compared to negative biases of –1.0±0.3 ms–1 (ascending) and –0.6±0.4 ms–1 (descending) in the Virgin\nIsland. As for random differences, Rayleigh-clear has an average STD of 4.8 ms–1, which varies only marginally between the Cal/Val sites and orbital nodes, ranging from 4.1 ms–1 to 5.3 ms–1. The overall SMAD is found to be slightly below at 4.3 ms–1. 295\nFor comparison with the ESA recommendation for random errors, we derived the random errors for Aeolus observations\nconsidering also the representativeness errors for the comparison and radiosonde observation errors according to Eq. 8 (table\n3). The random error at 2–16 km altitude of 3.8 – 4.3 ms–1 exceeds the threshold of 2.5 ms–1, while at 16–20 km altitude it\namounts to 4.3 – 4.8 ms–1, also exceeding the ESA threshold of 3 ms–1. The quality of Rayleigh-clear measurements primarily depends on the signal accumulation, which can vary with the thickness of the RBS and the horizontal accumulation length\n300\nas well as with the atmospheric path signal. The latter has been decreasing in recent years as a result of initial instrumental depends on the signal accumulation, which can vary with the thickness of the RBS and the horizontal accumulation length\n300\nas well as with the atmospheric path signal. 4.1.2\nSystematic and random errors using radiosondes The latter has been decreasing in recent years as a result of initial instrumental 11 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Table 2. Overview of the mean bias and uncertainty (μ, σμ; ms–1), Standard deviation (STD; ms–1), Scaled Median Absolute Deviation\n(SMAD; ms–1) and counts (COUNT) for the Rayleigh-clear and Mie-cloudy channels, orbital nodes and the different radiosonde locations. Due to the small amount of available data, Rayleigh-cloudy is not shown here. Table 2. Overview of the mean bias and uncertainty (μ, σμ; ms–1), Standard deviation (STD; ms–1), Scaled Median Absolute Deviation\n(SMAD; ms–1) and counts (COUNT) for the Rayleigh-clear and Mie-cloudy channels, orbital nodes and the different radiosonde locations. Due to the small amount of available data, Rayleigh-cloudy is not shown here. Region\nOrbital node\nRayleigh-clear\nMie-cloudy\nμ\nSTD\nSMAD\nCOUNT\nμ\nSTD\nSMAD\nCOUNT\nAscending\n0.6±0.4\n4.9\n4.4\n112\n-1±0.9\n2.9\n3.5\n15\nSal\nDescending\n-1.5±0.6\n4.6\n4.8\n55\n-1.6±0.8\n2.2\n2.1\n6\nAll\n-0.1±0.3\n4.9\n4.5\n167\n-1.2±0.7\n2.7\n3.2\n21\nAscending\n-1.0±0.3\n4.1\n3.7\n119\n-0.6±0.7\n2.9\n3.7\n16\nSCRX/PR\nDescending\n-0.6±0.4\n5.3\n4.3\n98\n-1.0±0.5\n2.9\n2.5\n22\nAll\n-0.8±0.3\n4.7\n4.3\n217\n-0.8±0.4\n2.9\n2.5\n38\nAscending\n-0.2±0.3\n4.6\n4.2\n231\n-0.8±0.6\n2.9\n3.3\n31\nSal/SCRX/PR\nDescending\n-0.9±0.4\n5.0\n4.6\n153\n-1.1±0.4\n2.8\n2.2\n28\nAll\n-0.5±0.2\n4.8\n4.3\n384\n-0.9±0.3\n2.9\n2.6\n59 misalignment, laser-induced contamination, as well as the wavefront error of the 1.5 m telescope. The solar background noise,\nwhich varies along the orbit and season, can also affect the quality of the Rayleigh-clear measurements. ESA’s specification and more uniform across regions and orbital nodes with a slightly larger bias in the descending orbits\n305\nand over Sal. Concerning the random differences, the measurements exhibit a total random error of 1.1 – 2.3 ms–1, which is\nbelow ESA’s 2–16 km recommendation, as most Mie-cloudy measurements are located underneath 16 km altitude. As with the\nbias, the STD and SMAD of Mie-cloudy are also quite independent of orbital and regional dependence. The overall accuracy\nof Mie-cloudy depends on the signal accumulation, the classification algorithm and the quality of the calibration data. The accuracy of Mie-cloudy winds is higher than that of Rayleigh-clear winds as particle backscatter is usually stronger than that\n310\nof clear air in addition to the fact that Mie backscatter is not subject to broadening induced by Rayleigh-Brillouin scattering\n(Witschas et al., 2012). https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Table 3. Overview of the total systematic (μ, σμ; ms–1) and random (σAeolus; ms–1) errors derived according to Eq. 8 for Rayleigh-clear\nand Mie-cloudy winds for altitudes ranges 2–16km and 16–20km, as well as the corresponding ESA’s error recommendations. The random\nerror σAeolus was computed for a representativeness error σrep ranging from 1.5 ms–1 to 2.5 ms–1. For Mie-cloudy, only the altitude range\n2–16km is shown for the random error, as Mie-cloudy does not sample sufficiently above 16km. Rayleigh-clear\nMie-cloudy\nσAeolus 2–16km\nσAeolus 16–20km\nμ\nσAeolus 2–16km\nμ\nAscending\n3.4 – 3.9\n4.0 – 4.4\n-0.2±0.3\n1.1 – 2.3\n-0.8±0.6\nDescending\n4.3 – 4.7\n4.4 – 4.9\n-0.9±0.4\n0.5 – 2.1\n-1.1±0.4\nAll\n3.8 – 4.3\n4.3 – 4.8\n-0.5±0.2\n1.1 – 2.3\n-0.9±0.3\nESA\n2.5\n3\n0.7\n2.5\n0.7 The difference in results is caused by the different altitudes at which the data are sampled, as the The difference in results is caused by the different altitudes at which the data are sampled, as the aircraft only samples the lower\n10 km portion of the troposphere, which is shown to be more noisy owing to the abundance of dust aerosols in this region. 320\nFor Mie-cloudy, the random error gives 2.9 ± 0.3 ms–1, which is similar to our radiosonde-based results as most Mie-cloudy\nscattering occurs at lower levels. 320 Comparing the results of different Cal/Val studies is tricky as the influence of geographical regions, atmospheric conditions,\ndecreasing laser energy, product baseline and quality control procedures on the result can be significant and must be considered. accuracy of Mie-cloudy winds is higher than that of Rayleigh-clear winds as particle backscatter is usually stronger than that\n310\nof clear air in addition to the fact that Mie backscatter is not subject to broadening induced by Rayleigh-Brillouin scattering\n(Witschas et al., 2012). Comparing the results of different Cal/Val studies is tricky as the influence of geographical regions, atmospheric conditions,\ndecreasing laser energy, product baseline and quality control procedures on the result can be significant and must be considered. In this analysis, comparisons are only made with statistics derived from AVATAR-T airborne-based measurements (Witschas\n315\net al., 2022; Lux et al., 2022b), as these were carried out in the framework of the same JATAC campaign. The statistical analysis\nof AVATAR-T shows systematic errors of –0.1 ± 0.3 ms–1 for Rayleigh-clear and –0.7 ± 0.2 ms–1 for Mie-cloudy, which are\nslightly smaller than for radiosondes. However, the random error of 7.1 ± 0.3 ms–1 for Rayleigh-clear is significantly higher. In this analysis, comparisons are only made with statistics derived from AVATAR-T airborne-based measurements (Witschas\n315\net al., 2022; Lux et al., 2022b), as these were carried out in the framework of the same JATAC campaign. The statistical analysis\nof AVATAR-T shows systematic errors of –0.1 ± 0.3 ms–1 for Rayleigh-clear and –0.7 ± 0.2 ms–1 for Mie-cloudy, which are\nslightly smaller than for radiosondes. However, the random error of 7.1 ± 0.3 ms–1 for Rayleigh-clear is significantly higher. 12 4.1.3\nOrbital bias in the Rayleigh-clear channel Figure 2 shows vertical profiles of the differences between Aeolus Rayleigh-clear observations and radiosonde measurements\nprojected along HLOS (O-RS; solid lines), and the corresponding ECMWF model equivalents (O-B; dotted lines) for both\n325\nascending (red) and descending (blue) orbits over Sal (a), PR and SCRX (b). The shading represents the bias uncertainty σμ. HLOS winds from the descending track are multiplied by -1 to conform with the sign convention of the model coordinate\nsystem. The vertical profiles illustrate the presence of an ascending/descending bias visible in both the O-B and O-RS profiles,\nreaching up to 2.5 ms–1 around 8 km altitude in both regions. The differences below 5 km altitude could be related to the greater amount of dust in Cabo Verde during this period, while above 17 km the differences could partly be related to the lack\n330\nof descending orbit data over Sal (Fig. 2a). This altitude- and orbit-dependent bias was already described by Borne et al. (2023)\nusing first-guess departure statistics over West Africa. greater amount of dust in Cabo Verde during this period, while above 17 km the differences could partly be related to the lack\n330\nof descending orbit data over Sal (Fig. 2a). This altitude- and orbit-dependent bias was already described by Borne et al. (2023)\nusing first-guess departure statistics over West Africa. using first-guess departure statistics over West Africa. This latitude consistent bias caused the zonal winds in the ECMWF analysis to accelerate in the morning and weaken in\nthe evening, affecting the African Easterly Jet (AEJ) and Tropical Easterly Jet (TEJ) in particular. Correcting this bias with This latitude consistent bias caused the zonal winds in the ECMWF analysis to accelerate in the morning and weaken in\nthe evening, affecting the African Easterly Jet (AEJ) and Tropical Easterly Jet (TEJ) in particular. Correcting this bias with a temperature-dependent approach helped to improve the representation of winds in the analysis and forecast fields (Borne\n335\net al., 2023). However, the cause of this bias remains unknown, as it has not been proven to be related to temperature, nor has\nany dependence on wind speed, SNR or useful signal been found (not shown here). Here, as both the O-B and O-RS profiles\nare very close to each other, with deviations below 0.5 ms–1, the existence of this bias can be confirmed observationally with\nradiosondes. As highlighted by Horányi et al. 4.1.3\nOrbital bias in the Rayleigh-clear channel Differences (dots) and average differences (lines) between ascending (red) and descending (blue) winds between Aeolus observa-\ntions (O) and radiosonde HLOS wind measurements (RS, solid line) along with ECMWF model equivalents (B, dotted line) over Sal (a) and\nPuerto Rico - Saint Croix (PR/SCRX, (b). The shadow represents the bias uncertainty σμ. To comply with the sign convention of the model\ncoordinate system, the HLOS winds from the descending orbit are multiplied by -1. Figure 2. Differences (dots) and average differences (lines) between ascending (red) and descending (blue) w Figure 2. Differences (dots) and average differences (lines) between ascending (red) and descending (blue) winds between Aeolus observa-\ntions (O) and radiosonde HLOS wind measurements (RS, solid line) along with ECMWF model equivalents (B, dotted line) over Sal (a) and\nPuerto Rico - Saint Croix (PR/SCRX, (b). The shadow represents the bias uncertainty σμ. To comply with the sign convention of the model\ncoordinate system, the HLOS winds from the descending orbit are multiplied by -1. 4.1.3\nOrbital bias in the Rayleigh-clear channel (2015), biases of the order of 1 ms–1 can already deteriorate forecast quality. a temperature-dependent approach helped to improve the representation of winds in the analysis and forecast fields (Borne\n335\net al., 2023). However, the cause of this bias remains unknown, as it has not been proven to be related to temperature, nor has\nany dependence on wind speed, SNR or useful signal been found (not shown here). Here, as both the O-B and O-RS profiles\nare very close to each other, with deviations below 0.5 ms–1, the existence of this bias can be confirmed observationally with\nradiosondes. As highlighted by Horányi et al. (2015), biases of the order of 1 ms–1 can already deteriorate forecast quality. a temperature-dependent approach helped to improve the representation of winds in the analysis and forecast fields (Borne\n335\net al., 2023). However, the cause of this bias remains unknown, as it has not been proven to be related to temperature, nor has\nany dependence on wind speed, SNR or useful signal been found (not shown here). Here, as both the O-B and O-RS profiles\nare very close to each other, with deviations below 0.5 ms–1, the existence of this bias can be confirmed observationally with\nradiosondes. As highlighted by Horányi et al. (2015), biases of the order of 1 ms–1 can already deteriorate forecast quality. 13 Figure 2. Differences (dots) and average differences (lines) between ascending (red) and descending (blue) winds between Aeolus observa-\ntions (O) and radiosonde HLOS wind measurements (RS, solid line) along with ECMWF model equivalents (B, dotted line) over Sal (a) and\nPuerto Rico\nSaint Croix (PR/SCRX (b) The shadow represents the bias uncertainty\nTo comply with the sign convention of the model Figure 2. Differences (dots) and average differences (lines) between ascending (red) and descending (blue) winds between Aeolus observa-\ntions (O) and radiosonde HLOS wind measurements (RS, solid line) along with ECMWF model equivalents (B, dotted line) over Sal (a) and\nPuerto Rico - Saint Croix (PR/SCRX, (b). The shadow represents the bias uncertainty σμ. To comply with the sign convention of the model\ncoordinate system, the HLOS winds from the descending orbit are multiplied by -1. Figure 2. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Rayleigh-clear and Rayleigh-cloudy Figure 3 shows the absolute difference between Aeolus and radiosonde measurement points |ΔdiffHLOS| as a function of EEtot\n(a), altitude (b), co-location radius (c) and co-location time (d) for the Rayleigh-clear (blue) and Rayleigh-cloudy (orange)\nobservation types. The solid and dashed blue lines show the Rayleigh-clear MADI and SMAD, respectively, with each value\ncalculated using a minimum sample size of 40 data points for panels a, b and d. Also shown are outliers (cross symbol +),\n350\nthat we define in this study as values with low EE (< 5 ms–1) and large absolute difference (> 10 ms–1), which are of particular\ninterest as they contribute the most to the wind quality degradation. The Rayleigh-clear outliers account for 13 observations,\ni.e. ∼3.4% of the data points. For Rayleigh-cloudy, no MADI and SMAD are computed due to the lack of data. Figure 3 shows the absolute difference between Aeolus and radiosonde measurement points |ΔdiffHLOS| as a function of EEtot\n(a), altitude (b), co-location radius (c) and co-location time (d) for the Rayleigh-clear (blue) and Rayleigh-cloudy (orange)\nobservation types. The solid and dashed blue lines show the Rayleigh-clear MADI and SMAD, respectively, with each value observation types. The solid and dashed blue lines show the Rayleigh-clear MADI and SMAD, respectively, with each value\ncalculated using a minimum sample size of 40 data points for panels a, b and d. Also shown are outliers (cross symbol +),\n350\nthat we define in this study as values with low EE (< 5 ms–1) and large absolute difference (> 10 ms–1), which are of particular\ninterest as they contribute the most to the wind quality degradation. The Rayleigh-clear outliers account for 13 observations,\ni.e. ∼3.4% of the data points. For Rayleigh-cloudy, no MADI and SMAD are computed due to the lack of data. In general, the MADI and SMAD between Rayleigh-clear and radiosonde wind measurements appear to be proportional to In general, the MADI and SMAD between Rayleigh-clear and radiosonde wind measurements appear to be proportional to\nthe Aeolus EEtot (Fig. 3a), with larger deviations associated with larger EEtots, as expected. However, on average, the mean\n355\nEEtot overestimates the MADI by 1 ms–1 for EEtot values below 6 ms–1 (see grey line). This discrepancy can be attributed to\nthe relatively small amount of data used in the study, as the EE is based on the Gaussian assumption of a large data set. 4.2\nError dependency\n340 In this section we examine the error dependency and associated error sources of the different Aeolus wind products. Firstly,\nwe investigate the error dependency as a function of co-location parameters, such as radius and time difference between two\nmeasurement points, to account for representativeness. Secondly, we explore the error dependency in relation to the presence\nof clouds and dust, as these supposedly influence the quality of Aeolus wind products. 14 Rayleigh-clear and Rayleigh-cloudy For\nRayleigh-cloudy measurements, it is difficult to establish a dependency although the absolute difference appears to be generally\nlarger owing to the large STD of 6.6 ms–1 for this observation type. Considering the altitude error dependency of Rayleigh- the Aeolus EEtot (Fig. 3a), with larger deviations associated with larger EEtots, as expected. However, on average, the mean\n355\nEEtot overestimates the MADI by 1 ms–1 for EEtot values below 6 ms–1 (see grey line). This discrepancy can be attributed to\nthe relatively small amount of data used in the study, as the EE is based on the Gaussian assumption of a large data set. For\nRayleigh-cloudy measurements, it is difficult to establish a dependency although the absolute difference appears to be generally\nlarger owing to the large STD of 6.6 ms–1 for this observation type. Considering the altitude error dependency of Rayleigh- clear (Fig. 3b), a general pattern emerges with MADI and SMAD reaching a minimum of 3 ms–1 and 2 ms–1 respectively on\n360\naverage in the middle troposphere at 10 km, while increasing above and below, with MADIs of 4–5 ms–1 and SMADs of almost\n6 ms–1 at 2.5 km and 19 km altitude. As we will see in the next subsection 4.2.2, this error pattern is inversely proportional\nto the Rayleigh backscattered useful signal, as it directly affects the SNR and thereby the quality of the measurement points. Rayleigh-clear outliers seem to occur at all altitudes and Rayleigh-cloudy measurements are primarily found in the lower clear (Fig. 3b), a general pattern emerges with MADI and SMAD reaching a minimum of 3 ms–1 and 2 ms–1 respectively on\n360\naverage in the middle troposphere at 10 km, while increasing above and below, with MADIs of 4–5 ms–1 and SMADs of almost\n6 ms–1 at 2.5 km and 19 km altitude. As we will see in the next subsection 4.2.2, this error pattern is inversely proportional\nto the Rayleigh backscattered useful signal, as it directly affects the SNR and thereby the quality of the measurement points. Rayleigh-clear outliers seem to occur at all altitudes and Rayleigh-cloudy measurements are primarily found in the lower troposphere, below 6 km. 365\nIn Fig. 3c we examine the error dependency with respect to the co-location radius, which extends up to 340 km, a distance\nthat is large relative to the 100 km specified in ESA’s recommendations. Rayleigh-clear and Rayleigh-cloudy However, the MADI and SMAD for Rayleigh-clear\ndo not increase with radius, but stagnate at an average of 3–4 ms–1 for radii above 100 km, while they are slightly higher\nbelow 100 km, reaching 4–5 ms–1. Furthermore, outliers appear across all co-location radii. This indicates that the use of a troposphere, below 6 km. 365\nIn Fig. 3c we examine the error dependency with respect to the co-location radius, which extends up to 340 km, a distance\nthat is large relative to the 100 km specified in ESA’s recommendations. However, the MADI and SMAD for Rayleigh-clear\ndo not increase with radius, but stagnate at an average of 3–4 ms–1 for radii above 100 km, while they are slightly higher\nbelow 100 km, reaching 4–5 ms–1. Furthermore, outliers appear across all co-location radii. This indicates that the use of a troposphere, below 6 km. 365\nIn Fig. 3c we examine the error dependency with respect to the co-location radius, which extends up to 340 km, a distance\nthat is large relative to the 100 km specified in ESA’s recommendations. However, the MADI and SMAD for Rayleigh-clear\ndo not increase with radius, but stagnate at an average of 3–4 ms–1 for radii above 100 km, while they are slightly higher\nbelow 100 km, reaching 4–5 ms–1. Furthermore, outliers appear across all co-location radii. This indicates that the use of a co-location distance up to 340 km is acceptable for the statistical comparison. Exploring the error dependency with respect to\n370\nthe time difference between the observations (Fig. 3d), there is indication for increasing difference for larger time-differences,\ngoing from 3–4 ms–1 at 0 minutes to 4–6 ms–1 above 30 minutes. There is also an asymmetry of the error dependence, with a\nlarger error magnitude for radiosonde observations preceding the Aeolus passage. Since most radiosondes were launched with\nthe objective of reaching the mid-troposphere during the satellite’s passage, the measurements preceding/following Aeolus of co-location distance up to 340 km is acceptable for the statistical comparison. Exploring the error dependency with respect to\n370\nthe time difference between the observations (Fig. 3d), there is indication for increasing difference for larger time-differences,\ngoing from 3–4 ms–1 at 0 minutes to 4–6 ms–1 above 30 minutes. There is also an asymmetry of the error dependence, with a\nlarger error magnitude for radiosonde observations preceding the Aeolus passage. Rayleigh-clear and Rayleigh-cloudy EEtot (a), altitude (b), co-location radius (c) and co-location time quantities expressed as a function of the absolute difference\nbetween radiosonde HLOS winds (HLOSRS) and Aeolus (HLOSAEOLUS) Rayleigh-clear (blue) and Rayleigh-cloudy (orange) observations. Outliers are defined as values with an EE below 5 ms–1 and absolute difference larger than 10 ms–1 and are represented by the cross symbol\n+. The solid blue lines indicate the MADI while the dotted blue lines represent the SMAD of Rayleigh-clear and each value is computed\nusing a minimum sample size of 40 data points. The grey line in panel a represents the diagonal at intercept 0 with slope 1. Due to the limited\namount of data, no MADI and SMAD are shown for Rayleigh-cloudy. Figure 3. EEtot (a), altitude (b), co-location radius (c) and co-location time quantities expressed as a function of the absolute difference\nbetween radiosonde HLOS winds (HLOSRS) and Aeolus (HLOSAEOLUS) Rayleigh-clear (blue) and Rayleigh-cloudy (orange) observations. Outliers are defined as values with an EE below 5 ms–1 and absolute difference larger than 10 ms–1 and are represented by the cross symbol\n+. The solid blue lines indicate the MADI while the dotted blue lines represent the SMAD of Rayleigh-clear and each value is computed\nusing a minimum sample size of 40 data points. The grey line in panel a represents the diagonal at intercept 0 with slope 1. Due to the limited\namount of data, no MADI and SMAD are shown for Rayleigh-cloudy. +. The solid blue lines indicate the MADI while the dotted blue lines represent the SMAD of Rayleigh-cle\nusing a minimum sample size of 40 data points. The grey line in panel a represents the diagonal at intercept 0\namount of data, no MADI and SMAD are shown for Rayleigh-cloudy. Rayleigh-clear and Rayleigh-cloudy Since most radiosondes were launched with\nthe objective of reaching the mid-troposphere during the satellite’s passage, the measurements preceding/following Aeolus of co-location distance up to 340 km is acceptable for the statistical comparison. Exploring the error dependency with respect to\n370\nthe time difference between the observations (Fig. 3d), there is indication for increasing difference for larger time-differences,\ngoing from 3–4 ms–1 at 0 minutes to 4–6 ms–1 above 30 minutes. There is also an asymmetry of the error dependence, with a\nlarger error magnitude for radiosonde observations preceding the Aeolus passage. Since most radiosondes were launched with\nthe objective of reaching the mid-troposphere during the satellite’s passage, the measurements preceding/following Aeolus of more than 30 minutes correspond mainly to measurements at lower/higher altitudes. The larger MADI and SMADI values for\n375\nthese time differences could hence be an indirect effect of the larger errors found at those altitudes (Fig. 3b). Again, no error\ndependency is observed for outliers, with most occurring below ±40 minutes time differences. more than 30 minutes correspond mainly to measurements at lower/higher altitudes. The larger MADI and SMADI values for\n375\nthese time differences could hence be an indirect effect of the larger errors found at those altitudes (Fig. 3b). Again, no error\ndependency is observed for outliers, with most occurring below ±40 minutes time differences. 15 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Figure 3. EEtot (a), altitude (b), co-location radius (c) and co-location time quantities expressed as a function of the absolute difference\nbetween radiosonde HLOS winds (HLOSRS) and Aeolus (HLOSAEOLUS) Rayleigh-clear (blue) and Rayleigh-cloudy (orange) observations. Outliers are defined as values with an EE below 5 ms–1 and absolute difference larger than 10 ms–1 and are represented by the cross symbol\n+. The solid blue lines indicate the MADI while the dotted blue lines represent the SMAD of Rayleigh-clear and each value is computed\nusing a minimum sample size of 40 data points. The grey line in panel a represents the diagonal at intercept 0 with slope 1. Due to the limited\namount of data, no MADI and SMAD are shown for Rayleigh-cloudy. Figure 3. Mie-cloudy Figure 4 shows the same error dependencies as in Fig. 3, but for the Mie-cloudy observation type. For Mie-cloudy, we define\noutliers as values exceeding an absolute error of 6 ms–1 along with EEs inferior to 3 ms–1. With a total of 3 data points, they\n380\naccount for ∼5% of the total Mie-cloudy observations. In panels a, b and d, each MADI and SMAD value is calculated using\na minimum sample size of 15 data points As shown in Fig. 4a, the absolute differences for Mie-cloudy measurements are generally smaller than for Rayleigh-clear,\nwith the largest deviations around 7–8 ms–1, while attaining 13–14 ms–1 for Rayleigh-clear. The MADI and SMAD remain\nbetween 2 and 3 ms–1, indicating an overestimation of the EEtot, especially for increasing EEtot. Regarding the altitude error\n385 16 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. dependency (Fig. 4b), most of the data are found within the 10-15 km layer, which is probably related to the presence of high-\nlevel clouds, and below 7 km, where low- and mid-level clouds and dust layers are found. Due to the sparseness of Mie-cloudy\ndata, both MADI and SMADI do not show a specific vertical error trend. While MADI and SMAD remain between 2.3 and 2.7\nms–1, respectively, they decrease to 1.8 and 2.3 between 1.5 and 3 km altitude before increasing to almost 3 ms–1 in the lowest\n1 km. Fig. 4c shows that similarly to Rayleigh-clear, Mie-cloudy reveals no error dependency with respect to co-location radii,\n390\nwith the mean absolute error and SMAD mainly ranging from 1.7 to 3.2 ms–1, and outliers found at all radii. Regarding the\nerror dependence on time difference (Fig. 4d), we find that most of the measurement differences occur at time intervals of\nless than ±40 minutes. MADIs and SMADs are generally higher for negative co-location times, corresponding to cases where\nradiosonde observations are sampled before those from Aeolus. Nevertheless, we do not notice a strong relationship between\nco location time and errors\n395 data, both MADI and SMADI do not show a specific vertical error trend. While MADI and SMAD remain between 2.3 and 2.7\nms–1, respectively, they decrease to 1.8 and 2.3 between 1.5 and 3 km altitude before increasing to almost 3 ms–1 in the lowest\n1 km. Fig. 4c shows that similarly to Rayleigh-clear, Mie-cloudy reveals no error dependency with respect to co-location radii,\n390\nwith the mean absolute error and SMAD mainly ranging from 1.7 to 3.2 ms–1, and outliers found at all radii. Regarding the\nerror dependence on time difference (Fig. 4d), we find that most of the measurement differences occur at time intervals of\nless than ±40 minutes. MADIs and SMADs are generally higher for negative co-location times, corresponding to cases where\nradiosonde observations are sampled before those from Aeolus. Nevertheless, we do not notice a strong relationship between\nl\nti\nti\nd\n395 1 km. Fig. 4c shows that similarly to Rayleigh-clear, Mie-cloudy reveals no error dependency with respect to co-location radii,\n390\nwith the mean absolute error and SMAD mainly ranging from 1.7 to 3.2 ms–1, and outliers found at all radii. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4.2.2\nCloud type and dust As already mentioned, the accuracy of Rayleigh-clear and, to a lesser extent, Mie-cloudy depends on the signal level and\nSNR. In general, the signal level depends on the range bin thickness, the horizontal accumulation length, the atmospheric\npath signal and the overall signal background level. In addition, Rayleigh-clear winds are sensitive to signal attenuation due\nto atmospheric conditions, with weaker signal return under optically thick clouds and dust-aerosol layers. Mie-cloudy is less\n400\nconcerned as backscatter from particles is stronger, although it is sensitive to weak backscatter, e.g. from dust layers. Because\nof its strong sensitivity on signal levels, the EE of Rayleigh-clear only considers Poisson noise and is therefore inversely\nproportional to the squared root of the useful signal. For Mie-cloudy, this rule of thumb is not true. In this context, we aim to\ninvestigate the quality of the Rayleigh-clear and Mie-cloudy winds and the reliability of the corresponding EE with respect to the presence of clouds and dust. 405 Rayleigh-clear Table 4 describes the error dependency of the Rayleigh-clear observations with respect to the presence of clouds and dust,\nwith cases below 50%, above 50% and above 75% of cloudiness, as well as sub-categories distinguishing the dust mixing\nratio above (Dust) and below (DustNO) 10–8 kgkg–1. Note that SMAD is not used for this analysis as this reliably removes Table 4 describes the error dependency of the Rayleigh clear observations with respect to the presence of clouds and dust,\nwith cases below 50%, above 50% and above 75% of cloudiness, as well as sub-categories distinguishing the dust mixing\nratio above (Dust) and below (DustNO) 10–8 kgkg–1. Note that SMAD is not used for this analysis as this reliably removes\noutliers, which ought to be quantified here. We note that the MADI, the STD, and the EEtot all increase with the amount of\n410\nclouds and dust along the track, presumably due to the reduced return signal. In non-dusty conditions (DustNO), we observe\nthat for low cloud cover (<50%), the MADI (3.3 ± 0.2 ms–1) is significantly lower than the EEtot (4.8 ms–1) with a difference\nof 1.5 ms–1, while for higher cloud cover, the difference between MADI and EEtot is much smaller (1.1 ms–1 and 1.0 ms–1\nfor above 50% and 75% of cloudiness, respectively). This phenomenon is further enhanced at higher dust concentrations, with\n1\n1 NO\ng g\ny\ny\noutliers, which ought to be quantified here. We note that the MADI, the STD, and the EEtot all increase with the amount of\n410\nclouds and dust along the track, presumably due to the reduced return signal. In non-dusty conditions (DustNO), we observe\nthat for low cloud cover (<50%), the MADI (3.3 ± 0.2 ms–1) is significantly lower than the EEtot (4.8 ms–1) with a difference\nof 1.5 ms–1, while for higher cloud cover, the difference between MADI and EEtot is much smaller (1.1 ms–1 and 1.0 ms–1\nfor above 50% and 75% of cloudiness, respectively). This phenomenon is further enhanced at higher dust concentrations, with the MADI reaching even higher values (5.7 ± 0.8 ms–1) than the EEtot (5.8 ms–1) for cloud cover above 75%. This highlights\n415\nhow the EEtot in clear sky conditions is well calibrated, while it is becoming gradually too low with the increasing presence of\nclouds and dust. Regarding the\nerror dependence on time difference (Fig. 4d), we find that most of the measurement differences occur at time intervals of\nless than ±40 minutes. MADIs and SMADs are generally higher for negative co-location times, corresponding to cases where\nradiosonde observations are sampled before those from Aeolus. Nevertheless, we do not notice a strong relationship between Figure 4. Same as for Fig. 3, but for Mie-cloudy. For Mie-cloudy (red), outliers are defined as values having an absolute error above 6 ms–1\nand an EE inferior to 3 ms–1. The MADI and the SMAD values are computed using a minimum sample size of 15 data points. Figure 4. Same as for Fig. 3, but for Mie-cloudy. For Mie-cloudy (red), outliers are defined as values having an absolute error above 6 ms–1\nand an EE inferior to 3 ms–1. The MADI and the SMAD values are computed using a minimum sample size of 15 data points. 17 17 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Table 4. Overview of the total Error Estimate (EEtot; ms–1), mean absolute difference and uncertainty (MADI, σμ; ms–1), Standard deviation\n(STD; ms–1) and counts (COUNT) for the Rayleigh-clear measurements under different cloud and dust conditions. This includes three\ncategories of cloud cover (< 25 %, > 50 %, > 75 %) for dust mixing ratios above (Dust) and below (DustNO) 109 kgkg–1 along the track. Table 4. Overview of the total Error Estimate (EEtot; ms–1), mean absolute difference and uncertainty (MADI, σμ; ms–1), Standard deviation\n(STD; ms–1) and counts (COUNT) for the Rayleigh-clear measurements under different cloud and dust conditions. This includes three\ncategories of cloud cover (< 25 %, > 50 %, > 75 %) for dust mixing ratios above (Dust) and below (DustNO) 109 kgkg–1 along the track. Cloud < 50 %\nCloud > 50 %\nCloud > 75 %\nDustNO\nDust\nDustNO\nDust\nDustNO\nDust\nEEtot\n4.8\n5.4\n5.0\n5.6\n5.3\n5.8\nMADI\n3.3±0.2\n4.4±0.6\n3.9±0.5\n5.0±0.5\n4.3±0.7\n5.7±0.8\nSTD\n4.3\n5.0\n5.1\n5.9\n5.6\n6.4\nCOUNT\n234\n28\n64\n52\n38\n24\nFigure 5. Altitude as a function of Rayleigh-clear absolute difference |ΔdiffHLOS| (a,e), EEtot (b,f), normalized useful signal (c,g) and SR\n(d,h), where the colouring is dependent on the percentage of SAF clouds (upper row) and CAMS dust mixing ratio (lower row) along the\ntrack. The cross symbol + stands for outliers and defines values with an EE below 5 ms–1 and an absolute difference of more than 10 ms–1. Panel (a) includes the MADI for each cloud cover percentage, with a minimum sample size of 10 data points used to compute each value. Cloud < 50 %\nCloud > 50 %\nCloud > 75 %\nDustNO\nDust\nDustNO\nDust\nDustNO\nDust\nEEtot\n4.8\n5.4\n5.0\n5.6\n5.3\n5.8\nMADI\n3.3±0.2\n4.4±0.6\n3.9±0.5\n5.0±0.5\n4.3±0.7\n5.7±0.8\nSTD\n4.3\n5.0\n5.1\n5.9\n5.6\n6.4\nCOUNT\n234\n28\n64\n52\n38\n24 Cloud < 50 %\nCloud > 50 %\nCloud > 75 %\nDustNO\nDust\nDustNO\nDust\nDustNO\nDust\nEEtot\n4.8\n5.4\n5.0\n5.6\n5.3\n5.8\nMADI\n3.3±0.2\n4.4±0.6\n3.9±0.5\n5.0±0.5\n4.3±0.7\n5.7±0.8\nSTD\n4.3\n5.0\n5.1\n5.9\n5.6\n6.4\nCOUNT\n234\n28\n64\n52\n38\n24 Figure 5. Rayleigh-clear The larger STD with increasing cloudiness and dust concentration suggests an increasingly perturbed pattern\nof Rayleigh-clear measurements,possibly owing to the lower signal levels or to a cross-talk. Figure 5 puts this phenomenon into perspective, by showing the altitude-dependent absolute difference |ΔdiffHLOS| (a,e), the EEtot (b,f), the normalized useful signal (c,g) and the SR (d,h), where the colouring depends on the percentage of SAF clouds\n420\n(upper row) and the CAMS dust mixing ratio (lower row) along the track. For reference, the values that did not pass the QC are\nshown transparently. In addition, panel 5a includes the MADI of four cloud cover percentage categories, where each MADI is\ncomputed with a minimum sample size of 10 values. The colourings in Fig. 5 are illustrative of the results summarised in Table\n4, with measurements showing generally greater MADI under high cloud cover (red, orange, Fig. 5a) than under lower cloud cover (blue, blue-green). Measurements in the lower troposphere are naturally more strongly affected by cloud cover compared\n425\nto higher levels. The same applies to dust (Fig. 5e), which also occurs mainly in the lower 5 km of the troposphere. As we have already shown in Fig. 3b, the absolute error is higher in the upper and lower troposphere and minimised in\nthe middle troposphere around 10 km altitude. This trend is well reflected in the EEtot in Fig. 5b, which is an indication of As we have already shown in Fig. 3b, the absolute error is higher in the upper and lower troposphere and minimised in\nthe middle troposphere around 10 km altitude. This trend is well reflected in the EEtot in Fig. 5b, which is an indication of 18 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. density. In the lower troposphere, the return signal is lower due to strong attenuation under clouds and dust layers. Interestingly,\nthe values with high EEtot and smaller useful signal in the mid-troposphere between 5 and 12.5 km in red likely correspond to\nmeasurements sampled under thick clouds, resulting in a strongly attenuated signal. They account for most of the measurements\nwith cloud cover greater than 75 % in this altitude range, while the cloud tops appear to be located between 12.5 and 15 km, as\n435\nthey exhibit a larger normalized useful signal and a SR greater than 1 (Fig. 5d,h). Finally, outliers are found under all types of\ncloud and dust conditions and affect different altitude ranges. They also occur for regular normalized useful signals, with most\nSRs lying around 1, which rules out a cause related to atmospheric particles. Altitude as a function of Rayleigh-clear absolute difference |ΔdiffHLOS| (a,e), EEtot (b,f), normalized useful signal (c,g) and SR\n(d,h), where the colouring is dependent on the percentage of SAF clouds (upper row) and CAMS dust mixing ratio (lower row) along the\ntrack. The cross symbol + stands for outliers and defines values with an EE below 5 ms–1 and an absolute difference of more than 10 ms–1. Panel (a) includes the MADI for each cloud cover percentage, with a minimum sample size of 10 data points used to compute each value. the generally good consistency between the EEtot and the absolute differences. As expected, this tendency fits inversely with\nthe normalized useful signal shown in Fig. 5c, with lower signal in the upper and lower troposphere. Indeed, in the higher\n430\ntroposphere the air is less dense and the thickness of the RB’s is not sufficient to compensate for the decrease in air molecule the generally good consistency between the EEtot and the absolute differences. As expected, this tendency fits inversely with\nthe normalized useful signal shown in Fig. 5c, with lower signal in the upper and lower troposphere. Indeed, in the higher\n430\ntroposphere the air is less dense and the thickness of the RB’s is not sufficient to compensate for the decrease in air molecule i\nthe normalized useful signal shown in Fig. 5c, with lower signal in the upper and lower troposphere. Indeed, in the higher\n430\ntroposphere the air is less dense and the thickness of the RB’s is not sufficient to compensate for the decrease in air molecule 19 Mie-cloudy 4b,\nmost backscatter occurs in two layers, i.e. within 10–15 km and below 7 km altitude. The majority of measurements have\nnormalized useful signals above 5e13 a.u. (Fig. 6c,g), which is overall above the normalized useful signal of the rejected\n450\nmeasurements shown in transparent. Furthermore, the SRs are generally above 1 (Fig. 6d,h), which is characteristic of Mie-\ncloudy measurements. More specifically, measurements sampled above 12.5 km have a cloud cover of more than 75 % along\nthe track and probably correspond to cloud tops, as they have stronger SRs between 1.5 and 3 (Fig. 6d,h). They exhibit good\nquality as well, with an average MADI of 1.5 ms–1 (Fig. 4b). Between 7.5 and 12.5 km altitude, most of the measurements\noccur with cloud cover less than 50 %, with SRs falling below 1.3. In this altitude range, there are also 2 outliers, which\n455\ninterestingly have SRs around 1 and a normalized useful signal in the same order of magnitude as the discarded ones. Their\npresence is unusual, as Mie-cloudy measurements are only obtainable for SRs above 1. Finally, below 7.5 km, the cloud cover\nis mainly above 50 %, while the dust concentration is mainly below 5 × 10–8 kgkg–1, showing that most of the Mie-cloudy\nbackscatter results from clouds and not from dust. As can be seen in Fig. 6g, measurements with high dust concentration\n(brown) are discarded (transparent) with normalized useful signals below 5e13 a.u. Surprisingly, measurements sampled at the\n460\nlower 1 km have the lowest normalized useful signals, mostly below 5e13 a.u. and are not discarded. These, however, tend to\nhave larger SRs between 1 and 2, which can compensate for the low normalized useful signal in the calculation of the EE. They\nalso correspond to the largest MADI scaling up to 4 ms–1 on average (i.e. Fig. 4d) in addition to relatively high EEtots (i.e. Fig. 6b,f). Two measurements also show negative SRs, which is an artifact, due to insufficient background signal corrections. The third outlier in the lower 1 km does not have abnormal characteristics compared to other measurements at this altitude. 465 normalized useful signals above 5e13 a.u. (Fig. 6c,g), which is overall above the normalized useful signal of the rejected\n450\nmeasurements shown in transparent. Furthermore, the SRs are generally above 1 (Fig. 6d,h), which is characteristic of Mie-\ncloudy measurements. Mie-cloudy More specifically, measurements sampled above 12.5 km have a cloud cover of more than 75 % along\nthe track and probably correspond to cloud tops, as they have stronger SRs between 1.5 and 3 (Fig. 6d,h). They exhibit good\nquality as well, with an average MADI of 1.5 ms–1 (Fig. 4b). Between 7.5 and 12.5 km altitude, most of the measurements normalized useful signals above 5e13 a.u. (Fig. 6c,g), which is overall above the normalized useful signal of the rejected\n450\nmeasurements shown in transparent. Furthermore, the SRs are generally above 1 (Fig. 6d,h), which is characteristic of Mie-\ncloudy measurements. More specifically, measurements sampled above 12.5 km have a cloud cover of more than 75 % along\nthe track and probably correspond to cloud tops, as they have stronger SRs between 1.5 and 3 (Fig. 6d,h). They exhibit good\nquality as well, with an average MADI of 1.5 ms–1 (Fig. 4b). Between 7.5 and 12.5 km altitude, most of the measurements normalized useful signals above 5e13 a.u. (Fig. 6c,g), which is overall above the normalized useful signal of the rejected\n450\nmeasurements shown in transparent. Furthermore, the SRs are generally above 1 (Fig. 6d,h), which is characteristic of Mie-\ncloudy measurements. More specifically, measurements sampled above 12.5 km have a cloud cover of more than 75 % along\nthe track and probably correspond to cloud tops, as they have stronger SRs between 1.5 and 3 (Fig. 6d,h). They exhibit good\nquality as well, with an average MADI of 1.5 ms–1 (Fig. 4b). Between 7.5 and 12.5 km altitude, most of the measurements occur with cloud cover less than 50 %, with SRs falling below 1.3. In this altitude range, there are also 2 outliers, which\n455\ninterestingly have SRs around 1 and a normalized useful signal in the same order of magnitude as the discarded ones. Their\npresence is unusual, as Mie-cloudy measurements are only obtainable for SRs above 1. Finally, below 7.5 km, the cloud cover\nis mainly above 50 %, while the dust concentration is mainly below 5 × 10–8 kgkg–1, showing that most of the Mie-cloudy\nbackscatter results from clouds and not from dust. As can be seen in Fig. 6g, measurements with high dust concentration occur with cloud cover less than 50 %, with SRs falling below 1.3. Mie-cloudy Table 5 shows the same as Table 4, but for Mie-cloudy. Due to the limited amount of data for Mie-cloudy winds, the inter-\n440\npretation of the results should be treated with caution. We find that, in contrast to Rayleigh-clear, the EE, MADI and STD\ndecrease with the percentage of cloud cover along the path. This is understandable as clouds provide the strongest backscatter\nsignal required for high quality Mie-cloudy measurements. However, the presence of dust for cloud cover below 50 % leads to\na decrease in EEtot, MADI and STD, while conversely there is an increase of these quantities in more dense cloudy conditions Table 5 shows the same as Table 4, but for Mie-cloudy. Due to the limited amount of data for Mie-cloudy winds, the inter-\n440\npretation of the results should be treated with caution. We find that, in contrast to Rayleigh-clear, the EE, MADI and STD\ndecrease with the percentage of cloud cover along the path. This is understandable as clouds provide the strongest backscatter\nsignal required for high quality Mie-cloudy measurements. However, the presence of dust for cloud cover below 50 % leads to\na decrease in EEtot, MADI and STD, while conversely there is an increase of these quantities in more dense cloudy conditions (>50 %, >75 %). A possible explanation is that in clear-sky conditions, the backscatter from dust layers is strong enough to\n445\nobtain high quality measurements, whereas in cloudy conditions, the attenuation by clouds weakens the backscatter return from\nthe dust. Figure 6 depicts the same as Fig. 5, but for Mie-cloudy. As mentioned in the previous section when discussing in Fig. 4b,\nmost backscatter occurs in two layers, i.e. within 10–15 km and below 7 km altitude. The majority of measurements have (>50 %, >75 %). A possible explanation is that in clear-sky conditions, the backscatter from dust layers is strong enough to\n445\nobtain high quality measurements, whereas in cloudy conditions, the attenuation by clouds weakens the backscatter return from\nthe dust. Figure 6 depicts the same as Fig. 5, but for Mie-cloudy. As mentioned in the previous section when discussing in Fig. 4b,\nmost backscatter occurs in two layers, i.e. within 10–15 km and below 7 km altitude. The majority of measurements have Figure 6 depicts the same as Fig. 5, but for Mie-cloudy. As mentioned in the previous section when discussing in Fig. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Table 5. Same as table 4, but for Mie-cloudy. Cloud < 50 %\nCloud > 50 %\nCloud > 75 %\nDustNO\nDust\nDustNO\nDust\nDustNO\nDust\nEEtot\n3.7\n3.6\n3.4\n3.5\n3.2\n3.4\nMADI\n2.8±0.5\n2.4±0.2\n1.8±0.3\n2.5±0.4\n1.6±0.3\n2.6±0.6\nSTD\n2.96\n1.53\n1.89\n2.95\n1.68\n3.18\nCOUNT\n11\n9\n16\n23\n8\n13\nFigure 6. Same as Fig. 5 but for Mie-cloudy. Here the cross symbol + defines values with an EE below 3 ms–1 and an absolute difference\nabove 6 ms–1. Table 5. Same as table 4, but for Mie-cloudy. Cloud < 50 %\nCloud > 50 %\nCloud > 75 %\nDustNO\nDust\nDustNO\nDust\nDustNO\nDust\nEEtot\n3.7\n3.6\n3.4\n3.5\n3.2\n3.4\nMADI\n2.8±0.5\n2.4±0.2\n1.8±0.3\n2.5±0.4\n1.6±0.3\n2.6±0.6\nSTD\n2.96\n1.53\n1.89\n2.95\n1.68\n3.18\nCOUNT\n11\n9\n16\n23\n8\n13 Figure 6 Same as Fig 5 but for Mie cloudy Here the cross symbol + defines values with an EE below 3 ms–1 and an absolute difference Figure 6. Same as Fig. 5 but for Mie-cloudy. Here the cross symbol + defines values with an EE below 3 ms–1 and an absolute difference\nabove 6 ms–1. Mie-cloudy In this altitude range, there are also 2 outliers, which\n455\ninterestingly have SRs around 1 and a normalized useful signal in the same order of magnitude as the discarded ones. Their\npresence is unusual, as Mie-cloudy measurements are only obtainable for SRs above 1. Finally, below 7.5 km, the cloud cover\nis mainly above 50 %, while the dust concentration is mainly below 5 × 10–8 kgkg–1, showing that most of the Mie-cloudy\nbackscatter results from clouds and not from dust. As can be seen in Fig. 6g, measurements with high dust concentration occur with cloud cover less than 50 %, with SRs falling below 1.3. In this altitude range, there are also 2 outliers, which\n455\ninterestingly have SRs around 1 and a normalized useful signal in the same order of magnitude as the discarded ones. Their\npresence is unusual, as Mie-cloudy measurements are only obtainable for SRs above 1. Finally, below 7.5 km, the cloud cover\nis mainly above 50 %, while the dust concentration is mainly below 5 × 10–8 kgkg–1, showing that most of the Mie-cloudy\nbackscatter results from clouds and not from dust. As can be seen in Fig. 6g, measurements with high dust concentration (brown) are discarded (transparent) with normalized useful signals below 5e13 a.u. Surprisingly, measurements sampled at the\n460\nlower 1 km have the lowest normalized useful signals, mostly below 5e13 a.u. and are not discarded. These, however, tend to\nhave larger SRs between 1 and 2, which can compensate for the low normalized useful signal in the calculation of the EE. They\nalso correspond to the largest MADI scaling up to 4 ms–1 on average (i.e. Fig. 4d) in addition to relatively high EEtots (i.e. Fig. 6b,f). Two measurements also show negative SRs, which is an artifact, due to insufficient background signal corrections. The third outlier in the lower 1 km does not have abnormal characteristics compared to other measurements at this altitude. 465 20 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Figure 7. Overview of the cloud-free case study for a radiosonde launched from Sal airport on 9th September 2021 at 18:45 UTC and the\nascending orbit of Aeolus between 19:23:56-19:24:31 UTC for a co-location radius of 180km. (a) Vertical radiosonde HLOS wind profile\n(black solid line) and projected onto Rayleigh-clear RBS (black stepped line), as well as averaged Rayleigh-clear observations (blue dots),\ncorresponding EE (error bars) and ECMWF model equivalents (Meq, stepped lines). (b) Vertical profile of the Rayleigh-clear EEtot (blue\nline), together with the EEtot of all profiles (grey solid lines) and their average (black solid line). (c), (d) Same as (b), but for normalised\nuseful signal and CAMS dust mixing ratio, respectively. (e) Horizontal map showing the SAFNWC CT at 19:00 UTC and the co-location\nperimeter (white solid line), the Aeolus track (red solid line) and the radiosonde launch site (red cross). Figure 7. Overview of the cloud-free case study for a radiosonde launched from Sal airport on 9th September 2021 at 18:45 UTC and the\nascending orbit of Aeolus between 19:23:56-19:24:31 UTC for a co-location radius of 180km. (a) Vertical radiosonde HLOS wind profile\n(black solid line) and projected onto Rayleigh-clear RBS (black stepped line), as well as averaged Rayleigh-clear observations (blue dots),\ncorresponding EE (error bars) and ECMWF model equivalents (Meq, stepped lines). (b) Vertical profile of the Rayleigh-clear EEtot (blue\nline), together with the EEtot of all profiles (grey solid lines) and their average (black solid line). (c), (d) Same as (b), but for normalised\nuseful signal and CAMS dust mixing ratio, respectively. (e) Horizontal map showing the SAFNWC CT at 19:00 UTC and the co-location\nperimeter (white solid line), the Aeolus track (red solid line) and the radiosonde launch site (red cross). passed over on an ascending orbit between 19:23:56 UTC and 19:24:31 UTC within a co-location radius of 180 km around the\nlaunch site. Figure 7a depicts the corresponding sampled radiosonde HLOS wind profile (black lines) as well as Rayleigh-clear\n(blue) wind measurement points with associated EEtot shown as error bars and ECMWF model equivalents shown as stepped\nlines. The corresponding Rayleigh-clear EEtot, normalized useful signal and CAMS dust mixing ratio profiles are shown in\n475\nblue in Figs. 4.2.3\nCase studies To further investigate the properties of the Aeolus wind errors, this section presents three case studies comparing Aeolus and\nradiosonde wind measurements under three different atmospheric conditions, namely clear sky, high cloud cover and high dust\nconcentration. 470 The first case-study illustrated in Fig. 7 presents a comparison between Aeolus and radiosonde wind measurements collected\nunder clear sky conditions. The radiosonde was launched over Sal Airport at 18:45 UTC on 9 September 2021, and Aeolus 21 7b, 7c and 7d, respectively, along with all other profiles in grey and the average of all profiles in black. Figure (blue) wind measurement points with associated EEtot shown as error bars and ECMWF model equivalents shown as stepped\nlines. The corresponding Rayleigh-clear EEtot, normalized useful signal and CAMS dust mixing ratio profiles are shown in\n475\nblue in Figs. 7b, 7c and 7d, respectively, along with all other profiles in grey and the average of all profiles in black. Figure 22 Beneath the cloud\nbase at 13 km altitude, however, it appears that the Rayleigh-clear measurements follow an irregular pattern, with most of the\nmeasurements and error bars not matching the radiosonde observations, reaching deviations higher than 10 ms–1. Accordingly,\nwe find that the EEtot (Fig. 8b) is larger in this altitude range mainly varying between 5 and 6 ms–1, which also corresponds to a sharp decrease of the normalized useful signal well below the average (Fig. 8c). Nonetheless, the ECMWF model-equivalents\n500\nin Fig. 8a remain fairly accurate relative to the radiosonde measurements. This result mirrors the findings presented in the\nprevious section, namely that the Rayleigh-clear EEtot is systematically underestimated when the normalized useful signal\nis strongly attenuated. It appears that the normalized useful signal further decreases below 2.5 km, presumably as a result\nof the increasing dust concentration at this height (Fig. 8d), which most likely leads to a QC rejection of the Rayleigh-clear sharp decrease of the normalized useful signal well below the average (Fig. 8c). Nonetheless, the ECMWF model-equivalents\n500\nin Fig. 8a remain fairly accurate relative to the radiosonde measurements. This result mirrors the findings presented in the\nprevious section, namely that the Rayleigh-clear EEtot is systematically underestimated when the normalized useful signal\nis strongly attenuated. It appears that the normalized useful signal further decreases below 2.5 km, presumably as a result\nof the increasing dust concentration at this height (Fig. 8d), which most likely leads to a QC rejection of the Rayleigh-clear measurements. 505\nLastly, Fig. 9 examines the influence of dust on the quality of Aeolus. In this case, the radiosonde was launched on 21\nSeptember 2021 at 06:50 UTC for a descending orbit of Aeolus, which passed over a co-location perimeter with a radius of 60\nkm between 07:28:44 UTC and 07:29:07 UTC. As can be seen in Fig. 9e, the atmospheric conditions in the co-location area\nwere completely cloud free at 07:30, with some low level cloud further south of the island. The radiosonde profile shown in Fig. measurements. 505\nLastly, Fig. 9 examines the influence of dust on the quality of Aeolus. In this case, the radiosonde was launched on 21\nSeptember 2021 at 06:50 UTC for a descending orbit of Aeolus, which passed over a co-location perimeter with a radius of 60\nkm between 07:28:44 UTC and 07:29:07 UTC. As can be seen in Fig. These high-clouds appear to be located between 13 km and 16 km altitude, as three Mie-cloudy (red) and two Rayleigh-\ncloudy (orange) measurements are found in this range, and where the normalized useful signal is found to have a maximum. In\nthis altitude range, all Rayleigh-clear, Rayleigh-cloudy and Mie-cloudy measurements exhibit good quality, with radiosonde co-location region between 07:28:32 UTC and 07:28:55 UTC, i.e. during the descending node. As can been seen in panel 8e,\n490\nwhich corresponds to SAFNWC CT at 07:30 UTC, Aeolus overpasses a variety of high clouds, mainly high semitransparent\nclouds. These high-clouds appear to be located between 13 km and 16 km altitude, as three Mie-cloudy (red) and two Rayleigh-\ncloudy (orange) measurements are found in this range, and where the normalized useful signal is found to have a maximum. In\nthis altitude range, all Rayleigh-clear, Rayleigh-cloudy and Mie-cloudy measurements exhibit good quality, with radiosonde co-location region between 07:28:32 UTC and 07:28:55 UTC, i.e. during the descending node. As can been seen in panel 8e,\n490\nwhich corresponds to SAFNWC CT at 07:30 UTC, Aeolus overpasses a variety of high clouds, mainly high semitransparent\nclouds. These high-clouds appear to be located between 13 km and 16 km altitude, as three Mie-cloudy (red) and two Rayleigh-\ncloudy (orange) measurements are found in this range, and where the normalized useful signal is found to have a maximum. In\nthis altitude range, all Rayleigh-clear, Rayleigh-cloudy and Mie-cloudy measurements exhibit good quality, with radiosonde measurements generally within the error bars. Above this cloud cover at 16 km, we only find Rayleigh-clear measurements\n495\nthat also perform well, with an EEtot (Fig. 8b) and normalized useful signal (Fig. 8c) close to average. Beneath the cloud\nbase at 13 km altitude, however, it appears that the Rayleigh-clear measurements follow an irregular pattern, with most of the\nmeasurements and error bars not matching the radiosonde observations, reaching deviations higher than 10 ms–1. Accordingly,\nwe find that the EEtot (Fig. 8b) is larger in this altitude range mainly varying between 5 and 6 ms–1, which also corresponds to a measurements generally within the error bars. Above this cloud cover at 16 km, we only find Rayleigh-clear measurements\n495\nthat also perform well, with an EEtot (Fig. 8b) and normalized useful signal (Fig. 8c) close to average. In this case study, the radiosonde was also launched from\nSal airport, this time at 07:00 UTC on 14 September 2021, with a co-location radius of 60 km. Aeolus passed across the\nco-location region between 07:28:32 UTC and 07:28:55 UTC, i.e. during the descending node. As can been seen in panel 8e,\n490\nwhich corresponds to SAFNWC CT at 07:30 UTC, Aeolus overpasses a variety of high clouds, mainly high semitransparent\nclouds. These high-clouds appear to be located between 13 km and 16 km altitude, as three Mie-cloudy (red) and two Rayleigh-\ncloudy (orange) measurements are found in this range, and where the normalized useful signal is found to have a maximum. In\nthis altitude range, all Rayleigh-clear, Rayleigh-cloudy and Mie-cloudy measurements exhibit good quality, with radiosonde\nmeasurements generally within the error bars. Above this cloud cover at 16 km, we only find Rayleigh-clear measurements\n495\nthat also perform well, with an EEtot (Fig. 8b) and normalized useful signal (Fig. 8c) close to average. Beneath the cloud\nbase at 13 km altitude, however, it appears that the Rayleigh-clear measurements follow an irregular pattern, with most of the\nmeasurements and error bars not matching the radiosonde observations, reaching deviations higher than 10 ms–1. Accordingly,\nwe find that the EEtot (Fig. 8b) is larger in this altitude range mainly varying between 5 and 6 ms–1, which also corresponds to a\nsharp decrease of the normalized useful signal well below the average (Fig. 8c). Nonetheless, the ECMWF model-equivalents\n500\nin Fig. 8a remain fairly accurate relative to the radiosonde measurements. This result mirrors the findings presented in the\nprevious section, namely that the Rayleigh-clear EEtot is systematically underestimated when the normalized useful signal\nis strongly attenuated. It appears that the normalized useful signal further decreases below 2.5 km, presumably as a result\nof the increasing dust concentration at this height (Fig. 8d), which most likely leads to a QC rejection of the Rayleigh-clear\nmeasurements. 505 co-location region between 07:28:32 UTC and 07:28:55 UTC, i.e. during the descending node. As can been seen in panel 8e,\n490\nwhich corresponds to SAFNWC CT at 07:30 UTC, Aeolus overpasses a variety of high clouds, mainly high semitransparent\nclouds. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 7e shows the SAFNWC CT over the Cape Verde region at 19:00 UTC. In the latter panel, it can be seen that conditions were\npredominantly cloud-free along the Aeolus track (red solid line) and within the co-location radius (white solid line), while\nsome low clouds can be found in the surrounding area. In these clear-sky conditions, it is not surprising to find that most of the\nmeasurements are of the Rayleigh-clear observation type, with no Mie-cloudy and Rayleigh-cloudy measurements (Fig. 7a). 480\nThroughout the atmosphere above 2.5 km, the quality of Rayleigh-clear is very good, with most error bars overlapping with\nradiosonde measurements and ECMWF model equivalents. In general, we found that the EEtot estimate (Fig. 7b) is below\naverage throughout the atmosphere, with a minimum of 3.5 ms–1 at 8 km altitude and a maximum above 5 ms–1 at 17.5 km\nand 2.5 km altitude. This is consistent with to a normalized useful signal (Fig. 7c) close to the average, except between 2.5 and 12.5 km, where it is higher, most likely due to the absence of cloud attenuation. In general, EEtot and normalized useful signal\n485\ndecrease below 5 km, which is accompanied by an increase in the dust mixing ratio. This increase reaches 1.2 kgkg–1 at about\n2 km altitude, below which no measurements are found, presumably filtered out during the QC procedure. Figure 8 shows the same as Fig. 7, but for cloudy conditions. In this case study, the radiosonde was also launched from\nSal airport, this time at 07:00 UTC on 14 September 2021, with a co-location radius of 60 km. Aeolus passed across the 12.5 km, where it is higher, most likely due to the absence of cloud attenuation. In general, EEtot and normalized useful signal\n485\ndecrease below 5 km, which is accompanied by an increase in the dust mixing ratio. This increase reaches 1.2 kgkg–1 at about\n2 km altitude, below which no measurements are found, presumably filtered out during the QC procedure. Figure 8 shows the same as Fig. 7, but for cloudy conditions. In this case study, the radiosonde was also launched from\nSal airport, this time at 07:00 UTC on 14 September 2021, with a co-location radius of 60 km. Aeolus passed across the Figure 8 shows the same as Fig. 7, but for cloudy conditions. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Figure 8. Same as Fig. 7, but for the case study with high cloud cover. Here, the radiosonde was launched from the Sal airport at 07:00 UTC\non the 14th September 2021, while Aeolus overpassed the co-location area, with radius of 60km, on a descending node between 07:28:32\nand 07:28:55 UTC. In panel a, the red and orange colours represent the averaged Mie-cloudy and Rayleigh-cloudy observations (points),\nrespectively, with the corresponding EE shown as error bars and ECMWF model equivalents (Meq) shown as stepped lines. The SAFNWC\nCT shown in (e) corresponds to 07:30 UTC. Figure 8. Same as Fig. 7, but for the case study with high cloud cover. Here, the radiosonde was launched from the Sal airport at 07:00 UTC\non the 14th September 2021, while Aeolus overpassed the co-location area, with radius of 60km, on a descending node between 07:28:32\nand 07:28:55 UTC. In panel a, the red and orange colours represent the averaged Mie-cloudy and Rayleigh-cloudy observations (points),\nrespectively, with the corresponding EE shown as error bars and ECMWF model equivalents (Meq) shown as stepped lines. The SAFNWC\nCT shown in (e) corresponds to 07:30 UTC. while outliers with EEs of less than 5 ms–1 (Fig. 9b) can be spotted above 15km and below 5km. This error structure is\nsurprising, as both the normalized useful signal and error estimation curves are similar to the one of the cloud-free case study\nin Fig. 7b and 7c. However, in panel 9c, we see that the Rayleigh-clear error pattern coincides with a strong peak in dust\nmixing ratio, reaching more than 2 10–7 kgkg–1 around 3.5 km altitude. The presence of dust seems to affect the quality of\n515\nRayleigh-clear measurements without influencing the normalized useful signal and thus leading to an underestimation of the\nEE. Reason could be linked to a cross-talk. while outliers with EEs of less than 5 ms–1 (Fig. 9b) can be spotted above 15km and below 5km. This error structure is\nsurprising, as both the normalized useful signal and error estimation curves are similar to the one of the cloud-free case study\nin Fig. 7b and 7c. 9e, the atmospheric conditions in the co-location area\nwere completely cloud free at 07:30, with some low level cloud further south of the island. The radiosonde profile shown in Fig. 9a indicates that Aeolus primarily measured in the Rayleigh channel along this orbital segment. Rayleigh-clear measurements\n510\nappear to be consistent with radiosonde wind measurements throughout the mid-troposphere between 5 km and 15 km altitude, 9a indicates that Aeolus primarily measured in the Rayleigh channel along this orbital segment. Rayleigh-clear measurements\n510\nappear to be consistent with radiosonde wind measurements throughout the mid-troposphere between 5 km and 15 km altitude, 23 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Figure 9. Same as Figs. 7 and 8 but for the case study with dust. Here, the radiosonde was launched from the Sal airport at 06:50 UTC on\nthe 21th September 2021, while Aeolus overpassed the co-location area, with radius of 60km, on a descending node between 07:28:44 UTC\nand 07:29:07 UTC. The SAFNWC CT shown in (e) corresponds to 07:30 UTC. Figure 9. Same as Figs. 7 and 8 but for the case study with dust. Here, the radiosonde was launched from the Sal airport at 06:50 UTC on\nthe 21th September 2021, while Aeolus overpassed the co-location area, with radius of 60km, on a descending node between 07:28:44 UTC\nand 07:29:07 UTC. The SAFNWC CT shown in (e) corresponds to 07:30 UTC. However, in panel 9c, we see that the Rayleigh-clear error pattern coincides with a strong peak in dust\nmixing ratio, reaching more than 2 10–7 kgkg–1 around 3.5 km altitude. The presence of dust seems to affect the quality of\n515\nRayleigh-clear measurements without influencing the normalized useful signal and thus leading to an underestimation of the\nEE. Reason could be linked to a cross-talk. 515 24 5\nConclusions In this study, we conducted a cross-Atlantic validation of Aeolus wind observations using radiosondes in the scope of the Joint\nAeolus Tropical Atlantic Campaign (JATAC). Of the total 20 radiosonde profiles included in this work, 11 were launched from\n520\nPuerto Rico and St. Croix in the Caribbean and 9 from Sal Airport on Cape Verde between August and September 2021. The\nadvantage of radiosondes is that they provide good vertical coverage, providing 384 Rayleigh-clear bin-to-bin comparisons\nfrom the surface to an altitude of 20 km and 59 Mie-cloudy comparisons, mainly restricted to the presence of clouds and\naerosols. After having applied several Quality Control (QC) and adaptation grid procedures, we quantified the quality of Aeolus Tropical Atlantic Campaign (JATAC). Of the total 20 radiosonde profiles included in this work, 11 were launched from\n520\nPuerto Rico and St. Croix in the Caribbean and 9 from Sal Airport on Cape Verde between August and September 2021. The\nadvantage of radiosondes is that they provide good vertical coverage, providing 384 Rayleigh-clear bin-to-bin comparisons\nfrom the surface to an altitude of 20 km and 59 Mie-cloudy comparisons, mainly restricted to the presence of clouds and\naerosols. After having applied several Quality Control (QC) and adaptation grid procedures, we quantified the quality of 25 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Rayleigh-clear, Mie-cloudy and to a lesser extent Rayleigh-cloudy observation types, with respect to co-location aspects as\n525\nwell as atmospheric conditions such as cloud cover and dust concentration. Rayleigh-clear, Mie-cloudy and to a lesser extent Rayleigh-cloudy observation types, with respect to co-location aspects as\n525\nwell as atmospheric conditions such as cloud cover and dust concentration. According to our statistical analysis, the total systematic error of Rayleigh-clear is –0.5 ± 0.2 ms–1, which is in agreement\nwith the ESA recommendation of 0.7 ms–1. The random error was calculated from the standard deviation of the difference\nbetween radiosonde and Aeolus measurements accounting for radiosonde observation errors estimated at 0 7±0 28 ms–1 and Rayleigh-clear, Mie-cloudy and to a lesser extent Rayleigh-cloudy observation types, with respect to co-location aspects as\n525\nwell as atmospheric conditions such as cloud cover and dust concentration. According to our statistical analysis, the total systematic error of Rayleigh-clear is –0.5 ± 0.2 ms–1, which is in agreement\nwith the ESA recommendation of 0.7 ms–1. The random error was calculated from the standard deviation of the difference\nbetween radiosonde and Aeolus measurements, accounting for radiosonde observation errors estimated at 0.7±0.28 ms–1 and between radiosonde and Aeolus measurements, accounting for radiosonde observation errors estimated at 0.7±0.28 ms–1 and\nrepresentativeness errors ranging from 1.5 to 2.5 ms–1. In the altitude range of 2–16 km and 16–20 km, the random error\n530\nis 3.8 – 4.3 ms–1 and 4.3 – 4.8 ms–1, respectively, which is above the ESA-specified values of 2.5 ms–1 and 3 ms–1, respec-\ntively. In general, Rayleigh-clear shows no error dependency with respect to co-location radius, even for distances reaching\n340 km, whilst being more sensitive to co-location time, especially if the radiosonde measurement is ahead of Aeolus’ over-\nflight time, which presumably corresponds to low altitude measurements. In addition, the systematic and random errors are representativeness errors ranging from 1.5 to 2.5 ms–1. In the altitude range of 2–16 km and 16–20 km, the random error\n530\nis 3.8 – 4.3 ms–1 and 4.3 – 4.8 ms–1, respectively, which is above the ESA-specified values of 2.5 ms–1 and 3 ms–1, respec-\ntively. In general, Rayleigh-clear shows no error dependency with respect to co-location radius, even for distances reaching\n340 km, whilst being more sensitive to co-location time, especially if the radiosonde measurement is ahead of Aeolus’ over-\nflight time, which presumably corresponds to low altitude measurements. In addition, the systematic and random errors are height-dependent, with larger errors occurring in the upper troposphere, mainly caused by the reduction in signal return from\n535\ndecreasing air density, and in lower levels, most likely caused by the signal attenuation by clouds and dust. The error estimate\nlikewise follows a similar form to the observed height error dependency, as it is inversely proportional to the squared root\nof the normalized useful signal. In cases where the normalized useful signal is strongly attenuated by clouds or dust, the er-\nror estimate is generally underestimated, with measurements exhibiting non physical features and departures from radiosonde height-dependent, with larger errors occurring in the upper troposphere, mainly caused by the reduction in signal return from\n535\ndecreasing air density, and in lower levels, most likely caused by the signal attenuation by clouds and dust. The error estimate\nlikewise follows a similar form to the observed height error dependency, as it is inversely proportional to the squared root\nof the normalized useful signal. In cases where the normalized useful signal is strongly attenuated by clouds or dust, the er-\nror estimate is generally underestimated, with measurements exhibiting non physical features and departures from radiosonde winds larger than the error estimate. A redefinition of the Rayleigh-clear error estimate could account for this underestimation\n540\nby including other sources of noise, such as detector noise or readout noise, which increase for reduced signal levels. Fur-\nthermore, a cross-talk, i.e. the leakage of the Mie signal into the Rayleigh receiver, could also explain this underestimation,\nespecially in the case of strong Mie returns. However, this supposition was not investigated in the context of this study. Outliers,\ndefined as measurements with small error estimate and large absolute differences, are found under all conditions, i.e. for all co-location radii, co-location times, altitudes as well as cloud and dust cover. Their origin does not appear to be correlated\n545\nwith low signal levels but seem to be inherent to the statistical nature of the error distribution. Taking other terms into account\nwhen defining the error estimate, such as the influence of temperature, pressure or scattering ratio on the Rayleigh response,\ncould certainly contribute to improving the error characterisation. The ECMWF model equivalents of Rayleigh-clear are found\nto have a significantly better agreement with the radiosonde wind measurements compared to the Rayleigh-clear observations. co-location radii, co-location times, altitudes as well as cloud and dust cover. Their origin does not appear to be correlated\n545\nwith low signal levels but seem to be inherent to the statistical nature of the error distribution. Taking other terms into account\nwhen defining the error estimate, such as the influence of temperature, pressure or scattering ratio on the Rayleigh response,\ncould certainly contribute to improving the error characterisation. The ECMWF model equivalents of Rayleigh-clear are found\nto have a significantly better agreement with the radiosonde wind measurements compared to the Rayleigh-clear observations. This is a further confirmation that the co-location parameters used for this validation study are appropriate and that the model\n550\nequivalents provide a suitable reference for validating Aeolus. In addition, we demonstrate the existence of an orbital- and\naltitude-dependent bias in the Rayleigh-clear channel, which is visible with respect to both radiosondes and ECMWF model\nequivalents. This bias has already been documented by Borne et al. (2023) in West Africa using model equivalents and is now\nconfirmed observationally. The underlying cause for this bias, however, remains unknown. In addition, we find that Rayleigh- For Mie-cloudy, the statistical analysis yielded a systematic negative deviation of –0.9±0.3 ms–1 within ESA specifications\nwhen uncertainty is taken into account, and it is consistent across all orbital nodes and Cal/Val sites. The random error between\n2–16 km is 1.1 – 2.3 ms–1, which falls within the ESA recommendations. The general quality of Mie-cloudy winds does not 26 Competing interests. The authors declare that they have no conflict of interest. Author contributions. MB, PK and MW conceptualized the study and developed the methodology. MB, PK, MW and BW carried out the\n580\ninvestigation and validation. PK, MW, PV and RR provided financial support for the project. MB, PV and RR were responsible for data\ncuration. MB conducted the formal analysis and wrote the original draft of the paper. MB, PK, MW, BW, CF, PV and RR reviewed and\nedited the paper. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. depend on the co-location radius, while it is more sensitive to temporal differences. The errors appear to be larger at 5 km and\n560\nabout 1 km altitude, typically at the upper and lower limits of the Saharan Air Layer, where clouds frequently occur. According\nto Lux et al. (2022a), the Mie fringe of the Fizeau interferometer can be distorted in the case of strong backscatter gradients, e.g. at cloud edges. Interestingly, Mie-cloudy does not seem to sample within dust layers, as most bins with high dust concentrations\nare rejected by the QC. Furthermore, the systematic and random Mie errors decrease with the percentage of cloud cover, while depend on the co-location radius, while it is more sensitive to temporal differences. The errors appear to be larger at 5 km and\n560\nabout 1 km altitude, typically at the upper and lower limits of the Saharan Air Layer, where clouds frequently occur. According\nto Lux et al. (2022a), the Mie fringe of the Fizeau interferometer can be distorted in the case of strong backscatter gradients, e.g. at cloud edges. Interestingly, Mie-cloudy does not seem to sample within dust layers, as most bins with high dust concentrations\nare rejected by the QC. Furthermore, the systematic and random Mie errors decrease with the percentage of cloud cover, while they increase in the presence of dust. This may be attributed to the generally weak backscatter of dust, increasing the error of\n565\nthe Mie-cloudy winds. Similar to Rayleigh-clear, outliers with small error estimate and large absolute differences can be found\nfor all co-location distance, co-location time, altitude, dust concentrations and cloud cover. An improvement of the Mie EE is\nexpected from an optimisation of the Mie core algorithm, such as the fitting function or the classification algorithm. The presented study determined the error dependencies of the different Aeolus observation types and error estimates with y\ny\nrespect to tropical clouds and dust. The acquired information are valuable to further improve the processing algorithms in order\n570\nto meet the requirements of the mission. respect to tropical clouds and dust. The acquired information are valuable to further improve the processing algorithms in order\n570\nto meet the requirements of the mission. Code availability. The analysis was conducted using the Python language and the code can be provided on request. Code availability. Acknowledgements. We would like to thank Thorsten Fehr for organising the Joint Aeolus Tropical Atlantic Campaign (JATAC), which\n585\nmade this cross-Atlantic study possible. We would also like to thank the German Aerospace Center (DLR), which assisted in the transport\nof radiosonde material and helium tanks from Germany to Sal in Cape Verde. Sincere gratitude also goes to the large teams involved in the\nradiosonde launches. For the Cape Verdean team, our special thanks go to Azusa Takeishi, Tanguy Jonville and Cedric Gacial for their hard\nwork and dedication. The analysis was conducted using the Python language and the code can be provided on request. Forecasts (ECMWF) model-equivalents are available in the ECMWF Meteorological Archival and Retrieval System (MARS) operational\n575\narchive. Dust mixing ratio data is openly available and can be downloaded in the Copernicus Atmosphere Monitoring Service (CAMS) Data\nStore. The NW SAF Cloud Type (CT) product can by obtained using the software package NWC/PPS available from http://www.nwcsaf.org/. The radiosonde data corresponding to launches from Puerto Rico are publicly available at http://dx.doi.org/10.5067/CPEXAW/DATA101. The other radiosonde data can be provided on request. Author contributions. MB, PK and MW conceptualized the study and developed the methodology. MB, PK, MW and BW carried out the\n580\ninvestigation and validation. PK, MW, PV and RR provided financial support for the project. MB, PV and RR were responsible for data\ncuration. MB conducted the formal analysis and wrote the original draft of the paper. MB, PK, MW, BW, CF, PV and RR reviewed and\nedited the paper. 27 References\n590 J., Langland, R.,\net al.: Lidar-measured wind profiles: The missing link in the global observing system, Bulletin of the American Meteorological Society, Baker, W. E., Atlas, R., Cardinali, C., Clement, A., Emmitt, G. D., Gentry, B. M., Hardesty, R. M., Källén, E., Kavaya, M. J., Langland, R.,\net al.: Lidar-measured wind profiles: The missing link in the global observing system, Bulletin of the American Meteorological Society,\n95, 543–564, 2014. 00 95, 543–564, 2014. 600\nBedka, K. M., Nehrir, A. R., Kavaya, M., Barton-Grimley, R., Beaubien, M., Carroll, B., Collins, J., Cooney, J., Emmitt, G. D., Greco, S.,\net al.: Airborne lidar observations of wind, water vapor, and aerosol profiles during the NASA Aeolus calibration and validation (Cal/Val)\ntest flight campaign, Atmospheric Measurement Techniques, 14, 4305–4334, 2021. Belanger, J., Jelinek, M., and Curry, J.: A climatology of easterly waves in the tropical Western Hemisphere, Geoscience Data Journal, 3,\n40–49, 2016. 605 Belanger, J., Jelinek, M., and Curry, J.: A climatology of easterly waves in the tropical Western Hemisphere, Geoscience Data Journal, 3,\n40–49, 2016. 605\nBelova, E., Kirkwood, S., Voelger, P., Chatterjee, S., Satheesan, K., Hagelin, S., Lindskog, M., and Körnich, H.: Validation of Aeolus winds 605 40–49, 2016. 605\nBelova, E., Kirkwood, S., Voelger, P., Chatterjee, S., Satheesan, K., Hagelin, S., Lindskog, M., and Körnich, H.: Validation of Aeolus winds\nusing ground-based radars in Antarctica and in northern Sweden, Atmospheric Measurement Techniques, 14, 5415–5428, 2021. Bormann, N., Saarinen, S., Kelly, G., and Thépaut, J.-N.: The spatial structure of observation errors in atmospheric motion vectors from\ngeostationary satellite data, Monthly Weather Review, 131, 706–718, 2003. Belova, E., Kirkwood, S., Voelger, P., Chatterjee, S., Satheesan, K., Hagelin, S., Lindskog, M., and Körnich, H.: Validation of Aeolus winds\nusing ground-based radars in Antarctica and in northern Sweden, Atmospheric Measurement Techniques, 14, 5415–5428, 2021. Bormann, N., Saarinen, S., Kelly, G., and Thépaut, J.-N.: The spatial structure of observation errors in atmospheric motion vectors from\ngeostationary satellite data, Monthly Weather Review, 131, 706–718, 2003. Borne, M., Knippertz, P., Weissmann, M., Martin, A., Rennie, M., and Cress, A.: Impact of Aeolus wind lidar observations on the rep-\n610\nresentation of the West African monsoon circulation in the ECMWF and DWD forecasting systems, Quarterly Journal of the Royal\nMeteorological Society, 2023. References\n590 Chen, S., Cao, R., Xie, Y., Zhang, Y., Tan, W., Chen, H., Guo, P., and Zhao, P.: Study of the seasonal variation in Aeolus wind product\nperformance over China using ERA5 and radiosonde data, Atmospheric Chemistry and Physics, 21, 11 489–11 504, 2021. Chen, S., Cao, R., Xie, Y., Zhang, Y., Tan, W., Chen, H., Guo, P., and Zhao, P.: Study of the seasonal variation in Aeolus wind product\nperformance over China using ERA5 and radiosonde data, Atmospheric Chemistry and Physics, 21, 11 489–11 504, 2021. Dabas, A., Denneulin, M., Flamant, P., Loth, C., Garnier, A., and Dolfi-Bouteyre, A.: Correcting winds measured with a Rayleigh Doppler\n615\nlidar from pressure and temperature effects, Tellus A: Dynamic Meteorology and Oceanography, 60, 206–215, 2008. Derrien, M. and Le Gléau, H.: MSG/SEVIRI cloud mask and type from SAFNWC, International Journal of Remote Sensing, 26, 4707–4732, Dabas, A., Denneulin, M., Flamant, P., Loth, C., Garnier, A., and Dolfi-Bouteyre, A.: Correcting winds measured with a Rayleigh Doppler\n615\nlidar from pressure and temperature effects, Tellus A: Dynamic Meteorology and Oceanography, 60, 206–215, 2008. Dabas, A., Denneulin, M., Flamant, P., Loth, C., Garnier, A., and Dolfi-Bouteyre, A.: Correcting winds measured with a Rayleigh Doppler\n615\nlidar from pressure and temperature effects, Tellus A: Dynamic Meteorology and Oceanography, 60, 206–215, 2008. Derrien, M. and Le Gléau, H.: MSG/SEVIRI cloud mask and type from SAFNWC, International Journal of Remote Sensing, 26, 4707–4732,\n2005. Derrien, M., Farki, B., Harang, L., LeGleau, H., Noyalet, A., Pochic, D., and Sairouni, A.: Automatic cloud detection applied to NOAA- p\np\n,\ny\ngy\ng p y,\n,\n,\nDerrien, M. and Le Gléau, H.: MSG/SEVIRI cloud mask and type from SAFNWC, International Journal of Remote Sensing, 26, 4707–4732,\n2005. 2005. Derrien, M., Farki, B., Harang, L., LeGleau, H., Noyalet, A., Pochic, D., and Sairouni, A.: Automatic cloud detection applied to NOAA- Derrien, M., Farki, B., Harang, L., LeGleau, H., Noyalet, A., Pochic, D., and Sairouni, A.: Automatic cloud detection applied to NOAA-\ni\ni\nf\ni\n6\n6\n6 Derrien, M., Farki, B., Harang, L., LeGleau, H., Noyalet, A., Pochic, D., and Sairouni, A.: Automatic cloud detection applied to NOAA-\n11/AVHRR imagery, Remote Sensing of Environment, 46, 246–267, 1993. 620 11/AVHRR imagery, Remote Sensing of Environment, 46, 246–267, 1993. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Acknowledgements. We would like to thank Thorsten Fehr for organising the Joint Aeolus Tropical Atlantic Campaign (JATAC), which\n585\nmade this cross-Atlantic study possible. We would also like to thank the German Aerospace Center (DLR), which assisted in the transport\nof radiosonde material and helium tanks from Germany to Sal in Cape Verde. Sincere gratitude also goes to the large teams involved in the\nradiosonde launches. For the Cape Verdean team, our special thanks go to Azusa Takeishi, Tanguy Jonville and Cedric Gacial for their hard\nwork and dedication. 28 References\n590 Abril-Gago, J., Ortiz-Amezcua, P., Bermejo-Pantaleón, D., Andújar-Maqueda, J., Bravo-Aranda, J. A., Granados-Muñoz, M. J., Navas-\nGuzmán, F., Alados-Arboledas, L., Foyo-Moreno, I., and Guerrero-Rascado, J. L.: Validation Activities of Aeolus Wind Products in the\nSoutheastern Iberian Peninsula, Atmospheric Chemistry and Physics Discussions, pp. 1–32, 2022. Alonso Lasheras, O., Sanz Diaz, A., and Lopez Cotin, L.: The Initial Operations Phase of the EUMETSAT’s SAF to Support Nowcasting Abril-Gago, J., Ortiz-Amezcua, P., Bermejo-Pantaleón, D., Andújar-Maqueda, J., Bravo-Aranda, J. A., Granados-Muñoz, M. J., Navas- Guzmán, F., Alados-Arboledas, L., Foyo-Moreno, I., and Guerrero-Rascado, J. L.: Validation Activities of Aeolus Wind Products in the\nSoutheastern Iberian Peninsula, Atmospheric Chemistry and Physics Discussions, pp. 1–32, 2022. Alonso Lasheras, O., Sanz Diaz, A., and Lopez Cotin, L.: The Initial Operations Phase of the EUMETSAT’s SAF to Support Nowcasting\n(NWC SAF) s, ESA Special Publication, 584, 4, 2005. 595 Alonso Lasheras, O., Sanz Diaz, A., and Lopez Cotin, L.: The Initial Operations Phase of the EUMETSAT o so\nas e as, O., Sa\na ,\n., a d\nope Cot ,\n.:\ne\nt a Ope at o s\nase o t e\nU\nS\ns S\nto Suppo t Nowcast g\n(NWC SAF) s, ESA Special Publication, 584, 4, 2005. (NWC SAF) s, ESA Special Publication, 584, 4, 2005. (NWC SAF) s, ESA Special Publication, 584, 4, 2005. 595\nBaars, H., Herzog, A., Heese, B., Ohneiser, K., Hanbuch, K., Hofer, J., Yin, Z., Engelmann, R., and Wandinger, U.: Validation of Aeolus\nwind products above the Atlantic Ocean, Atmospheric Measurement Techniques, 13, 6007–6024, 2020. Baker, W. E., Atlas, R., Cardinali, C., Clement, A., Emmitt, G. D., Gentry, B. M., Hardesty, R. M., Källén, E., Kavaya, M. J., Langland, R.,\net al.: Lidar-measured wind profiles: The missing link in the global observing system, Bulletin of the American Meteorological Society, Baars, H., Herzog, A., Heese, B., Ohneiser, K., Hanbuch, K., Hofer, J., Yin, Z., Engelmann, R., and Wandinger, U.: Validation of Aeolus\nwind products above the Atlantic Ocean, Atmospheric Measurement Techniques, 13, 6007–6024, 2020. ,\n,\ng,\n,\n,\n,\n,\n,\n,\n,\n,\n,\n,\n,\ng\n,\n,\ng ,\nwind products above the Atlantic Ocean, Atmospheric Measurement Techniques, 13, 6007–6024, 2020. Baker, W. E., Atlas, R., Cardinali, C., Clement, A., Emmitt, G. D., Gentry, B. M., Hardesty, R. M., Källén, E., Kavaya, M. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Folger, K. and Weissmann, M.: Height correction of Atmospheric Motion Vectors using satellite lidar observations from CALIPSO, Journal\nof Applied Meteorology and Climatology, 53, 1809–1819, https://doi.org/10.1175/JAMC-D-13-0337.1, 2014. Folger, K. and Weissmann, M.: Height correction of Atmospheric Motion Vectors using satellite lidar observations from CALIPSO, Journal\nof Applied Meteorology and Climatology, 53, 1809–1819, https://doi.org/10.1175/JAMC-D-13-0337.1, 2014. Garrett, K., Liu, H., Ide, K., Hoffman, R. N., and Lukens, K. E.: Optimization and impact assessment of Aeolus HLOS wind assimilation in of Applied Meteorology and Climatology, 53, 1809–1819, https://doi.org/10.1175/JAMC-D-13-0337.1, 2014. Garrett, K., Liu, H., Ide, K., Hoffman, R. N., and Lukens, K. E.: Optimization and impact assessment of Aeolus HLOS wind assimilation in Garrett, K., Liu, H., Ide, K., Hoffman, R. N., and Lukens, K. E.: Optimization and impact assessment of Aeo Garrett, K., Liu, H., Ide, K., Hoffman, R. N., and Lukens, K. E.: Optimization and impact assessment of Aeolus HLOS wind assimilation in\nNOAA’s global forecast system, Quarterly Journal of the Royal Meteorological Society, 148, 2703–2716, 2022. 630 g\ny\ny\ny\ng\ny\nGuo, J., Liu, B., Gong, W., Shi, L., Zhang, Y., Ma, Y., Zhang, J., Chen, T., Bai, K., Stoffelen, A., et al.: First comparison of wind observations\nfrom ESA’s satellite mission Aeolus and ground-based radar wind profiler network of China, Atmospheric Chemistry and Physics, 21,\n2945–2958, 2021. Horányi, A., Cardinali, C., Rennie, M., and Isaksen, L.: The assimilation of horizontal line-of-sight wind information into the ECMWF data\nassimilation and forecasting system. Part II: The impact of degraded wind observations, Quarterly Journal of the Royal Meteorological\n635\nSociety, 141, 1233–1243, 2015. Horányi, A., Cardinali, C., Rennie, M., and Isaksen, L.: The assimilation of horizontal line-of-sight wind information into the ECMWF data\ni il i\nd f\ni\nh i\nf d\nd d\ni d\nb\ni\nl\nl\nf h\nl\nl\ni\nl Horányi, A., Cardinali, C., Rennie, M., and Isaksen, L.: The assimilation of horizontal line-of-sight wind information into the ECMWF data\nassimilation and forecasting system. Part II: The impact of degraded wind observations, Quarterly Journal of the Royal Meteorological\n635\nSociety, 141, 1233–1243, 2015. References\n590 620\nDirksen, R., Sommer, M., Immler, F., Hurst, D., Kivi, R., and Vömel, H.: Reference quality upper-air measurements: GRUAN data processing\nfor the Vaisala RS92 radiosonde, Atmospheric Measurement Techniques, 7, 4463–4490, 2014. Flemming, J., Huijnen, V., Arteta, J., Bechtold, P., Beljaars, A., Blechschmidt, A.-M., Diamantakis, M., Engelen, R. J., Gaudel, A., Inness,\nA., et al.: Tropospheric chemistry in the Integrated Forecasting System of ECMWF, Geoscientific model development, 8, 975–1003, 2015. g y\ng\nDirksen, R., Sommer, M., Immler, F., Hurst, D., Kivi, R., and Vömel, H.: Reference quality upper-air measurements: GRUAN data processing\nfor the Vaisala RS92 radiosonde, Atmospheric Measurement Techniques, 7, 4463–4490, 2014. Flemming, J., Huijnen, V., Arteta, J., Bechtold, P., Beljaars, A., Blechschmidt, A.-M., Diamantakis, M., Engelen, R. J., Gaudel, A., Inness,i Dirksen, R., Sommer, M., Immler, F., Hurst, D., Kivi, R., and Vömel, H.: Reference quality upper-air measurements: GRUAN data processing\nfor the Vaisala RS92 radiosonde, Atmospheric Measurement Techniques, 7, 4463–4490, 2014. Flemming, J., Huijnen, V., Arteta, J., Bechtold, P., Beljaars, A., Blechschmidt, A. M., Diamantakis, M., Engelen, R. J., Gaudel, A., Inness,\nA., et al.: Tropospheric chemistry in the Integrated Forecasting System of ECMWF, Geoscientific model development, 8, 975–1003, 2015. Flesia, C. and Korb, C. L.: Theory of the double-edge molecular technique for Doppler lidar wind measurement, Applied Optics, 38, 432–440,\n625\n1999. Flesia, C. and Korb, C. L.: Theory of the double-edge molecular technique for Doppler lidar wind measurement, Applied Optics, 38, 432–440,\n625\n1999. 29 Huijnen, V., Pozzer, A., Arteta, J., Brasseur, G., Bouarar, I., Chabrillat, S., Christophe, Y., Doumbia, T., Flemming, J., Guth, J., et al.:\nQuantifying uncertainties due to chemistry modelling–evaluation of tropospheric composition simulations in the CAMS model (cycle\n43R1), Geoscientific Model Development, 12, 1725–1752, 2019. Huijnen, V., Pozzer, A., Arteta, J., Brasseur, G., Bouarar, I., Chabrillat, S., Christophe, Y., Doumbia, T., Flemming, J., Guth, J., et al.:\nQuantifying uncertainties due to chemistry modelling–evaluation of tropospheric composition simulations in the CAMS model (cycle\n43R1), Geoscientific Model Development, 12, 1725–1752, 2019. Iglewicz, B. and Hoaglin, D. C.: How to detect and handle outliers, vol. 16, Asq Press, 1993. 640\nIngmann, P. and Straume, A.: ADM-AEOLUS mission requirements document, Centre ESRaT, 2016. Inness, A., Ades, M., Agustí-Panareda, A., Barré, J., Benedictow, A., Blechschmidt, A.-M., Dominguez, J. J., Engelen, R., Eskes, H., Flem-\nming, J., et al.: The CAMS reanalysis of atmospheric composition, Atmospheric Chemistry and Physics, 19, 3515–3556, 2019. Iwai, H., Aoki, M., Oshiro, M., and Ishii, S.: Validation of Aeolus Level 2B wind products using wind profilers, ground-based Doppler wind Iglewicz, B. and Hoaglin, D. C.: How to detect and handle outliers, vol. 16, Asq Press, 1993. 640\nIngmann, P. and Straume, A.: ADM-AEOLUS mission requirements document, Centre ESRaT, 2016. Inness, A., Ades, M., Agustí-Panareda, A., Barré, J., Benedictow, A., Blechschmidt, A.-M., Dominguez, J. J., Engelen, R., Eskes, H., Flem\nming, J., et al.: The CAMS reanalysis of atmospheric composition, Atmospheric Chemistry and Physics, 19, 3515–3556, 2019. gmann, P. and Straume, A.: ADM-AEOLUS mission requirements document, Centre ESRaT, 2016. Inness, A., Ades, M., Agustí-Panareda, A., Barré, J., Benedictow, A., Blechschmidt, A.-M., Dominguez, J. J., Engelen, R., Eskes, H., Flem-\nming, J., et al.: The CAMS reanalysis of atmospheric composition, Atmospheric Chemistry and Physics, 19, 3515–3556, 2019. Iwai, H., Aoki, M., Oshiro, M., and Ishii, S.: Validation of Aeolus Level 2B wind products using wind profilers, ground-based Doppler wind\nlidars, and radiosondes in Japan, Atmospheric Measurement Techniques, 14, 7255–7275, 2021. 645 Iwai, H., Aoki, M., Oshiro, M., and Ishii, S.: Validation of Aeolus Level 2B wind products using wind profile Iwai, H., Aoki, M., Oshiro, M., and Ishii, S.: Validation of Aeolus Level 2B wind products using wind profilers, ground-based Doppler wind lidars, and radiosondes in Japan, Atmospheric Measurement Techniques, 14, 7255–7275, 2021. 645\nLux, O., Lemmerz, C., Weiler, F., Marksteiner, U., Witschas, B., Rahm, S., Geiß, A., and Reitebuch, O.: Intercomparison of wind observa-\ntions from the European Space Agency’s Aeolus satellite mission and the ALADIN Airborne Demonstrator, Atmospheric Measurement\nTechniques, 13, 2075–2097, 2020. L\nO L\nC W il\nF M k\ni\nU Wi\nh\nB R h\nS G iß A S h fl\nA\nd R i b\nh O R\ni\nl i Lux, O., Lemmerz, C., Weiler, F., Marksteiner, U., Witschas, B., Rahm, S., Geiß, A., and Reitebuch, O.: Intercomparison of wind observa-\ntions from the European Space Agency’s Aeolus satellite mission and the ALADIN Airborne Demonstrator, Atmospheric Measurement\nTechniques, 13, 2075–2097, 2020. Lux, O., Lemmerz, C., Weiler, F., Marksteiner, U., Witschas, B., Rahm, S., Geiß, A., Schäfler, A., and Reitebuch, O.: Retrieval improvements\nfor the ALADIN Airborne Demonstrator in support of the Aeolus wind product validation, Atmospheric Measurement Techniques, 15,\n650\n1303–1331, 2022a. Lux, O., Lemmerz, C., Weiler, F., Marksteiner, U., Witschas, B., Rahm, S., Geiß, A., Schäfler, A., and Reitebuch, O.: Retrieval improvements Lux, O., Lemmerz, C., Weiler, F., Marksteiner, U., Witschas, B., Rahm, S., Geiß, A., Schäfler, A., and Reitebuch, O.: Retrieval improvements\nfor the ALADIN Airborne Demonstrator in support of the Aeolus wind product validation, Atmospheric Measurement Techniques, 15,\n650\n1303–1331, 2022a. Lux, O., Witschas, B., Geiß, A., Lemmerz, C., Weiler, F., Marksteiner, U., Rahm, S., Schäfler, A., and Reitebuch, O.: Quality control and error 650 for the ALADIN Airborne Demonstrator in support of the Aeolus wind product validation, Atmospheric Measurement Techniques, 15,\n650\n1303–1331, 2022a. Lux, O., Witschas, B., Geiß, A., Lemmerz, C., Weiler, F., Marksteiner, U., Rahm, S., Schäfler, A., and Reitebuch, O.: Quality control and error\nassessment of the Aeolus L2B wind results from the Joint Aeolus Tropical Atlantic Campaign, Atmospheric Measurement Techniques,\n15, 6467–6488, 2022b. Lux, O., Witschas, B., Geiß, A., Lemmerz, C., Weiler, F., Marksteiner, U., Rahm, S., Schäfler, A., and Reitebuch, O.: Quality control and error\nassessment of the Aeolus L2B wind results from the Joint Aeolus Tropical Atlantic Campaign, Atmospheric Measurement Techniques,\n15, 6467–6488, 2022b. Martin, A., Weissmann, M., Reitebuch, O., Rennie, M., Geiß, A., and Cress, A.: Validation of Aeolus winds using radiosonde observations\n655\nand numerical weather prediction model equivalents, Atmospheric Measurement Techniques, 14, 2167–2183, 2021. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Portabella, M. and Stoffelen, A.: On scatterometer ocean stress, Journal of Atmospheric and Oceanic Technology, 26, 368–382, 2009. 665 Portabella, M. and Stoffelen, A.: On scatterometer ocean stress, Journal of Atmospheric and Oceanic Technology, 26, 368–382, 2009. 665\nRatynski, M., Khaykin, S., Hauchecorne, A., Wing, R., Cammas, J.-P., Hello, Y., and Keckhut, P.: Validation of Aeolus wind profiles using\nground-based lidar and radiosonde observations at La Réunion Island and the Observatoire de Haute Provence, EGUsphere, pp. 1–33,\n2022. Reitebuch, O.: The spaceborne wind lidar mission ADM-Aeolus, in: Atmospheric Physics, pp. 815–827, Springer, 2012. ground-based lidar and radiosonde observations at La Réunion Island and the Observatoire de Haute Provence, EGUsphere, pp. 1–33,\n2022. Reitebuch, O.: The spaceborne wind lidar mission ADM-Aeolus, in: Atmospheric Physics, pp. 815–827, Spr Reitebuch, O., Marksteiner, U., Rompel, M., Meringer, M., Schmidt, K., Huber, D., Nikolaus, I., Dabas, A., Marshall, J., de Bruin, F., et al.:\n670\nAeolus End-to-End Simulator and Wind Retrieval Algorithms up to Level 1B, in: EPJ Web of Conferences, vol. 176, p. 02010, EDP\nSciences, 2018. Rémy S Kipling Z Flemming J Boucher O Nabat P Michou M Bozzo A Ades M Huijnen V Benedetti A et al : Description Reitebuch, O., Marksteiner, U., Rompel, M., Meringer, M., Schmidt, K., Huber, D., Nikolaus, I., Dabas, A., Marshall, J., de Bruin, F., et al.:\n670\nAeolus End-to-End Simulator and Wind Retrieval Algorithms up to Level 1B, in: EPJ Web of Conferences, vol. 176, p. 02010, EDP\nSciences, 2018. Rémy, S., Kipling, Z., Flemming, J., Boucher, O., Nabat, P., Michou, M., Bozzo, A., Ades, M., Huijnen, V., Benedetti, A., et al.: Description\nand evaluation of the tropospheric aerosol scheme in the European Centre for Medium-Range Weather Forecasts (ECMWF) Integrated Rémy, S., Kipling, Z., Flemming, J., Boucher, O., Nabat, P., Michou, M., Bozzo, A., Ades, M., Huijnen, V., Benedetti, A., et al.: Description\nand evaluation of the tropospheric aerosol scheme in the European Centre for Medium-Range Weather Forecasts (ECMWF) Integrated\nForecasting System (IFS-AER, cycle 45R1), Geoscientific Model Development, 12, 4627–4659, 2019. 75 Forecasting System (IFS-AER, cycle 45R1), Geoscientific Model Development, 12, 4627–4659, 2019. 675\nRennie, M., Isaksen, L., et al.: The NWP impact of Aeolus Level-2B winds at ECMWF, ECMWF Technical Memoranda, 864, 2020. Rennie, M. Martin, A., Weissmann, M., Reitebuch, O., Rennie, M., Geiß, A., and Cress, A.: Validation of Aeolus winds using radiosonde observations\n655\nand numerical weather prediction model equivalents, Atmospheric Measurement Techniques, 14, 2167–2183, 2021. Martin, A., Weissmann, M., and Cress, A.: Impact of assimilating Aeolus observations in the global model ICON: A global statistical\noverview, https://ucloud.univie.ac.at/index.php/s/DIP0XxSgKA6eXek, 2022a. Martin, A., Weissmann, M., and Cress, A.: Investigation of dynamical scenarios leading to particularly high impact of Aeolus on NWP\nforecasts EGUsphere pp 1 23 2022b\n660 Martin, A., Weissmann, M., Reitebuch, O., Rennie, M., Geiß, A., and Cress, A.: Validation of Aeolus winds using radiosonde observations\n655\nand numerical weather prediction model equivalents, Atmospheric Measurement Techniques, 14, 2167–2183, 2021. Martin, A., Weissmann, M., and Cress, A.: Impact of assimilating Aeolus observations in the global model ICON: A global statistical\noverview, https://ucloud.univie.ac.at/index.php/s/DIP0XxSgKA6eXek, 2022a. Martin, A., Weissmann, M., and Cress, A.: Impact of assimilating Aeolus observations in the global model ICON: A global statistical\noverview, https://ucloud.univie.ac.at/index.php/s/DIP0XxSgKA6eXek, 2022a. Martin, A., Weissmann, M., and Cress, A.: Investigation of dynamical scenarios leading to particularly high impact of Aeolus on NWP\nforecasts, EGUsphere, pp. 1–23, 2022b. 60 660 Martin, C. and Suhr, I.: NCAR/EOL Atmospheric Sounding Processing ENvironment (ASPEN) software, Version 3.4. 3, 2021. McKay, J. A.: Assessment of a multibeam Fizeau wedge interferometer for Doppler wind lidar, Applied opti ssessment of a multibeam Fizeau wedge interferometer for Doppler wind lidar, Applied optics, 41, 1760–1767 Naderi, F. M., Freilich, M. H., and Long, D.: Spaceborne radar measurement of wind velocity over the ocean-an overview of the NSCAT\nscatterometer system, Proceedings of the IEEE, 79, 850–866, 1991. 30 and Chaphalkar, N.: Impact of outlier detection on neural networks based property value prediction, in: Information systems E.: Observing the Diurnal Cycle of Coastal Rainfall over Western Puerto Rico in Collaboration with University of Puerto Rico Students,\n680\nBulletin of the American Meteorological Society, 104, E305–E324, 2023. Bulletin of the American Meteorological Society, 104, E305–E324, 2023. Sandbhor, S. and Chaphalkar, N.: Impact of outlier detection on neural networks based property value prediction, in: Information systems\ndesign and intelligent applications, pp. 481–495, Springer, 2019. Saunders, R. W. and Kriebel, K. T.: An improved method for detecting clear sky and cloudy radiances from AVHRR data, International\nJournal of Remote Sensing, 9, 123–150, 1988. 685 Journal of Remote Sensing, 9, 123–150, 1988. 685\nSchillinger, M., Morancais, D., Fabre, F., and Culoma, A. J.: ALADIN: the LIDAR instrument for the AEOLUS mission, in: Sensors,\nSystems, and Next-Generation Satellites VI, vol. 4881, pp. 40–51, SPIE, 2003. Stoffelen, A., Pailleux, J., Källén, E., Vaughan, J. M., Isaksen, L., Flamant, P., Wergen, W., Andersson, E., Schyberg, H., Culoma, A., et al.:\nThe atmospheric dynamics mission for global wind field measurement, Bulletin of the American Meteorological Society, 86, 73–88, 2005. Schillinger, M., Morancais, D., Fabre, F., and Culoma, A. J.: ALADIN: the LIDAR instrument for the AEOLUS mission, in: Sensors,\nSystems, and Next-Generation Satellites VI, vol. 4881, pp. 40–51, SPIE, 2003. Stoffelen A Pailleux J Källén E Vaughan J M Isaksen L Flamant P Wergen W Andersson E Schyberg H Culoma A et al : Systems, and Next-Generation Satellites VI, vol. 4881, pp. 40–51, SPIE, 2003. Stoffelen, A., Pailleux, J., Källén, E., Vaughan, J. M., Isaksen, L., Flamant, P., Wergen, W., Andersson, E., Schyberg, H., Culoma, A., et al.:\nThe atmospheric dynamics mission for global wind field measurement, Bulletin of the American Meteorological Society, 86, 73–88, 2005. Stoffelen, A., Pailleux, J., Källén, E., Vaughan, J. M., Isaksen, L., Flamant, P., Wergen, W., Andersson, E., Schyberg, H., Culoma, A., et al.:\nThe atmospheric dynamics mission for global wind field measurement, Bulletin of the American Meteorological Society, 86, 73–88, 2005. Stowe, L. L., Davis, P. A., and McClain, E. P.: Scientific basis and initial evaluation of the CLAVR-1 global clear/cloud classification\n690\nalgorithm for the Advanced Very High Resolution Radiometer, Journal of atmospheric and oceanic technology, 16, 656–681, 1999. P., Isaksen, L., Weiler, F., de Kloe, J., Kanitz, T., and Reitebuch, O.: The impact of Aeolus wind retrievals on ECMWF global\nweather forecasts, Quarterly Journal of the Royal Meteorological Society, 147, 3555–3586, 2021. Rios-Berrios, R., Sakaeda, N., Jimenez-González, H. J., Nieves-Jimenez, A., Zayas, Y., Martin, E., Wu, S.-N., Homeyer, C. R., and Rodríguez, Forecasting System (IFS-AER, cycle 45R1), Geoscientific Model Development, 12, 4627–4659, 2019. 675\nRennie, M., Isaksen, L., et al.: The NWP impact of Aeolus Level-2B winds at ECMWF, ECMWF Technical Memoranda, 864, 2020. Rennie, M. P., Isaksen, L., Weiler, F., de Kloe, J., Kanitz, T., and Reitebuch, O.: The impact of Aeolus wind retrievals on ECMWF global\nweather forecasts Quarterly Journal of the Royal Meteorological Society 147 3555 3586 2021 Rennie, M., Isaksen, L., et al.: The NWP impact of Aeolus Level-2B winds at ECMWF, ECMWF Technical Memoranda, 864, 2020. Rennie, M. P., Isaksen, L., Weiler, F., de Kloe, J., Kanitz, T., and Reitebuch, O.: The impact of Aeolus wind retrievals on ECMWF global Rennie, M. P., Isaksen, L., Weiler, F., de Kloe, J., Kanitz, T., and Reitebuch, O.: The impact of Aeolus wind retrievals on ECMWF global\nweather forecasts, Quarterly Journal of the Royal Meteorological Society, 147, 3555–3586, 2021. ,\n,\n,\n,\n,\n,\n,\n,\n,\n,\n,\np\ng\nweather forecasts, Quarterly Journal of the Royal Meteorological Society, 147, 3555–3586, 2021. Rios-Berrios, R., Sakaeda, N., Jimenez-González, H. J., Nieves-Jimenez, A., Zayas, Y., Martin, E., Wu, S.-N., Homeyer, C. R., and Rodríguez, Rios-Berrios, R., Sakaeda, N., Jimenez-González, H. J., Nieves-Jimenez, A., Zayas, Y., Martin, E., Wu, S.-N., Homeyer, C. R., and Rodríguez, E.: Observing the Diurnal Cycle of Coastal Rainfall over Western Puerto Rico in Collaboration with University of Puerto Rico Students,\n680\nBulletin of the American Meteorological Society, 104, E305–E324, 2023. Sandbhor, S. and Chaphalkar, N.: Impact of outlier detection on neural networks based property value prediction, in: Information systems\ndesign and intelligent applications, pp. 481–495, Springer, 2019. Saunders, R. W. and Kriebel, K. T.: An improved method for detecting clear sky and cloudy radiances from AVHRR data, International E.: Observing the Diurnal Cycle of Coastal Rainfall over Western Puerto Rico in Collaboration with University of Puerto Rico Students,\n680\nBulletin of the American Meteorological Society, 104, E305–E324, 2023. Sandbhor, S. St\nA G R\ni\nM I\nk\nL d Kl\nJ M\nill\nG J St ff l\nA Fl\nt T Sti\nlit\nH D b\nA H b\nD\nt l Stowe, L. L., Davis, P. A., and McClain, E. P.: Scientific basis and initial evaluation of the CLAVR-1 global clear/cloud classification\n690\nalgorithm for the Advanced Very High Resolution Radiometer, Journal of atmospheric and oceanic technology, 16, 656–681, 1999. Straume, A.-G., Rennie, M., Isaksen, L., de Kloe, J., Marseille, G.-J., Stoffelen, A., Flament, T., Stieglitz, H., Dabas, A., Huber, D., et al.:\nESA’s space-based Doppler wind lidar mission Aeolus–First wind and aerosol product assessment results, in: EPJ Web of Conferences,\nvol. 237, p. 01007, EDP Sciences, 2020. Straume, A.-G., Rennie, M., Isaksen, L., de Kloe, J., Marseille, G.-J., Stoffelen, A., Flament, T., Stieglitz, H., Dabas, A., Huber, D., et al.:\nESA’s space-based Doppler wind lidar mission Aeolus–First wind and aerosol product assessment results, in: EPJ Web of Conferences,\nvol. 237, p. 01007, EDP Sciences, 2020. Tan, D. G., Andersson, E., Kloe, J. D., Marseille, G.-J., Stoffelen, A., Poli, P., Denneulin, M.-L., Dabas, A., Huber, D., Reitebuch, O., et al.:\n695\nThe ADM-Aeolus wind retrieval algorithms, Tellus A: Dynamic Meteorology and Oceanography, 60, 191–205, 2008. Tan, D. G., Andersson, E., Kloe, J. D., Marseille, G.-J., Stoffelen, A., Poli, P., Denneulin, M.-L., Dabas, A., Huber, D., Reitebuch, O., et al.:\n695\nThe ADM-Aeolus wind retrieval algorithms, Tellus A: Dynamic Meteorology and Oceanography, 60, 191–205, 2008. Tripathy, S. S., Saxena, R. K., and Gupta, P. K.: Comparison of statistical methods for outlier detection in proficiency testing data on analysis\nof lead in aqueous solution, American Journal of Theoretical and Applied Statistics, 2, 233–242, 2013. Weiler, F., Kanitz, T., Wernham, D., Rennie, M., Huber, D., Schillinger, M., Saint-Pe, O., Bell, R., Parrinello, T., and Reitebuch, O.: Charac-\nterization of dark current signal measurements of the ACCDs used on board the Aeolus satellite, Atmospheric Measurement Techniques,\n700\n14, 5153–5177, 2021a. Tan, D. G., Andersson, E., Kloe, J. D., Marseille, G.-J., Stoffelen, A., Poli, P., Denneulin, M.-L., Dabas, A., Huber, D., Reitebuch, O., et al.:\n695\nThe ADM-Aeolus wind retrieval algorithms, Tellus A: Dynamic Meteorology and Oceanography, 60, 191–205, 2008. Tripathy, S. S., Saxena, R. K., and Gupta, P. K.: Comparison of statistical methods for outlier detection in proficiency testing data on analysis\nof lead in aqueous solution, American Journal of Theoretical and Applied Statistics, 2, 233–242, 2013. Tripathy, S. S., Saxena, R. K., and Gupta, P. K.: Comparison of statistical methods for outlier detection in proficiency testing data on analysis\nof lead in aqueous solution, American Journal of Theoretical and Applied Statistics, 2, 233–242, 2013. Weiler, F., Kanitz, T., Wernham, D., Rennie, M., Huber, D., Schillinger, M., Saint-Pe, O., Bell, R., Parrinello, T., and Reitebuch, O.: Charac-\nterization of dark current signal measurements of the ACCDs used on board the Aeolus satellite, Atmospheric Measurement Techniques,\n700\n14, 5153–5177, 2021a. 31 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Weiler, F., Rennie, M., Kanitz, T., Isaksen, L., Checa, E., de Kloe, J., Okunde, N., and Reitebuch, O.: Correction of wind bias for the lidar on\nboard Aeolus using telescope temperatures, Atmospheric Measurement Techniques, 14, 7167–7185, 2021b. Weiler, F., Rennie, M., Kanitz, T., Isaksen, L., Checa, E., de Kloe, J., Okunde, N., and Reitebuch, O.: Correction of wind bias for the lidar on\nboard Aeolus using telescope temperatures, Atmospheric Measurement Techniques, 14, 7167–7185, 2021b. Weissmann, M., Busen, R., Dörnbrack, A., Rahm, S., and Reitebuch, O.: Targeted observations with an airborne wind lidar, Journal of Weissmann, M., Busen, R., Dörnbrack, A., Rahm, S., and Reitebuch, O.: Targeted observations with an airborne wind lidar, Journal of\nAtmospheric and Oceanic Technology, 22, 1706–1719, 2005. Atmospheric and Oceanic Technology, 22, 1706–1719, 2005. 705\nWitschas, B., Lemmerz, C., and Reitebuch, O.: Horizontal lidar measurements for the proof of spontaneous Rayleigh–Brillouin scattering in\nthe atmosphere, Applied Optics, 51, 6207–6219, 2012. Witschas, B., Lemmerz, C., Geiß, A., Lux, O., Marksteiner, U., Rahm, S., Reitebuch, O., and Weiler, F.: First validation of Aeolus wind\nobservations by airborne Doppler wind lidar measurements, Atmospheric Measurement Techniques, 13, 2381–2396, 2020. Witschas, B., Lemmerz, C., and Reitebuch, O.: Horizontal lidar measurements for the proof of spontaneous Rayleigh–Brillouin scattering in\nthe atmosphere, Applied Optics, 51, 6207–6219, 2012. Witschas, B., Lemmerz, C., Geiß, A., Lux, O., Marksteiner, U., Rahm, S., Reitebuch, O., and Weiler, F.: First validation of Aeolus wind Witschas, B., Lemmerz, C., and Reitebuch, O.: Horizontal lidar measurements for the proof of spontaneous Rayleigh–Brillouin scattering in\nthe atmosphere, Applied Optics, 51, 6207–6219, 2012. Witschas, B., Lemmerz, C., Geiß, A., Lux, O., Marksteiner, U., Rahm, S., Reitebuch, O., and Weiler, F.: First validation of Aeolus wind\nobservations by airborne Doppler wind lidar measurements, Atmospheric Measurement Techniques, 13, 2381–2396, 2020. Witschas, B., Lemmerz, C., Geiß, A., Lux, O., Marksteiner, U., Rahm, S., Reitebuch, O., Schäfler, A., and Weiler, F.: Validation of the Aeolus\n10\nL2B wind product with airborne wind lidar measurements in the polar North Atlantic region and in the tropics, Atmospheric Measurement\nTechniques, 15, 7049–7070, 2022. Witschas, B., Lemmerz, C., Geiß, A., Lux, O., Marksteiner, U., Rahm, S., Reitebuch, O., Schäfler, A., and Weiler, F.: Validation of the Aeolus\n710\nL2B wind product with airborne wind lidar measurements in the polar North Atlantic region and in the tropics, Atmospheric Measurement\nTechniques, 15, 7049–7070, 2022. Zuo, H., Hasager, C. B., Karagali, I., Stoffelen, A., Marseille, G.-J., and De Kloe, J.: Evaluation of Aeolus L2B wind product with wind\nprofiling radar measurements and numerical weather prediction model equivalents over Australia, Atmospheric Measurement Techniques, Techniques, 15, 7049–7070, 2022. Zuo, H., Hasager, C. B., Karagali, I., Stoffelen, A., Marseille, G.-J., and De Kloe, J.: Evaluation of Aeolus L2B wind product with wind\nprofiling radar measurements and numerical weather prediction model equivalents over Australia, Atmospheric Measurement Techniques, Zuo, H., Hasager, C. B., Karagali, I., Stoffelen, A., Marseille, G.-J., and De Kloe, J.: Evaluation of Aeolus L2B wind product with wind\nprofiling radar measurements and numerical weather prediction model equivalents over Australia, Atmospheric Measurement Techniques,\n15, 4107–4124, 2022. 715 15, 4107–4124, 2022. 715 15, 4107–4124, 2022. 715 32"
|
https://openalex.org/W2008392372
|
https://bmcbioinformatics.biomedcentral.com/counter/pdf/10.1186/1471-2105-11-372
|
English
| null |
Beyond co-localization: inferring spatial interactions between sub-cellular structures from microscopy images
|
BMC bioinformatics
| 2,010
|
cc-by
| 11,343
|
RESEARCH ARTICLE Open Access Open Access * Correspondence: ivos@ethz.ch
1Institute of Theoretical Computer Science, ETH Zurich, Universitätstrasse 6,
8092 Zürich, Switzerland © 2010 Helmuth 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: Sub-cellular structures interact in numerous direct and indirect ways in order to fulfill cellular
functions. While direct molecular interactions crucially depend on spatial proximity, other interactions typically result
in spatial correlations between the interacting structures. Such correlations are the target of microscopy-based co-
localization analysis, which can provide hints of potential interactions. Two complementary approaches to co-
localization analysis can be distinguished: intensity correlation methods capitalize on pattern discovery, whereas
object-based methods emphasize detection power. Results: We first reinvestigate the classical co-localization measure in the context of spatial point pattern analysis. This allows us to unravel the set of implicit assumptions inherent to this measure and to identify potential
confounding factors commonly ignored. We generalize object-based co-localization analysis to a statistical
framework involving spatial point processes. In this framework, interactions are understood as position co-
dependencies in the observed localization patterns. The framework is based on a model of effective pairwise
interaction potentials and the specification of a null hypothesis for the expected pattern in the absence of
interaction. Inferred interaction potentials thus reflect all significant effects that are not explained by the null
hypothesis. Our model enables the use of a wealth of well-known statistical methods for analyzing experimental
data, as demonstrated on synthetic data and in a case study considering virus entry into live cells. We show that
the classical co-localization measure typically under-exploits the information contained in our data. Conclusions: We establish a connection between co-localization and spatial interaction of sub-cellular structures
by formulating the object-based interaction analysis problem in a spatial statistics framework based on nearest-
neighbor distance distributions. We provide generic procedures for inferring interaction strengths and quantifying
their relative statistical significance from sets of discrete objects as provided by image analysis methods. Within our
framework, an interaction potential can either refer to a phenomenological or a mechanistic model of a physico-
chemical interaction process. This increased flexibility in designing and testing different hypothetical interaction
models can be used to quantify the parameters of a specific interaction model or may catalyze the discovery of
functional relations. Beyond co-localization: inferring spatial
interactions between sub-cellular structures from
microscopy images Jo A Helmuth1,2, Grégory Paul1,2 and Ivo F Sbalzarini1,2* Jo A Helmuth1,2, Grégory Paul1,2 and Ivo F Sbalzarini1,2* Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Background The actual physical or che-
mical interaction between sub-cellular objects can be of
short temporal duration and they can quickly separate
thereafter. In such situations, high thresholds can
increase the detection power, but only at the expense of
increased false-positive rates. When interactions take
place over long distances, the choice of threshold impli-
citly determines a range limit of the analysis. Apart from fixing the interaction scale a priori, using a
hard distance threshold also implies a binary distinction
of pair-wise distances: either they are below the threshold
and hence the objects are assumed to interact - or they
don’t. A co-localization percentage thus corresponds to
an indirect measure for the preference of “interaction”
over “non-interaction”. This preference reflects the
strength of the interaction. However, it also depends on
the frequency of possible distances that the population of
objects can assume. y
f
Such inference, however, entails a trade-off between
the objectives of pattern discovery and statistical detec-
tion power. According to these objectives, two comple-
mentary approaches to co-localization analysis can be
distinguished: Intensity correlation methods capitalize on
pattern discovery [1], whereas object-based methods [2]
emphasize detection power. Intensity correlation meth-
ods quantify correlations in the intensities of different
color channels on individual pixels. Intensity correlation
methods are straightforward to implement and use. The
results, however, may be difficult to interpret since inter-
actions need to be inferred from correlations in intensity
space, which is sensitive to the blurring and noise inher-
ent to microscopic imaging systems [3]. Object-based
methods quantify the spatial relationships between sets
of discrete objects. This requires reducing the image to a
set of geometric objects using, e.g., image segmentation
or fitting of structure models. Object-based approaches
infer interactions from correlations in physical space,
which allows constructing intuitive and simple co-
localization measures, such as counting the number of
overlapping objects [2]. More specifically, the cellular context in which the inter-
actions take place is a confounding factor. A high co-loca-
lization percentage can, for example, be observed in a cell
with densely packed sub-cellular structures of interest,
irrespective of their interaction strength. This artifact
needs to be considered in statistical tests [7] or corrected
for in order to construct an interaction score [8]. Taken together, object-based approaches provide
intuitive co-localization measures whose statistical inter-
pretation, however, is not straightforward. Background interaction as the collection of all effects that cause sig-
nificant (above the level predicted by a null hypothesis)
correlations in the positions of the participating objects. interaction as the collection of all effects that cause sig-
nificant (above the level predicted by a null hypothesis)
correlations in the positions of the participating objects. Over the last decades, advances in fluorescent markers
have enabled probing interactions of sub-cellular struc-
tures in the microscope, either directly or indirectly. The direct approach relies on experiments that generate
a signal upon the proximity required for molecular
interaction. Indirect approaches are based on indepen-
dently imaging two populations of interest, and A general biological principle states that cellular func-
tion results from the combined interactions of sub-cellu-
lar structures in space and time. Interactions typically
manifest themselves through statistical dependencies in
the spatial distributions of the involved structures. Here,
we adopt this general definition and we understand Over the last decades, advances in fluorescent markers
have enabled probing interactions of sub-cellular struc-
tures in the microscope, either directly or indirectly. The direct approach relies on experiments that generate
a signal upon the proximity required for molecular
interaction. Indirect approaches are based on indepen-
dently imaging two populations of interest, and * Correspondence: ivos@ethz.ch
1Institute of Theoretical Computer Science, ETH Zurich, Universitätstrasse 6,
8092 Zürich, Switzerland Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Page 2 of 12 searching for clues of interaction in their spatial distri-
butions. This approach is based on the paradigm that
spatial proximity (or co-localization) is a hallmark of
many types of physical and chemical interactions
between sub-cellular structures. If two or more struc-
tures interact, their spatial distributions hence appear
correlated. The reverse, however, is not necessarily true. Presence or absence of significant co-localization does
not imply presence or absence of interaction. The rea-
son is that co-localization depends on the specific inter-
action mechanism: An unobserved third structure may
act as a confounding factor (in the statistical sense),
making the observed structures appear co-localized even
though they do not interact. Furthermore, one can ima-
gine interaction mechanisms that lead to spatial distri-
butions with correlations that are not captured by
simple co-localization measures. Hence, the interaction
has to be statistically inferred from the data. that can be reliably detected. Background Here, we
establish a connection between co-localization and the
notion of interaction as used in spatial statistics [9],
namely the non-independence of the relative positions
of objects under study. This is based on modeling the
nearest-neighbor distance distribution between the
observed objects. These distances are the result of inter-
actions, measurement inaccuracies, and the geometry of
the domain in which the objects are distributed. This
modeling provides generic procedures for inferring
interaction strengths and quantifying their statistical sig-
nificance. Our approach helps formalizing design deci-
sions in co-localization and interaction studies and
shows how they translate to biological hypotheses. Stan-
dard object-based co-localization analysis is included as
a special case, which makes explicit the connections
between interaction and co-localization. After develop-
ing and characterizing the statistical interaction analysis
framework, we exemplify its utility in a biological study
of virus entry. The intensity-based approach is limited to interactions
on a spatial scale on the order of the resolution of the
microscope. While the object-based approach is not
necessarily limited to any particular length scale (note
that the localization accuracy for an isolated object is
not limited by the spatial resolution of the microscope,
but rather the signal-to-noise ratio [4-6]), a spatial scale
is nevertheless assumed in practice. Many object-based
co-localization methods rely on a hard threshold for the
distances between objects in order to distinguish
between “co-localized” and “not co-localized” for each
individual pair of objects [2]. The choice of distance
threshold greatly influences the types of interactions Basic scenario: co-localization analysis Basic scenario: co-localization analysis We review the basic concepts of classical object-based
co-localization analysis and its interpretation in terms of
interactions. Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Page 3 of 12 Page 3 of 12 Figure 1 Illustration of co-localization analysis and cellular
context. (A) Illustration of co-localization analysis based on nearest
neighbor distances (arrows) between point-like objects X = {xi}N
i=1
(dots) and circular objects Y = {yj}M
j=1 (solid circles). For all distances
d, the state density q(d) is proportional to the total length of the
d-isoline (dashed lines) in Ω. The expected co-localization in the
absence of interactions, Ct
0, is proportional to the area enclosed by
the t-isoline (gray region). (B)-(D) Effect of the positioning of the
objects Y on q(d), illustrating the influence of the cellular context. Ct = 1
N
N
i=1
1(di < t)
N→∞
−−−→
t
−∞
p(d)dd ,
(2) (2) where 1(·) is the indicator function and t an applica-
tion-specific distance threshold. The form of Eq. 2
implies assumptions about how the objects in X and Y
interact. The interaction process is considered to be
translation- and rotation-invariant since only the dis-
tance between interacting objects is taken into account. Based on this distance only two categories of positions
of the objects in X are distinguished: either they are suf-
ficiently close to any object in Y to be considered inter-
acting, or they are not. Furthermore, objects in X
interact with at most one object in Y and they do not
experience the presence of any yj unless they cross the
distance threshold t. The choice of t reflects an assump-
tion about the length scale of the interaction to be
detected. Based on this distance only two categories of positions
of the objects in X are distinguished: either they are suf-
ficiently close to any object in Y to be considered inter-
acting, or they are not. Furthermore, objects in X
interact with at most one object in Y and they do not
experience the presence of any yj unless they cross the
distance threshold t. The choice of t reflects an assump-
tion about the length scale of the interaction to be
detected. Basic scenario: co-localization analysis Inferring interactions from an observed co-localization
measure Ct is not trivial since Ct > 0 does not necessa-
rily imply any interaction between the objects. This is
because spatial correlations can also be caused by con-
founding factors, such as the cellular context {Ω, Y}. Even if the objects in X and Y do not interact there is a
finite probability that any possible distance in an interval
Δd about di is observed. We arbitrarily choose Y as a
reference in order to compute the relative frequency of
possible distances (state density) as: Figure 1 Illustration of co-localization analysis and cellular
context. (A) Illustration of co-localization analysis based on nearest
N Figure 1 Illustration of co-localization analysis and cellular
context. (A) Illustration of co-localization analysis based on nearest
neighbor distances (arrows) between point-like objects X = {xi}N
i=1
(dots) and circular objects Y = {yj}M
j=1 (solid circles). For all distances
d, the state density q(d) is proportional to the total length of the
d-isoline (dashed lines) in Ω. The expected co-localization in the
absence of interactions, Ct
0, is proportional to the area enclosed by
the t-isoline (gray region). (B)-(D) Effect of the positioning of the
objects Y on q(d), illustrating the influence of the cellular context. Figure 1 Illustration of co-localization analysis and cellular
context. (A) Illustration of co-localization analysis based on nearest
neighbor distances (arrows) between point-like objects X = {xi}N
i=1
(dots) and circular objects Y = {yj}M
j=1 (solid circles). For all distances
d, the state density q(d) is proportional to the total length of the
d-isoline (dashed lines) in Ω. The expected co-localization in the
absence of interactions, Ct
0, is proportional to the area enclosed by
the t-isoline (gray region). (B)-(D) Effect of the positioning of the
objects Y on q(d), illustrating the influence of the cellular context. Object-based co-localization measures are typically
constructed for two sets of objects X = {xi}N
i=1 and
Y = {yj}M
j=1. These objects are located in a bounded region
Ω ⊂Rn with boundary ∂Ω and dimensionality n
(usually 2 or 3; see Fig. 1). Each object i (j) is repre-
sented by a feature vector xi (yj) that comprises infor-
mation about the object’s position and, if available, its
dimension and shape. These features vectors are
extracted from image data by means of image segmenta-
tion or fitting of structure models. Basic scenario: co-localization analysis q(d) =
lim
d→0
Pr(di ∈[d, d + d]|“no interaction”, Y)
d
. (3) q(d) =
lim
d→0
Pr(di ∈[d, d + d]|“no interaction”, Y)
d
. (3) q(d) = q(d) = (3) This density q(d) is determined by the positions,
dimensions, and number density of the objects in Y (see
Fig. 1). Independent random positions will result in a
relatively wide density q(d) (Fig. 1C). With regularly
placed objects Y, large distances do not occur (Fig. 1B). Clustering increases the frequency of long distances at
the expense of short distances (Fig. 1D). Objects with
large surfaces or a large number density give rise to
shorter distances. In case there are interactions between
the objects in X and Y, some of the possible distances
are additionally favored over others, deforming the den-
sity q(d) to p(d). Suppose one wishes to investigate the interaction
between the objects in X and Y, one can define for each
xi the distance to the nearest neighbor (NN) in Y, di = min
j
d
xi, yj
. (1) (1) The function d(·) is a suitable distance function in fea-
ture space, for example the Euclidean distance between
point-like objects or the minimum distance between
outlines of more complex objects. A nearest-neighbor
distance distribution p(d) can then be estimated from
the set of distances D = {di}N
i=1. The classical overlap or
nearest-neighbor-distance co-localization measure Ct fol-
lows by counting [8]: The co-localization measure Ct is, therefore, not suffi-
cient to separate the contributions from the cellular
context and the interactions. Information about the
interactions is only contained in the deviation from an
expected base-level in the absence of interactions. This
base level, Ct
0, is the co-localization measure that would
be observed under the hypothesis H0: “no interaction” Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Page 4 of 12 Page 4 of 12 p (X|, Y) =
N
i=1
p (xi|, Y) ∝
N
i=1
exp (−φ (di)) ,
(6) (obtained by letting p(d) = q(d) and numerical evalua-
tion of the integral in Eq. 2). But how does a certain
deviation from the base level Ct
0 relate to interactions
between the objects, and what deviations can be consid-
ered significant? We address this question in the follow-
ing sections by generalizing co-localization analysis to
interaction analysis. Generalization: interaction analysis (7) Spatial point process analysis [9-11] is a standard sta-
tistical framework for studying the spatial distribution
of interacting objects. Our interaction analysis is
derived from the general binary Gibbs process with
fixed number of objects. Its central component is an
effective pair-wise interaction potential F(·). In many
applications, “interaction” is an abstraction of the dif-
ferent effects that collectively cause an observed spatial
pattern. Nevertheless, the mathematical form of the
Gibbs process corresponds to physical models of inter-
acting objects. The potential associates an energy level
with each pair {i, j} of interacting objects. The prob-
ability density of the Gibbs process for two sets of
interacting objects, X and Y, has the shape of a Boltz-
mann distribution: The normalization constant Z (the partition function)
renders p(d|q) a true probability density function. The normalization constant Z (the partition function)
renders p(d|q) a true probability density function. So far, we have not specified any particular shape for
the interaction potential (·), which can be a parametric
or non-parametric model. A specific choice constitutes a
hypothesis or assumption about the range, strength, and
distance dependence of the interaction. These three
aspects of the interaction are represented independently
in our parameterization: φ(d) = ϵ f
d −t
σ
. (8) (8) is the strength, f encodes the shape, s defines the
length-scale, and t is a shift along the distance axis of
the interaction potential. Using Eqs. 7 and 8 we find the
joint probability density of observations D: is the strength, f encodes the shape, s defines the
length-scale, and t is a shift along the distance axis of
the interaction potential. Using Eqs. 7 and 8 we find the
joint probability density of observations D: p (X, Y) ∝exp
⎛
⎝−
N
i=1
M
j=1
xi, yj
⎞
⎠,
(4) (4) p
D|q
= Z−N
N
i=1
q(di) exp
−ϵf
di −t
σ
. (9) i.e., states with lower energy occur with higher prob-
ability. Eq. 4 implies mutual independence of the objects
within the same set X or Y, in agreement with the
assumptions formulated in the previous section. For
nearest-neighbor interactions, the corresponding inter-
action potential is given by: (9) This is the central class of models that we use to
extend co-localization analysis to interaction analysis. Basic scenario: co-localization analysis Ideally, an interaction score is inde-
pendent of the cellular context and reflects variations of
the interaction strength in a monotonous fashion. The
first step toward constructing such a score is a precise
definition of the term interaction strength in the context
of an interaction model. (6) where, unlike in Eq. 4, an explicit dependence of the
potential on xi is no longer present. The probability of observing a certain xi is propor-
tional to exp (-(di)). The probability of observing a cer-
tain di, however, also depends on how frequently an
arbitrary object x is a distance di away from its NN in
the given cellular context. This frequency is given by the
state density q(d) as stated in Eq. 3. Straightforward
calculations yield: p (d|, Y) = p
d|q
= Z−1q(d) exp (−φ (d)) . (7) Generalization: interaction analysis All interaction models will be formulated as specific
instances of such a model.
xi, yj
=
φ (di) if yj is NN of xi
0
else ,
(5) The assumptions underlying the simple overlap co-
localization measure can, for example, be formalized in
a specific interaction potential. Only two categories of
distances (d <t and d ≥t) are distinguished (Eq. 2). This
implies a step function for the shape f(z) of the interac-
tion potential (d) (taking s = 1): (5) where the function (d) specifies the distance depen-
dence of the interaction. Assume a cellular context {Ω,Y} is given. The prob-
ability density p(X|Ω,Y) for the potential in Eq. 5 then
only depends on the di. An inner sum over all j, as in
Eq. 4, is then not required. The mutual independence
within X allows factorizing p(X|Ω,Y) into terms that
only depend on a single di: φst(d) = ϵ f st(d −t)
with
f st(z) =
−1 if z < 0
0
else . (10) φst(d) = ϵ f st(d −t)
with (10) Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Page 5 of 12 Figure 2 Power analysis for a step potential. Minimum Ct (A)
and ˆϵ (B) that allows rejecting H0: “no interaction” (a = 0.05) as a
function of the base-level Ct
0. In A, the expected value of Ct under
H0 is indicated by a dashed line. (C) Statistical power (1 - b) for
detecting interactions of a true strength = 1. Red, green, and blue
lines correspond to N = 10, 100, and 1000, respectively, in all three
panels. Using the integral definition in Eq. 2, the co-localiza-
tion measure Ct can then be expressed as a function of
the interaction strength. Inserting Eq. 10 into Eq. 7 and
Eq. 2 and solving for yields an estimator ˆϵ of the
model interaction strength: ˆϵ = ˆϵ(N, q) = log
Ct
1 −Ct
−log
Ct
0
1 −Ct
0
. (11) (11) The quantity ˆϵ corrects for the cellular context and,
therefore, fulfills our requirements for a valid interaction
score. Eq. 11 relates the purely descriptive co-localiza-
tion measure Ct to an interaction model between the
objects in X and Y. Hypothesis testing and power analysis for the step
potential In the parameterization of our interaction model (Eqs. 8
and 9), the presence of an interaction is equivalent to
≠0. Since ˆϵ is an estimator, it is a random variable. Even if the hypothesis H0: “no interaction” is true, a
non-zero ˆϵ can occur with finite probability (ˆϵ ≠0 does
not imply ≠0). Inference about interactions requires
finding a critical estimated interaction strength above
which one can reject H0 on a prescribed significance
level a. Figure 2 Power analysis for a step potential. Minimum Ct (A)
and ˆϵ (B) that allows rejecting H0: “no interaction” (a = 0.05) as a
function of the base-level Ct
0. In A, the expected value of Ct under
H0 is indicated by a dashed line. (C) Statistical power (1 - b) for
detecting interactions of a true strength = 1. Red, green, and blue
lines correspond to N = 10, 100, and 1000, respectively, in all three
panels. Figure 2 Power analysis for a step potential. Minimum Ct (A) and ˆϵ (B) that allows rejecting H0: “no interaction” (a = 0.05) as a
function of the base-level Ct
0. In A, the expected value of Ct under
H0 is indicated by a dashed line. (C) Statistical power (1 - b) for
detecting interactions of a true strength = 1. Red, green, and blue
lines correspond to N = 10, 100, and 1000, respectively, in all three
panels. critical value does, however, not guarantee that it will
always be detected by the test (type II error: b). Further-
more, a weak interaction may lead to unwanted rejec-
tion of H0. The behavior of the test critically depends
on the effect size, which quantifies the departure from
H0. Here, effect size refers to the true interaction
strength = a > 0. The statistical “power” (1 - b) quan-
tities the probability of rejecting H0 when H1: “ = st,
= a“ is true. In Fig. 2C, the detection power for a true
strength of a = 1 is shown as a function of Ct
0. As
expected from Fig. 2B, the power is low at the extremes
of Ct
0, eventually dropping significantly below the
recommended value of 0.8, even for N = 100. Weak
interactions are harder to detect, requiring larger sample
sizes to yield a certain power. Generalization: interaction analysis It builds a bridge between patterns
in the data (the cellular context summarized in q and
the measure Ct) and functional relationships (interac-
tions) between sub-cellular components. Whether an observed estimate ˆϵ is indicative of the
actual presence of an interaction, however, has to be
addressed using statistical inference as presented in the
following section. Hypothesis testing and power analysis for the step
potential This critical interaction strength is determined by the
distribution of ˆϵ under H0 (null distribution), which
depends on the sample size N, q, and the prescribed a. Under H0, CtN is binomially distributed with parameters
(Ct
0, N). Hence, the critical Ct can be computed from
the (numerically) inverted cumulative distribution func-
tion of the binomial distribution. The corresponding cri-
tical ˆϵ follows from Eq. 11. The dependence of the critical Ct and ˆϵ on Ct
0 and N
is shown in Fig. 2A and 2B. It can be seen that the
minimum significant excess over Ct
0 varies only weakly
with Ct
0 (Fig. 2A). Obviously, large values of Ct
0 in con-
junction with small N do not allow rejecting H0, even if
Ct = 1. The critical value of ˆϵ is highest at the two
extremes of Ct
0 and lowest for Ct
0 ≈0.4 (Fig. 2B). As for
Ct, it can be seen that for large Ct
0 and small N no finite
ˆϵ is sufficiently large to allow rejecting H0. In the design of experimental interaction studies, a key
objective is to maximize the robustness and reliability of
detecting effects of unknown size. Power can be
increased by optimizing the experimental design or the
subsequent statistical analysis. While increasing the
sample size might be possible, controlling the cellular The curves in Fig. 2B show the decision of the statisti-
cal test based on the estimated interaction strength ˆϵ. A
true interaction with a strength greater than this Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Page 6 of 12 Figure 3 Power analysis for non-step potentials. (A) Black line:
state density q(d) for M = 100 circular objects Y with radius R = 3.57
randomly placed in a square domain of size 200 × 200; R is chosen to
yield a circle-covered area fraction of 0.1; Colored lines: resulting
distance distribution p(d) for the three potentials shown in B. (B)
Plummer potential (Eq. 12) with = 1 and varying scale parameter. (C) Monte-Carlo estimates of 80%-power isolines in the N-a-plane;
dashed lines: tests based on Tst, solid lines: tests based on Tpl. Note
that larger kinks in the dashed lines are due to the discreteness of Tst
and are statistically significant. Hypothesis testing and power analysis for the step
potential Colors in A-C indicate scale parameters
of the true potential; red: s = 0.2, green: s = 1.0, and blue: s = 5.0. context is not feasible in most situations. Our analysis is
based on the interaction model introduced in the pre-
vious section. It allows specifying different shapes f(·)
and scales s of the interaction potential. Power could
potentially be increased by better modeling the interac-
tion potential. In the next section, we thus quantify the
influence of alternative model potentials on statistical
power. Improving statistical power with non-step interaction
potentials Constructing statistical tests as described above requires
assuming a specific shape and scale of the interaction
potential. In the absence of prior knowledge, however,
this model potential can be arbitrarily different from the
true potential of the actual biological interactions under
observation. Test statistics that are based on a model
potential close to the real one may achieve greater
power. We quantify the influence of the discrepancy between
the model and the true potential by considering a scenario
where N objects {xi} are distributed in the square region Ω
containing M randomly placed circular objects {yi} with
identical radii R. Fig. 3A shows the corresponding state
density q(d). The objects in X interact with the objects in
Y according to the Plummer potential (with t = 0): φpl(d) = ϵ f pl
d
σ
with
f pl (z) =
−
z2 + 1
−0.5 if z > 0
−1
else . (12) (12) Figure 3 Power analysis for non-step potentials. (A) Black line:
state density q(d) for M = 100 circular objects Y with radius R = 3.57
randomly placed in a square domain of size 200 × 200; R is chosen to
yield a circle-covered area fraction of 0.1; Colored lines: resulting
distance distribution p(d) for the three potentials shown in B. (B)
Plummer potential (Eq. 12) with = 1 and varying scale parameter. (C) Monte-Carlo estimates of 80%-power isolines in the N-a-plane;
dashed lines: tests based on Tst, solid lines: tests based on Tpl. Note
that larger kinks in the dashed lines are due to the discreteness of Tst
and are statistically significant. Colors in A-C indicate scale parameters
of the true potential; red: s = 0.2, green: s = 1.0, and blue: s = 5.0. Figure 3 Power analysis for non-step potentials. (A) Black line:
state density q(d) for M = 100 circular objects Y with radius R = 3.57
randomly placed in a square domain of size 200 × 200; R is chosen to
yield a circle-covered area fraction of 0.1; Colored lines: resulting
distance distribution p(d) for the three potentials shown in B. (B)
Plummer potential (Eq. 12) with = 1 and varying scale parameter. (C) Monte-Carlo estimates of 80%-power isolines in the N-a-plane;
dashed lines: tests based on Tst, solid lines: tests based on Tpl. Improving statistical power with non-step interaction
potentials Note
that larger kinks in the dashed lines are due to the discreteness of Tst
and are statistically significant. Colors in A-C indicate scale parameters
of the true potential; red: s = 0.2, green: s = 1.0, and blue: s = 5.0. Figure 3 Power analysis for non-step potentials. (A) Black line:
state density q(d) for M = 100 circular objects Y with radius R = 3.57 Figure 3 Power analysis for non-step potentials. (A) Black line:
state densit q(d) for M
100 circ lar objects Y
ith radi s R
3 57 This potential has an overall 1 = d-shape, but finite
value and slope everywhere. The parameter again con-
trols the interaction strength (potential depth). The
parameter s sets the length scale of the interaction
(potential range) and allows gradually changing (d)
from a step-like shape to a potential that causes signifi-
cant attraction toward the objects in Y over large dis-
tances (see Fig. 3B). For a set of distances D, distributed according to Eq. 9
with (d) = pl(d), a test for the presence of interactions
can thus be constructed based on Tpl = −N
i=1 f pl(di/σ)
under H0: “no interaction”, where the scale parameter s
is assumed to be known. The null-distribution can be
approximated by i.i.d. Monte Carlo (MC) samples
{Tpl
k }K
k=1 (see Materials and Methods). An observed value
of Tpl is then ranked among the {Tpl
k }K
k=1. If it ranks
higher than ⌈(1 - a)K⌉-th, H0 is rejected on the signifi-
cance level a [12]. The statistical power of this test to
reject H0 when H1: = pl, = a“ is true, can be For such more general potentials, algebraic expres-
sions for ˆϵ (such as Eq. 11 for the step potential) can in
general not be derived. Statistical tests for the presence
of interactions can nevertheless be constructed using a
different statistic. Since Eq. 9 describes a member of the
exponential family, T = −
N
i=1
f
di −t
σ
,
(13) (13) is a sufficient test statistic for [12]. Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Page 7 of 12 endocytosis and a marker for early endosomes. These
dynamic, lipid-bounded organelles are formed by invagi-
nations of the plasma membrane. They are the first sort-
ing compartment of clathrin-derived cargo [13]. Improving statistical power with non-step interaction
potentials However, Ad2 exhibits significant interaction with endo-
somes in half of the cells (a = 0.05). Like Ad2, TS1 is known to enter the cell by clathrin-
mediated endocytosis, but the mutation inhibits escape
from endosomes [14,15]. This should be reflected in a
deviation of the empirical distribution of observed dis-
tances D from the null distribution p(d) = q(d), which is
stronger for TS1 than for Ad2. In our framework, this
translates to a non-flat interaction potential between
virus centroids and outlines of Rab5-positive endosomes. Fig. 3C shows the number of samples required to
reach 80% power as a function of the strength a of the
true interaction potential. It can be seen that the power
of a test based on the true interaction potential (solid
lines) is higher than the power of a test based on a step
potential (dashed lines). Moreover, this difference
strongly increases with increasing potential range s: for
s = 5 (blue lines) using the step model potential
requires 4 times more samples. If the true potential is
close to a step potential (s = 0.2, red lines), both tests
perform comparably well. Moreover, the figure also
shows that interactions over longer distances are harder
to detect. We therefore conclude that one needs to be
careful when assuming a step potential (as implicitly
done in traditional co-localization analysis). Controlling
power requires prior knowledge about the interaction
potential. Such prior knowledge can easily be included
in the present framework by choosing t, s, and f(·). Before modeling an interaction potential, we test H0:
“(d) = 0” against H1: “(d) ≠0” for each imaged cell
using a non-parametric statistical test (see Materials and
Methods). This test does not assume any specific shape
of the interaction potential, which allows detecting any
type of interaction, albeit with reduced power. The
results are summarized in Table 1. The fraction of cells
for which H0 has to be rejected is significantly higher
for TS1 than for Ad2, irrespective of the significance
level and despite the on average smaller sample sizes N. However, Ad2 exhibits significant interaction with endo-
somes in half of the cells (a = 0.05). Example: virus trafficking These results indicate that the interaction potential is
non-zero for many cells. They do not, however, permit
any conclusions about the shape or strength of the interac-
tion potential, for which, in addition, no prior information
is available. We therefore apply a non-parametric estima-
tion procedure for the interaction potential to get a sketch
of its strength and distance-dependence. Subsequently we
can specify and identify parametric potentials. Ignoring,
for now, possible variability between cells and virus types, The uptake and intracellular transport of virus particles
is a complex process that involves temporary association
with membrane receptors and multiple organelles of the
endocytic machinery, such as early and late endosomes
[13]. In many cases, fluorescence microscopy allows
resolving the involved entities as discrete objects. This
has previously motivated the use of object-based co-
localization measures to quantify association kinetics
and unravel infection pathways. Here, we show how the
generalized framework of interaction analysis presented
above can be applied in a practical experimental situa-
tion, and how it enables using a large toolbox of well-
known statistical techniques. Improving statistical power with non-step interaction
potentials Either
fluorescently tagged Adenovirus serotype 2 (Ad2) or its
temperature sensitive mutant (TS1) were recorded in
the red color channel. Images were taken between 2 and
46 min post infection. The same data have already been
used in a previous study [5]. Virus positions and endo-
some outlines were extracted from the images as
described in the Materials and Methods section. Based
on these object representations, the set D of virus-to-
nearest-endosome distances and the state density q(d)
were computed for each of the imaged cells. estimated with additional MC simulations: For a fixed
effect size a > 0, one draws N distances di from p(d),
computes Tpl, and conducts the test as described above
[12]. This procedure is repeated many times and the
fraction of tests rejected serves as an estimator of the
power. In order to quantify the influence of the model poten-
tial on statistical power, we test H0 against H1 and H2:
“ = st, = a“ on data generated under H1 for varying
s (see Fig. 3B for the true interaction potentials under
H1). Testing H0 against H2 makes use of the sufficient
statistic Tst = −N
i=1 f st(di), which is proportional to Ct
with t = 0. As opposed to Tpl, this statistic only contains
information about the signs of the di and should thus
yield a less powerful test. p
g
Like Ad2, TS1 is known to enter the cell by clathrin-
mediated endocytosis, but the mutation inhibits escape
from endosomes [14,15]. This should be reflected in a
deviation of the empirical distribution of observed dis-
tances D from the null distribution p(d) = q(d), which is
stronger for TS1 than for Ad2. In our framework, this
translates to a non-flat interaction potential between
virus centroids and outlines of Rab5-positive endosomes. Before modeling an interaction potential, we test H0:
“(d) = 0” against H1: “(d) ≠0” for each imaged cell
using a non-parametric statistical test (see Materials and
Methods). This test does not assume any specific shape
of the interaction potential, which allows detecting any
type of interaction, albeit with reduced power. The
results are summarized in Table 1. The fraction of cells
for which H0 has to be rejected is significantly higher
for TS1 than for Ad2, irrespective of the significance
level and despite the on average smaller sample sizes N. Table 1 Results of non-parametric statistical tests for
interaction in the virus trafficking data Scale parameters ˆσ ∗of potentials as found by maximum-likelihood
estimation, and the corresponding maximized pooled log-likelihoods max l*
for the different potentials (Eq. 17.) which take into account the smaller number of free
parameters. With a difference in log-likelihood of > 103
to second-best fit, the Hermquist potential is by far the
best fit. It is also subjectively most similar to the non-
parametric potential identified above. Fig. 5 shows an
example of an imaged cell, infected with TS1, together
with the empirical and estimated distance distributions
and the corresponding Hermquist potential. The images
of Ad2-infected cells are visually indistinguishable from
those of TS1-infected cells and are hence not shown. Despite fitting only one independent parameter (s* is
fixed from the estimate over all cells), the estimated
model distribution captures the features of the data
remarkably well. The parameters of the potentials are found by maxi-
mum likelihood estimation (MLE). In order to exclude
cell-to-cell variations of the potential range, we do not
determine the pairs (k, sk) for each cell separately. Rather, we estimate for a given potential a single scale
parameter sk = s* common to all cells, while the inter-
action strengths k may vary between cells. The resulting
(Ncells+1)-dimensional estimation problem is solved with
a nested ML algorithm (see Materials and Methods). The common scale ˆσ ∗and the maximum of the pooled
log-likelihood l* for the four potentials are reported in
Table 2. As a reference, the values are also given for a
step potential with distance threshold t = 0. The estimated interaction strength ˆϵ of the Hermquist
potential varies within and between the two groups of
infected cells. The within-group variability comprises
statistical fluctuations and natural variations between
cells. Since virus internalization and transport is a
dynamic process, the time at which a cell was imaged
(time post infection) is a further source of in-group
variability. Fig. 6 shows the estimated interaction
strength of a Hermquist potential for all cells infected
with Ad2 (blue crosses) and TS1 (red circles) as a func-
tion of the time post infection. Throughout the observa-
tion period, the interaction strength for TS1 is
significantly larger than that for Ad2, confirming the
trend reported in Table 1. Furthermore, a temporal
maximum of the interaction strength is apparent for
TS1, while for Ad2 no significant variation over time
can be resolved. Table 1 Results of non-parametric statistical tests for
interaction in the virus trafficking data These results indicate that TS1 and
Ad2 use different uptake pathways or exhibit signifi-
cantly different escape kinetics from Rab5-positive
endosomes. The potentials are ranked according to their log-likeli-
hood. It can be seen that the step potential is outper-
formed by all others. This remains unchanged even if
one compares Akaike or Bayesian information criteria, Figure 4 Non-parametric estimate of the interaction potential. The non-parametric estimate of the interaction potential based on
all imaged cells. Table 1 Results of non-parametric statistical tests for
interaction in the virus trafficking data Table 1 Results of non-parametric statistical tests for
interaction in the virus trafficking data
#cells
p < 0.05
p < 0.01
N
Ad2
135
70 (52%)
25 (19%)
180 ± 50
TS1
139
128 (92%)
100 (72%)
157 ± 59
First column: number of cells analyzed; second and third columns: number
and percentage of cells for which H0 was rejected on the indicated
significance levels; forth column: mean and standard deviation of the
observed number of virus particles per cell. We consider a set of 274 two-color fluorescence
microscopy images of single HER-911 cells expressing
the small GTPase Rab5 tagged with enhanced green
fluorescent protein (EGFP), recorded in the green color
channel. Rab5 is a regulator of clathrin-mediated Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Page 8 of 12 Table 2 Comparison of estimated scale parameters of
interaction potentials for the virus trafficking data
ˆσ ∗
max l*
rank
Hermquist
3.96
-1.2247·105
1
Linear, type 1
4.14
-1.2362·105
2
Linear, type 2
6.61
-1.2427·105
4
Plummer
1.15
-1.2374·105
3
Step
(t = 0)
-1.2632·105
5
Scale parameters ˆσ ∗of potentials as found by maximum-likelihood
estimation, and the corresponding maximized pooled log-likelihoods max l*
for the different potentials (Eq. 17.) Table 2 Comparison of estimated scale parameters of
interaction potentials for the virus trafficking data we pool all data and estimate a common non-parametric
potential φn.p.(d) (see Materials and Methods). The esti-
mated ˆφn.p.(d) is shown in Fig. 4. Its shape is notably differ-
ent from a step function. The slow decay suggests that
viruses interact with endosomes over distances of about
10 pixels (1 μm) from their center. The estimated non-parametric potential serves as a
template for the shape of parametric models. Parametric
potentials can be identified more robustly from sets of
observed distances of individual cells. This allows corre-
lating their parameters with co-variates such as the virus
type or the time at which a cell was imaged after infec-
tion. We consider four different potentials, two that
resemble the shape in Fig. 4 (Hermquist and Linear type
1) and two that are generalizations of the step potential
with a plateau below d = 0 (Linear type 2 and Plum-
mer). For all potentials, we fix the threshold to t = 0. Definitions of the potential shapes f(·) are given in the
Materials and Methods section. Conclusions Nearest-endosome-distance
isolines (dashed red lines) are shown in the magnified inset. (B)
State density q(d) for the shown cell (dashed black line), observed
virus-to-nearest-endosome distances (marks and histogram, N =
143), and estimated distance distribution from the model p(d) (solid
black line). (C) Estimated Hermquist potential (ˆϵ = 3.90, ˆσ ∗= 3.96)
of the interactions between viruses and nearest endosomes. Figure 5 Interaction analysis applied to virus trafficking. Figure 5 Interaction analysis applied to virus trafficking. Interaction analysis for a single cell infected with TS1, imaged 27
min post infection. (A) Imaged endosomes (Rab5-EGFP) with
overlaid outlines (solid red lines) and virus centroid positions (blue
crosses, virus channel not shown). Nearest-endosome-distance
isolines (dashed red lines) are shown in the magnified inset. (B)
State density q(d) for the shown cell (dashed black line), observed
virus-to-nearest-endosome distances (marks and histogram, N =
143), and estimated distance distribution from the model p(d) (solid
black line). (C) Estimated Hermquist potential (ˆϵ = 3.90, ˆσ ∗= 3.96)
of the interactions between viruses and nearest endosomes. This limitation can be relaxed by affording more gen-
eral shapes of the interaction potential, which naturally
extends co-localization analysis to (spatial) co-distribu-
tion analysis without requiring any additional assump-
tions. The additional flexibility allows capturing
information about a wider range of sub-cellular interac-
tions. This was demonstrated by statistical power analysis
of the classical and generalized measures. Our results
highlight that the probability of detecting an interaction
strongly depends on the cellular context. We furthermore
illustrated the influence of the range of an interaction on
its detectability. Test statistics that include knowledge
about the shape of the true interaction potential can
greatly reduce the number of samples required to achieve
a certain target power. Physico-chemical models might
provide such prior knowledge. Alternatively, a non-para-
metric phenomenological potential can be estimated
from the data as demonstrated here. This potential can
then serve as a template for the parametric potentials
used in subsequent analyses. In addition, the present fra-
mework enables comparison of the likelihoods of spatial statistics framework based on nearest-neighbor
distance distributions. The present framework provides
generic procedures for inferring interaction strengths
and quantifying their statistical significance. Standard
object-based co-localization analysis is included as a
limit case, making explicit the connections between the
present framework and more classical approaches. Conclusions (A) Imaged endosomes (Rab5-EGFP) with
overlaid outlines (solid red lines) and virus centroid positions (blue
crosses, virus channel not shown). Nearest-endosome-distance
isolines (dashed red lines) are shown in the magnified inset. (B)
State density q(d) for the shown cell (dashed black line), observed
virus-to-nearest-endosome distances (marks and histogram, N =
143), and estimated distance distribution from the model p(d) (solid
black line). (C) Estimated Hermquist potential (ˆϵ = 3.90, ˆσ ∗= 3.96)
of the interactions between viruses and nearest endosomes. Figure 6 Time-resolved interaction analysis of the trafficking of
two strains of viruses. Estimated strength of a Hermquist potential
(scale s* = 3.96) for the interaction between endosomes and virus
particles versus the time post infection. Red circles: TS1; blue
crosses: Ad2. The time course of the mean (solid lines) and the ± 1
standard deviation interval (shaded bands) are estimated using a
Nadaraya-Watson kernel estimator with bandwidth of 5 min. Figure 6 Time-resolved interaction analysis of the trafficking of
two strains of viruses. Estimated strength of a Hermquist potential
(scale s* = 3.96) for the interaction between endosomes and virus
particles versus the time post infection. Red circles: TS1; blue
crosses: Ad2. The time course of the mean (solid lines) and the ± 1
standard deviation interval (shaded bands) are estimated using a
Nadaraya-Watson kernel estimator with bandwidth of 5 min. Figure 6 Time-resolved interaction analysis of the trafficking of
two strains of viruses. Estimated strength of a Hermquist potential
(scale s* = 3.96) for the interaction between endosomes and virus
particles versus the time post infection. Red circles: TS1; blue
crosses: Ad2. The time course of the mean (solid lines) and the ± 1
standard deviation interval (shaded bands) are estimated using a
Nadaraya-Watson kernel estimator with bandwidth of 5 min. dependence of the interaction. We have shown that
classical co-localization analysis amounts to estimating
the parameters of a step potential. This requires a
notion of “inside” and “outside”, either naturally defined
by the physical extent of the objects or imposed through
the step function’s distance threshold. For point-like
objects, or weak correlations between object positions,
the choice of distance threshold is arbitrary. Figure 5 Interaction analysis applied to virus trafficking. Interaction analysis for a single cell infected with TS1, imaged 27
min post infection. (A) Imaged endosomes (Rab5-EGFP) with
overlaid outlines (solid red lines) and virus centroid positions (blue
crosses, virus channel not shown). Conclusions We have introduced a statistical inference framework
for robustly estimating interaction parameters from
experimentally observed object distributions. Figure 4 Non-parametric estimate of the interaction potential. The non-parametric estimate of the interaction potential based on
all imaged cells. This allowed establishing a connection between spatial
co-distributions of objects and interaction, by formulat-
ing the object-based interaction analysis problem in a Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Page 9 of 12 Figure 5 Interaction analysis applied to virus trafficking. Interaction analysis for a single cell infected with TS1, imaged 27
min post infection. (A) Imaged endosomes (Rab5-EGFP) with
overlaid outlines (solid red lines) and virus centroid positions (blue
crosses, virus channel not shown). Nearest-endosome-distance
isolines (dashed red lines) are shown in the magnified inset. (B)
State density q(d) for the shown cell (dashed black line), observed
virus-to-nearest-endosome distances (marks and histogram, N =
143), and estimated distance distribution from the model p(d) (solid
black line). (C) Estimated Hermquist potential (ˆϵ = 3.90, ˆσ ∗= 3.96)
of the interactions between viruses and nearest endosomes. dependence of the interaction. We have shown
classical co-localization analysis amounts to estim
the parameters of a step potential. This requi
notion of “inside” and “outside”, either naturally de
by the physical extent of the objects or imposed thr
the step function’s distance threshold. For poin
objects, or weak correlations between object posit
the choice of distance threshold is arbitrary. This limitation can be relaxed by affording more
eral shapes of the interaction potential, which natu
extends co-localization analysis to (spatial) co-dist
tion analysis without requiring any additional assu
tions. The additional flexibility allows captu
information about a wider range of sub-cellular int
tions. This was demonstrated by statistical power an
of the classical and generalized measures. Our re
Figure 6 Time-resolved interaction analysis of the traffickin
two strains of viruses. Estimated strength of a Hermquist pote
(scale s* = 3.96) for the interaction between endosomes and v
particles versus the time post infection. Red circles: TS1; blue
crosses: Ad2. The time course of the mean (solid lines) and the
standard deviation interval (shaded bands) are estimated using
Nadaraya-Watson kernel estimator with bandwidth of 5 min. Figure 5 Interaction analysis applied to virus trafficking. Interaction analysis for a single cell infected with TS1, imaged 27
min post infection. Conclusions We empirically found that the strengths of
the potentials estimated from the projected 2D data
may be smaller than those estimated directly on the
raw 3D data (data not shown). Although all distances
D are systematically reduced by the projection, this
effect is overcompensated by the non-linear distortion
of q(d), which is strongest for intermediate distances,
but negligible for very small and large distances. Besides projection artifacts, errors in the image proces-
sing may also influence the estimated co-localization
measures. Depending on the accuracy of the image
segmentation method used, object sizes can be under-
or overestimated, or entire objects can be missed alto-
gether. This problem is inherent to all forms of co-
localization or distribution analysis. We have assessed
the sensitivity of our method with respect to image
segmentation errors by successively eroding or dilating
the endosomes from the presented case study. The
results show that the mean of the estimated strength
of the Hermquist potential remains unaffected, yet the
variance of the estimate increases for strong erosion
when entire endosomes start to be missed (data not
shown). This robustness of the present method is due
to the state density q(d) correcting for size errors. The
classical co-localization measure, naively corrected for
the cellular context by subtracting the amount of
unspecific co-localization C0, significantly changes
when under- or over-estimating object sizes. For
strong erosion, leading to very small and frequently All of these limitations could be relaxed by using posi-
tion-dependent interaction potentials or allowing for
many-body interactions as described by general Gibbs
processes. Considering such processes, however, is theo-
retically and numerically challenging. The presented fra-
mework could also be extended by including additional
confounding factors, such as imaging artifacts causing
spurious co-localization. Temporal plasticity of interac-
tions, cell-to-cell variations, and experiment-to-experi-
ment variations could be accounted for through
additional co-variates (time, cell index, experiment
index) in the statistical model. Already in its present
form, the statistical framework can be used to test more
general hypotheses, such as “interactions are stronger in
strain A than in strain B”. The interpretation of fitted potentials is limited to
their relative strengths. In the absence of a mechanistic
or physical model of the process that has created the
observed spatial pattern, biophysical interpretation of
the identified parameter values is difficult or misleading. Conclusions In the present framework, two novel key quantities
emerge: (i) the state density q(d), which is the distribu-
tion of nearest-neighbor distances expected under the
null hypothesis of no interaction, and (ii) the interaction
potential (d), which defines the strength and distance Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Page 10 of 12 Page 10 of 12 different hypothetical physico-chemical interaction mod-
els directly on the original image data. missing objects, it even drops to a meaningless value
of zero (data not shown). Since image segmentation
errors are always present in practical applications, we
consider the robustness of our method one of its
major advantages over classical measures. The present approach enables applying a wide range
of established statistical tools for analyzing experimen-
tal data, from parameter identification to model selec-
tion. This workflow was illustrated by studying the
spatial patterns of endosomes and viruses infecting live
human cells. In this case study, the experimental data
were very well explained using only a single free para-
meter per cell. Among the five potentials considered,
the step potential (corresponding to the classical co-
localization measure) was worst in explaining the data. This highlights the benefit of the present method over
classical co-localization analysis. Moreover, the fitted
potentials provided additional quantitative readouts
that could be used in subsequent machine learning
analyses. The presented framework is limited by the same
assumptions that also underlie classical co-localization
analysis: (i) spatial homogeneity and (ii) isotropy of the
interaction within the observation window, and (iii)
exclusively nearest-neighbor interactions between objects
of different classes. Assumption (i) is, e.g., violated if
large areas of the analyzed images do not contain any
objects. In this case, estimation of q(d) is not robust. Assumption (iii) imposes limits on admissible distances
between objects: If objects X are attracted toward objects
Y, the distances between the objects within the set Y
need to be larger than the typical interaction range. y
For simplicity the case study was done on 2D projec-
tions of 3D images. The presented approach, however,
is equally applicable in three dimensions without any
changes, provided three-dimensional object detection
and segmentation is available. Projecting the data into
two dimensions alters the estimated potentials (as it
also does for any other co-localization measure), since
it distorts both the distance data D and the state den-
sity q(d). Conclusions This is because the fitted interaction potentials reflect
the collection of all intracellular phenomena that lead to
the observed point pattern. Interestingly, however, a
relation between the steady-state distribution of a diffu-
sion process with added deterministic forces and the
distribution of the Gibbs process (Eq. 4) exists: If the
deterministic force acting between the diffusing objects
is given by -∂/∂d, the two distributions become identi-
cal (in appropriate units). This fact points a possibility
of connecting fitted interaction potentials with biophysi-
cal processes. Image acquisition and processing Endosomes and virus particles were imaged with a high-
resolution spinning disk confocal microscope (NA 1.35,
100× objective plus additional 1.6× lens, 100 nm pixel Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Page 11 of 12 The parametric tests used in sections “Hypothesis test-
ing and power analysis for the step potential” and
“Improving statistical power with non-step interaction
potentials” followed a simpler protocol. The ranking was
directly performed among the scalar test statistics Tst
and Tpl, avoiding the detour via U. A priori estimation
of the expectation and variance of Tst and Tpl was there-
fore not required. size) as described [5]. We acquired z-stacks of 8 images
each with a 400 nm z-spacing. Stacks were maximum
projected prior to image analysis. Endosome outlines
were represented as piece-wise linear closed splines in
the focal plane. Outlines were estimated from images
using a specialized model-based image analysis techni-
que [5], yielding sub-pixel localization accuracy and pre-
cision. Virus particles were modeled as points and
represented by estimated intensity centroid positions [6]. Prior to distance measurement, relative shifts between
virus and endosome positions due to chromatic aberra-
tion were corrected using an empirical calibration func-
tion [5,16]. The boundary ∂Ω of the region Ω was
defined as the cell boundary. An approximation of it
was found by low-pass filtering and thresholding of the
endosome images. ML estimation of potentials For a given potential , the log-likelihood of its para-
meters Θ given the observations Dk in cell k is: l(|Dk, k) =
Nk
i=1
pφ(dk,i|qk) = −Nk log
Zk()
+
Nk
i=1
log
qk
dk,i
−φ
dk,i;
. (16) (16) Measuring q(d) The state density q(d) was determined from the
objects {yi} contained in the region Ω. Positions x in
Ω were sampled exhaustively on a uniform Cartesian
grid with spacing h = 0.25 pixel. For each x, the dis-
tance di to the nearest neighbor in Y was computed. Using this finite sample of distances D = {di}i, an
approximation of q(d) was found by Gaussian kernel
smoothing density estimation using the MATLAB
(The MathWorks, Inc.) function ksdensity.m with
default settings. Simultaneous estimation of the common scale s* and
independent strengths k of a set of Ncells cells was done
by maximizing the pooled log-likelihood: l∗({k}|{Dk}) =
Ncells
k=1
l(k|Dk, k)
(17) (17) with respect to the parameters {k} = {(ϵk, σ ∗)}. This
was done by numerically maximizing (using Nelder-
Mead simplex) the sum of maxima maxϵkl((ϵk, σ ∗)|Dk, k)
with respect to s*. Test for interaction Greber (University of Zurich) for providing experimental data. JAH
further thanks Rajesh Ramaswamy (ETH Zurich) for encouraging comments on
early results and Christian Müller (ETH Zurich) for his help with CMA-ES. and Urs F. Greber (University of Zurich) for providing experimental data. JAH
further thanks Rajesh Ramaswamy (ETH Zurich) for encouraging comments on
early results and Christian Müller (ETH Zurich) for his help with CMA-ES. Implementation Stat Sci 2000, 15:61-78. 11. Diggle PJ: Statistical Analysis of Spatial Point Patterns A Hodder Arnold
Publication, 2nd edition 2003. 12. Assunção R: Score test for pairwise interaction parameters of Gibbs point
processes. Braz J Probab Stat 2003, 17:169-178. 12. Assunção R: Score test for pairwise interaction parameters of Gibbs point
processes. Braz J Probab Stat 2003, 17:169-178. 13. Mellman I, Warren G: The road taken: Past and future foundations of
membrane trafic. Cell 2000, 100:99-112. 13. Mellman I, Warren G: The road taken: Past and future foundations of
membrane trafic. Cell 2000, 100:99-112. 14. Imelli N, Ruzsics Z, Puntener D, Gastaldelli M, Greber UF: Genetic
reconstitution of the human Adenovirus type 2 temperature-sensitive 1
mutant defective in endosomal escape. Virol J 2009, 6(174). 14. Imelli N, Ruzsics Z, Puntener D, Gastaldelli M, Greber UF: Genetic
reconstitution of the human Adenovirus type 2 temperature-sensitive 1
mutant defective in endosomal escape. Virol J 2009, 6(174). Additional material p
15. Gastaldelli M, Imelli N, Boucke K, Amstutz B, Meier O, Greber UF: Infectious
Adenovirus Type 2 Transport Through Early but not Late Endosomes. Trafic 2008, 9(12):2265-2278. 15. Gastaldelli M, Imelli N, Boucke K, Amstutz B, Meier O, Greber UF: Infectious
Adenovirus Type 2 Transport Through Early but not Late Endosomes. Trafic 2008, 9(12):2265-2278. Additional file 1: MATLAB source code. ZIP archive containing the
MATLAB source code for potentials, likelihood functions, and statistical
tests, as well as sample scripts and sample data at the time of writing. 16. Kozubek M, Matula P: An efficient algorithm for measurement and
correction of chromatic aberrations in fluorescence microscopy. J Microsc
2000, 200:206-217. 16. Kozubek M, Matula P: An efficient algorithm for measurement and
correction of chromatic aberrations in fluorescence microscopy. J Microsc
2000, 200:206-217. 17. Heikkinen J, Penttinen A: Bayesian smoothing in the estimation of the
pair potential function of Gibbs point processes. Bernoulli 1999,
5(6):1119-1136. 17. Heikkinen J, Penttinen A: Bayesian smoothing in the estimation of the
pair potential function of Gibbs point processes. Bernoulli 1999,
5(6):1119-1136. • Plummer potential: defined in Eq. 12. 6. Sbalzarini IF, Koumoutsakos P: Feature point tracking and trajectory
analysis for video imaging in cell biology. J Struct Biol 2005,
151(2):182-195. Author details
1 1Institute of Theoretical Computer Science, ETH Zurich, Universitätstrasse 6,
8092 Zürich, Switzerland. 2Swiss Institute of Bioinformatics, ETH Zurich,
Universitätstrasse 6, 8092 Zürich, Switzerland. Potentials were parameterized as (d) = f((d - t)/s)
with interaction strength , length scale s, and threshold
t = 0. Their shapes f(·) were defined as: References 1. Costes SV, Daelemans D, Cho EH, Dobbin Z, Pavlakis G, Lockett S:
Automatic and quantitative measurement of protein-protein
colocalization in live cells. Biophys J 2004, 86(6):3993-4003. 2. Bolte S, Cordelieres FP: A guided tour into subcellular colocalization
analysis in light microscopy. J Microsc 2006, 224:213-232. 3. Anlauf E, Derouiche A: A practical calibration procedure for fluorescence
colocalization at the single organelle level. J Microsc 2009, 233:225-233. 4. Thompson RE, Larson DR, Webb WW: Precise nanometer localization
analysis for individual fluorescent probes. Biophys J 2002, 82(5):2775-2783. 5. Helmuth JA, Burckhardt CJ, Greber UF, Sbalzarini IF: Shape reconstruction
of subcellular structures from live cell fluorescence microscopy images. J
Struct Biol 2009, 167:1-10. 1. Costes SV, Daelemans D, Cho EH, Dobbin Z, Pavlakis G, Lockett S:
Automatic and quantitative measurement of protein-protein
colocalization in live cells. Biophys J 2004, 86(6):3993-4003. 2. Bolte S, Cordelieres FP: A guided tour into subcellular colocalization
analysis in light microscopy. J Microsc 2006, 224:213-232. 3. Anlauf E, Derouiche A: A practical calibration procedure for fluorescence
colocalization at the single organelle level. J Microsc 2009, 233:225-233. 4. Thompson RE, Larson DR, Webb WW: Precise nanometer localization
analysis for individual fluorescent probes. Biophys J 2002, 82(5):2775-2783. 5. Helmuth JA, Burckhardt CJ, Greber UF, Sbalzarini IF: Shape reconstruction
of subcellular structures from live cell fluorescence microscopy images. J
Struct Biol 2009, 167:1-10. • Linear potential, type 2: f 12(z) =
⎧
⎨
⎩
0
if z > 1
−1
if z < 0
−(1 −z) else . (22) (22) • Plummer potential: defined in Eq. 12. Test for interaction The piece-wise linear non-parametric potential
φn.p.(d) was defined as a weighted sum of kernel func-
tions (·) centered on the support points dp: Following [12], a non-parametric test for interaction was
constructed using the distance counts T = (T1, . . . , TL)t ,
Tl = N
i=1 1(tl < di ≤tl+1) ,
(14) (14) φn.p.(d) = P
p=1 wp κ(d −dp) with
κ(z) =
|z|/h if |z| < h
0
else . (18) (18) in L = 20 equi-sized bins defined by L + 1 strictly
increasing thresholds tl that span the entire non-zero
range of q(d) for a given cell. First, a Monte Carlo sam-
ple {Tk}K
k=1 from the null distribution of T was obtained
by sampling N = |D| distances di from q(d), computing
Tk, and repeating this procedure K times. This sample
allowed approximating the expectation E0(T) and co-
variance matrix Cov0(T) of the null distribution. The
test statistic U was defined as P = 21 support points dp were distributed between -5
and 95 with constant spacing h = 5 pixel. Setting wP = 0
enforced φn.p.(d) = 0 for all d ≥95. Setting = n.p. the remaining weights were estimated by numerically
maximizing (using CMA-ES) the penalized joint log-
likelihood [17]: U = (E0(T) −T)t Cov0(T)−1 (E0(T) −T) . (15) (15) pl(|{Dk}) =
Ncells
k=1
l(|Dk, k)
+
P−1
p=1
wp −wp+1
s
2
,
(19) Second, T and U were computed for the set D of
observed distances. U was then ranked among the
{Uk}K
k=1 obtained from an additional Monte Carlo sample
{Tk}K
k=1, generated as described above. If it ranked higher
than ⌈(1 - a)K⌉-th, H0 was rejected on the significance
level a. (19) k=1
+
P−1
p=1
wp −wp+1
s
2
,
(19) with respect to Θ = (w1,...,wP-1). Smoothness of n.p. was
controlled by the parameter s = 2. The quadratic penalty in with respect to Θ = (w1,...,wP-1). Smoothness of n.p. was
controlled by the parameter s = 2. The quadratic penalty in Page 12 of 12 Page 12 of 12 Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Helmuth et al. BMC Bioinformatics 2010, 11:372
http://www.biomedcentral.com/1471-2105/11/372 Eq. 19 corresponded to a Gaussian prior with zero mean
and standard deviation s on the differences wp - wp+1. and Urs F. Implementation p
All software was implemented in MATLAB version 7.9
(The Mathworks, Inc.) and run on a 2.66 GHz Intel
Core2 Duo machine. Estimation of two-parameter
potentials (Eqs. 12 and 20 to 22) took a few milliseconds
per cell. Computation of q(d) took about one second. This time, however, strongly depended on the sampling
resolution used. The non-parametric test for interaction
took about half a second per cell. The time needed to
estimate the common scale parameter for all cells was
around ten minutes. A constantly updated version of
the developed software is freely available from the web
site of the authors http://www.mosaic.ethz.ch/Down-
loads. The MATLAB functions, scripts, and sample data
at the time of writing are contained in additional file 1. 7. Zhang B, Chenouard N, Olivio-Marin JC, Meas-Yedid V: Statistical
Colocalization in Biological Imaging with False Discovery Control. Proceedings of the 2008 IEEE International Symposium on Biomedical Imaging:
From Nano to Macro: 14-17 May 2008; Paris, France 2008, 1327-1330. 7. Zhang B, Chenouard N, Olivio-Marin JC, Meas-Yedid V: Statistical
Colocalization in Biological Imaging with False Discovery Control. Proceedings of the 2008 IEEE International Symposium on Biomedical Imaging:
From Nano to Macro: 14-17 May 2008; Paris, France 2008, 1327-1330. 8. Lachmanovich E, Shvartsman DE, Malka Y, Botvin C, Henis YI, Weiss AM: Co-
localization analysis of complex formation among membrane proteins
by computerized fluorescence microscopy: application to
immunofluorescence co-patching studies. J Microsc 2003, 212(2):122-131. 9. Møller J, Waagepetersen R: Statistical inference and simulation for spatial
point processes CRC Press 2004. Colocalization in Biological Imaging with False Discovery Control. Proceedings of the 2008 IEEE International Symposium on Biomedical Imaging:
From Nano to Macro: 14-17 May 2008; Paris, France 2008, 1327-1330. 8. Lachmanovich E, Shvartsman DE, Malka Y, Botvin C, Henis YI, Weiss AM: Co-
localization analysis of complex formation among membrane proteins
by computerized fluorescence microscopy: application to
immunofluorescence co-patching studies. J Microsc 2003, 212(2):122-131. 9. Møller J, Waagepetersen R: Statistical inference and simulation for spatial
point processes CRC Press 2004. y
8. Lachmanovich E, Shvartsman DE, Malka Y, Botvin C, Henis YI, Weiss AM: Co-
localization analysis of complex formation among membrane proteins
by computerized fluorescence microscopy: application to
immunofluorescence co-patching studies. J Microsc 2003, 212(2):122-131. p
g
9. Møller J, Waagepetersen R: Statistical inference and simulation for spatial
point processes CRC Press 2004. 10. Stoyan D, Penttinen A: Recent applications of point process methods in
forestry statistics. Authors’ contributions JAH and GP developed the theory and analyzed the virus trafficking data. JAH designed, conducted, and analyzed numerical experiments and drafted
the manuscript. GP participated in designing and analyzing the numerical
experiments and helped in writing the manuscript. IFS participated in
designing the theory and numerical experiments, helped writing and editing
the manuscript, and coordinated the project. All authors read and approved
the final manuscript. • Hermquist potential: • Hermquist potential: f he(z) =
−(z + 1)−1 if z > 0
−(1 −z)
else . (20) (20) • Linear potential, type 1: • Linear potential, type 1: Received: 10 March 2010 Accepted: 7 July 2010 Published: 7 July 2010 Received: 10 March 2010 Accepted: 7 July 2010 Published: 7 July 2010 f 11(z) =
0
if z > 1
−(1 −z) else . (21) f 11(z) =
0
if z > 1
−(1 −z) else . (21) Acknowledgements g
JAH was financed by the ETH Research Commission under grant TH-1007-1. GP
was funded through CTI grant 9325.2 PFLS-LS from the Swiss Federal
Commission for Technology and Innovation. This project was also supported
with a grant from the Swiss SystemsX.ch initiative, grant LipidX-2008/011 to IFS. The authors thank Christoph J. Burckhardt (Harvard University, Cambridge, MA) doi:10.1186/1471-2105-11-372
Cite this article as: Helmuth et al.: Beyond co-localization: inferring
spatial interactions between sub-cellular structures from microscopy
images. BMC Bioinformatics 2010 11:372.
|
https://openalex.org/W3094810024
|
https://hal.science/hal-00361567/document
|
English
| null |
Two-phase damage and plate generation in a 2-D model of mantle convection
|
HAL (Le Centre pour la Communication Scientifique Directe)
| 2,008
|
cc-by
| 21,374
|
Two-phase damage and plate generation in a 2-D model
of mantle convection William Landuyt, David Bercovici, Yanick Ricard To cite this version:
William Landuyt, David Bercovici, Yanick Ricard. Two-phase damage and plate generation in
a 2-D model of mantle convection. Geophysical Journal International, 2008, 10.1111/j.1365-
246X.2008.03844. hal-00361567 To cite this version: William Landuyt, David Bercovici, Yanick Ricard. Two-phase damage and plate generation in
a 2-D model of mantle convection. Geophysical Journal International, 2008, 10.1111/j.1365-
246X.2008.03844. hal-00361567 Distributed under a Creative Commons Attribution 4.0 International License S U M M A R Y The formation of narrow, rapidly deforming plate boundaries separating strong plate interiors
are integral components of the generation of plate tectonics from mantle convection. The
development of narrow plate boundaries requires the interaction of a non-linear rheology and
convection. One such non-linear rheology is two-phase damage theory which employs a non-
equilibrium relation between interfacial surface energy, pressure and viscous deformation,
thereby forming a theoretical model for void generation. Two-phase damage theory was
recently extended to allow for deformational work to increase the fineness (reduce the grain
size) of the matrix phase. We present results testing two-phase damage theory in a 2-D
convectively driven system where we allow for (1) pure void-generating damage, (2) pure
fineness-generating damage and (3) combined void- and fineness-generating damage. Pure
void-generating damage is found to be unsuccessful at producing plate-like features. Fineness-
generating damage is successful at inducing plate-like behaviour in certain circumstances,
including increasing viscosity sensitivity to fineness and certain regimes of damage input and
healing rate. Cases with combined void- and fineness-generating damage produce significantly
more localization than the end-members due to the apparent increase of deformational work
input into fineness generation. The interaction of microcracks and grain size reduction in
two-phase damage theory suggests a rheological model for shear localization necessary for
the formation of plate tectonic boundaries. GJI Tectonics and geodynamics Key words: Planetary tectonics; Dynamics of lithosphere and mantle. Key words: Planetary tectonics; Dynamics of lithosphere and mantle. some of the most challenging features for plate generation studies to
explain (Bercovici 2003). Initiation of subduction (King 2001) and
the creation of passive ridges (Ricard & Froidevaux 1986; Tackley
2000b) are also vital aspects of plate tectonics to be understood. HAL Id: hal-00361567
https://hal.science/hal-00361567v1
Submitted on 17 Oct 2021 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 doi: 10.1111/j.1365-246X.2008.03844.x Geophys. J. Int. (2008) 174, 1065–1080 Plate generation and two-phase damage theory in a model
of mantle convection W. Landuyt,1 D. Bercovici1 and Y. Ricard2
1Department of Geology and Geophysics, Yale University, New Haven, CT, USA. E-mail: william.landuyt@yale.edu
2Laboratoire des Sciences de la Terre, CNRS, Ecole Normale Superieure de Lyon, Lyon, France W. Landuyt,1 D. Bercovici1 and Y. Ricard2
1Department of Geology and Geophysics, Yale University, New Haven, CT, USA. E-mail: william.landuyt@yale.edu
2Laboratoire des Sciences de la Terre, CNRS, Ecole Normale Superieure de Lyon, Lyon, France Accepted 2008 May 6. Received 2008 February 2; in original form 2007 June 1 C⃝2008 The Authors
Journal compilation C⃝2008 RAS 1 I N T RO D U C T I O N These models have
been able to generate narrow shear zones, but the connection be-
tween the assumed damage parameter and the underlying physics of
damage creation is often prescribed rather than derived (Bercovici
2003). Two-phase dynamics has been richly studied in geophysics,
with many people considering its application to problems in melt-
dynamics (McKenzie 1984; Spiegelman 1993a,b; Katz et al. 2006). Two-phase damage theory was developed in order to take a first-
principles approach to studying the partitioning of deformational
work into both dissipative heating and the generation of surface
energy (Bercovici et al. 2001a). The generation of surface energy
represents internal damage within the medium, and the manifesta-
tion of surface energy in the medium is through the creation and/or
expansion/dilation of voids. Two-phase theory is, therefore, devel-
oped in order to track both the rock and void phases. Since voids
are suggested to represent damage or weakness in the material,
tracking the voids essentially boils down to tracking the damage in
the system. The theory was generalized to allow for deformational
work to go into generating fineness (grain size reduction) in the rock
phase (or essentially pulverizing the rock) in addition to void gen-
eration, and pulverizing the rock acts to create more surface energy
in the medium (Bercovici & Ricard 2005). This theory avoids the
rather nebulous approach of assuming that damage exists as some
state variable without discerning how the damage is manifested
in the system. An important question to ask though is how these
manifestations of damage are related to the observed microphysics
of damage. Void generation or Mode I cracks are well observed
in brittle behaviour (and possibly brittle–ductile behaviour) at low
pressures (Kohlstedt et al. 1995). At greater depths (∼15–18 km)
the overburden pressure acts to suppress rock dilation (Kohlstedt
et al. 1995), so the relevance of void generating damage to mid to
deep lithospheric depths is of questionable importance (unless these
voids were somehow filled with fluid which seems difficult given
the petrologically inferred lack of water in the lithosphere). The
relevance of grain size (and specifically the reduction in grain size)
to the rheological properties of rocks and shear localization is well
demonstrated (Karato 1983; Jin et al. 1998). The cause of reduction
in grain size is generally associated with dynamic recrystallization
(Karato et al. 1980). 1 I N T RO D U C T I O N Plate tectonics is the unifying theory of geology, yet much remains
to be understood about the development of plate tectonics on Earth
as well as its absence on the other terrestrial planets (Bercovici et al. 2000). Plate tectonics on Earth has been determined to have been
in operation for at least two billion years and may well have been
in operation much earlier (Cawood et al. 2006). These observations
place an important constraint on numerical models in that Earth-
like simulations of plate motion must be non-sporadic and long-
lasting. The collection of studies labelled as plate generation refer
to the attempt to understand how plate tectonics (or the surface
manifestation of mantle convection) arise self-consistently within
planetary convection, as opposed to viewing plate tectonics and
planetary convection as separate entities. One of the most prominent
features of plate tectonics that plate generation studies attempt to
address is the existence of broad, strong plate interiors with weak,
rapidly deforming boundaries (Weinstein & Olson 1992; Moresi &
Solomatov 1998). The rapidly deforming boundaries tend to be very
narrow features and are synonymous with shear localization. Also,
the existence of strike-slip boundaries and toroidal motion remain The features of plate tectonics discussed above imply that the
plates do not deform according to a simple Newtonian rheology. Experimental work on upper mantle rocks has shown that a signif-
icant portion of the upper mantle deforms according to dislocation
creep with a power law of approximately three (e.g. Karato & Wu
1993). Incorporation of non-Newtonian rheologies into plate gen-
eration studies has not shown a significant improvement in gener-
ating plate-like behaviours, especially in producing toroidal motion
(Christensen & Harder 1991; Weinstein & Olson 1992; Bercovici
1993, 1995). Implementation of more exotic rheologies (e.g. stick-
slip rheologies) has had more success at generating plate-like be-
haviour (Bercovici 1993; Tackley 1998, 2000b), but the relevance of
such rheologies to the actual lithosphere is questionable (Bercovici
2003). The longevity of plate boundaries (and their ability to be
reactivated) implies that the low viscosity zone that constitutes
the boundary is a time-dependent quantity that only decays after
very long times (Gurnis et al. 2000). Therefore, tracking the time 1065 C⃝2008 The Authors
Journal compilation C⃝2008 RAS W. Landuyt, D. Bercovici and Y. 1 I N T RO D U C T I O N Ricard 1066 to add another level of sophistication to previous work, but is still
obviously a simplification of fully variable viscosity convection
and hence convection in Earth. Our goal though is to ascertain the
general plate behaviours (i.e. narrow boundary zones separated by
rigid plate interiors) of two-phase damage theory in a convective
system were we assume that the rheology is controlled by a single
strong layer in the lithosphere. A simple but important addition to
the evolution equation for fineness (inverse grain size) is added to
the theory by the incorporation of a healing term. Healing in the
fineness equation is representative of coarsening or grain growth in
a poly-crystalline rock and is a well studied phenomena in Earth
materials and in general (Karato 1989). One expects that increas-
ing the fraction of deformational work which goes into generating
damage as well as increasing the sensitivity of viscosity to grain
size will allow for greater shear localization. In contrast, we expect
that increasing the healing parameter will result in moderating the
amount of localization within the lithosphere but potentially allow
for a strengthening of the plate interior with respect to the bound-
aries. While we do find that increasing the sensitivity of viscosity
to grain size results in greater shear localization, the effect of in-
creasing the healing parameter and fraction of deformational work
stored as damage exhibits some behaviours different than expected. We also develop a series of scaling analyses for how the convergent
and divergent boundary widths depend upon the model parameters
and what damage inputs control the boundary behaviour. Scaling
analyses are useful guides for exploring parameter space and un-
derstanding the different localization behaviours demonstrated by
the numerical experiments. In this study, we will inspect the abil-
ity of both void and fineness generating damage in a convectively
driven system to produce Earth-like features and determine what
implications this may have for plate tectonics on Earth. evolution of the viscosity (or its controlling parameter) is impera-
tive for plate generation studies. Previous studies have attempted to
incorporate a time-dependent damage parameter (e.g. void or defect
creation) to induce severe weakening in the plate that would address
some of the deficiencies of the previous models (Bercovici 1998;
Tackley 2000b; Auth et al. 2003; Ogawa 2003). 1 I N T RO D U C T I O N The relation between deformational work par-
titioning into grain size reduction in the two-phase damage theory
and the microphysics of dynamic recrystallization is not currently
understood and remains an important and essential consideration
for understanding this type of damage (Bercovici & Ricard 2005). Two-phase damage theory was recently incorporated by Bercovici
& Ricard (2005) into a simple source-sink flow to determine the
efficacy of both void and fineness generating damage to produce
plate like flow (solid body translation, toroidal motion) in a 2-D
sheet. Their results showed that fineness generating damage was
very successful at generating plate like flow, while void generating
damage merely enhanced the dipolar source-sink flow field and was,
therefore, highly unplate-like (Bercovici & Ricard 2005). In this study, we will extend the previous work done on two- C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS 2 M O D E L F O R M U L AT I O N The goal of this paper is to determine the effectiveness of different
damage formulations in producing plate-like flow in a 2-D convec-
tively driven system; this will involve determining how convergent
and divergent zones as well as slowly deforming ‘plate-like’ zones
respond to the given damage parameters. To that end we will em-
ploy a lithosphere–mantle coupling model whereby a Newtonian
mantle is overlain by a two-phase damage rheology lithospheric
layer (Fig. 1); this model is similar to the formulation of Weinstein
& Olson (1992). The lithospheric layer is of constant thickness h
and is much smaller than the thickness of the underlying mantle
D, (h ≪D). Material which flows across the boundary between 0
L
D
D + h
h/D << 1
x
z
Newtonian Mantle
vz , σxz = 0
vz , σxz , σzz
viscosity(z)
μm
Non-Linear Rheology , μL (x)
μL
μL > μm
μm
Figure 1. The thin sheet model employed in this study along with the
vertical viscosity profile. In this study, we will extend the previous work done on two-
phase damage theory (Bercovici et al. 2001b; Ricard et al. 2001;
Ricard & Bercovici 2003; Bercovici & Ricard 2003, 2005) by in-
corporating two-phase damage physics into a coupled plate-mantle
model that is convectively driven. Our model formulation attempts Figure 1. The thin sheet model employed in this study along with the
vertical viscosity profile. C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection 1067 the lithosphere and mantle undergoes an instantaneous rheologi-
cal transformation to the rheological properties of the region into
which it flows. The boundary between the two regions is a no-slip
surface such that stresses transmitted from the Newtonian layer to
the lithosphere drive the flow in the surface layer. In the context
of the thin sheet approximation that will be described hereafter,
the temperature at the surface of the Newtonian mantle is assumed
to be uniform and the thin layer is isothermal. With the assumed
thermal profile of the lithosphere it follows that the top layer has no
buoyancy variations to drive flow in the model. 2.1 Mantle convection model where the subscript m denotes variables in the mantle, and
Pm, vm, wm and θ are the mantle non-hydrostatic pressure, ve-
locity, vertical velocity and temperature perturbation, respectively
(Schubert et al. 2001). The constant Ra is the Rayleigh number
for the system. The convection equations above have also been
non-dimensionalized by x = Dx′, t = (D2/κ)t′, vm = (κ/D)v′
m
and P m = (μm κ/D2)P′
m, and the primes have subsequently been
dropped. The constants D, κ and μm are the depth of the convect-
ing system, thermal diffusivity and mantle viscosity, respectively. The above equations with the applied boundary conditions (to be
discussed later) determine the behaviour of the Newtonian mantle
in our model. where Aref is a reference value for fineness and μo is reference
viscosity of the lithosphere. The viscosity exponent m is a dimen-
sionless positive constant, and here we consider a reasonable range
m (i.e. 1 ≤m ≤3) assuming a grain size sensitive deformation
mechanism (e.g. diffusion creep) is in operation. Given the above
momentum equations (eq. 5) we find that the effective matrix vis-
cosity is given by where Aref is a reference value for fineness and μo is reference
viscosity of the lithosphere. The viscosity exponent m is a dimen-
sionless positive constant, and here we consider a reasonable range
m (i.e. 1 ≤m ≤3) assuming a grain size sensitive deformation
mechanism (e.g. diffusion creep) is in operation. Given the above
momentum equations (eq. 5) we find that the effective matrix vis-
cosity is given by μeff = (1 −φ)μL = μo(1 −φ)
Aref
A
m
,
(9) (9) where the (1 −φ) factor in the viscosity essentially arises from the
deviatoric stress term in (eq. 5). 2.1 Mantle convection model α = Aη(φ) , η(φ) = φa(1 −φ)b,
(7) (7) In order to focus on the effect of the various forms of damage on
convection we employ a rather simple model for mantle convection,
that is isoviscous, Boussinesq, infinite Prandtl number, Rayleigh–
Benard convection. The equations for mass, momentum and energy
are where A is the inverse void or grain size (depending on the curvature
of the interface), η(φ) is a dimensionless function of porosity and
a, b are constants ≤1. The above equation satisfies the requirement
that interfacial area goes to zero at the different limits of porosity,
and simple micromechanical models have shown how A relates to
inverse grain/void size (Bercovici et al. 2001a). Following the previ-
ous formulations of the matrix rheology we assume the lithospheric
viscosity is given by where A is the inverse void or grain size (depending on the curvature
of the interface), η(φ) is a dimensionless function of porosity and
a, b are constants ≤1. The above equation satisfies the requirement
that interfacial area goes to zero at the different limits of porosity,
and simple micromechanical models have shown how A relates to
inverse grain/void size (Bercovici et al. 2001a). Following the previ-
ous formulations of the matrix rheology we assume the lithospheric
viscosity is given by ∇· vm = 0
(1)
0 = −∇Pm + ∇2vm + Raθ ˆz
(2)
∂θ
∂t + vm · ∇θ −wm = ∇2θ,
(3) ∇· vm = 0 (1) 0 = −∇Pm + ∇2vm + Raθ ˆz (2) ∂θ
∂t + vm · ∇θ −wm = ∇2θ,
(3) μL = μo
Aref
A
m
,
(8) (3) (8) where the subscript m denotes variables in the mantle, and
Pm, vm, wm and θ are the mantle non-hydrostatic pressure, ve-
locity, vertical velocity and temperature perturbation, respectively
(Schubert et al. 2001). The constant Ra is the Rayleigh number
for the system. The convection equations above have also been
non-dimensionalized by x = Dx′, t = (D2/κ)t′, vm = (κ/D)v′
m
and P m = (μm κ/D2)P′
m, and the primes have subsequently been
dropped. The constants D, κ and μm are the depth of the convect-
ing system, thermal diffusivity and mantle viscosity, respectively. The above equations with the applied boundary conditions (to be
discussed later) determine the behaviour of the Newtonian mantle
in our model. 2.2.3 Energy The two-phase damage equations originate from a series of papers
(Bercovici et al. 2001a,b; Ricard et al. 2001), with subsequent
papers refining various aspects of the theory (Bercovici & Ricard
2003, 2005; Ricard & Bercovici 2003). The equations presented
below are in the geologically applicable ‘void limit’ as discussed
in Ricard & Bercovici (2003), whereby the void phase has zero
density, pressure and viscosity. The energy equation is separated into two coupled equations which
govern the evolution of thermal energy and the rate of work done on
the interface by pressure, surface tension and deformational work
(Bercovici & Ricard 2005). The evolution of thermal energy is (1 −φ)ρc DT
Dt −T D
Dt
α dσ
dT
−T α dσ
dT ∇· v =
Q −∇· q + B
Dφ
Dt
2
+ σηkA Ap + (1 −f ),
(10) (10) 2.2.2 Momentum The momentum equations for the matrix are The momentum equations for the matrix are 0 = −∇[(1 −φ)P −σα] + ∇· [(1 −φ)τ] −(1 −φ)ρgˆz,
(5) (5) where P is the matrix pressure,σ is the surface tension, α is the
interfacial area density, τ is the deviatoric matrix stress and ρ is the
density of the matrix phase. The deviatoric stress is given by τ = μL
∇v + [∇v]t −2
3(∇· v)I
,
(6) (6) where I is the identity matrix and μL is the top layer viscosity. The
interfacial area density has been suggested to go as 2 M O D E L F O R M U L AT I O N Contrary to simi-
lar model formulations (Weinstein & Olson 1992; Weinstein 1996)
the lithosphere is recycled back into the mantle by calculating the
vertical velocity at the plate-mantle interface, this avoids the rather
unphysical situation where lithospheric material would build up at
a subduction zone and become depleted at a ridge. the same as the matrix velocity, hence we do not need to solve
independent mass and momentum equations for the void phase. 2.2.2 Momentum C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS (16) where f ∗is the maximum permissible f φ, γ controls the vari-
ability of f φ and f φ is assumed to depend on an even power of
Dφ/Dt since it must be positive definite. As previously discussed in
Bercovici et al. (2001b) and Bercovici & Ricard (2003), the above
equation for f φ precludes singular solutions of the porosity damage
equation (eq. 14) in areas of zero void growth (i.e. Dφ/Dt = 0). 2.2.1 Mass where T is the temperature, c is the matrix heat capacity,
−bσ/dT is the interfacial entropy per unit area, q is an energy
flux vector (e.g. heat diffusion) and Q is an intrinsic heat source. The quantity B is positive, has units of viscosity, and is related to the
bulk viscosity term in the two-phase theory of McKenzie (1984);
the term proportional to B represents irreversible work done during The equation for mass conservation is The equation for mass conservation is Dφ
Dt = ∂φ
∂t + v · ∇φ = (1 −φ)∇· v,
(4) (4) where v is the matrix (or rock phase) velocity and φ is the porosity. In the void limit the velocity of the void (or secondary) phase is where v is the matrix (or rock phase) velocity and φ is the porosity. In the void limit the velocity of the void (or secondary) phase is C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS 1068 W. Landuyt, D. Bercovici and Y. Ricard this paper) the healing term would be representative of coarsening
or grain growth (Karato 1989). Whether A represents inverse grain
or void size depends upon the values of a and b in (eq. 7) as
discussed previously. Similar to Bercovici & Ricard (2005) we find
that variations in these parameters do not significantly affect the
outcome of our results, and making the values of a smaller (which
puts the fineness parameter in the inverse grain size regime) are
easily offset by increasing f A. We will, therefore, associate the
fineness parameter with inverse grain size in this paper and attempt
to use constraints from grain size evolution to determine parameters
in our evolution equation. isotropic compression or dilation. Following previous formulations
for B we arrive at B =
Kμo
φ(1 −φ),
(11) B =
Kμo
φ(1 −φ), (11) where K is a dimensionless factor accounting for pore or grain ge-
ometry and is typically O(1) (Bercovici et al. 2001a; Bercovici &
Ricard 2005). The (1 −φ) in denominator of (eq. 11) may not be
necessary in the void limit, but the range of porosities in our study
make this term negligible anyways. The quantity k A represents the
rate of grain growth (Karato 1989) and η(φ) is a dimensionless
function of porosity (see eq. 2.2.1 Mass 7); the term proportional to k A rep-
resents the contribution of irreversible loss of interfacial area (via
grain growth) to the evolution of thermal energy. The exponent p in
the term proportional to k A is related to surface tension driven grain
growth which will be further elaborated on later in this section. The
viscous deformational work is given by where K is a dimensionless factor accounting for pore or grain ge-
ometry and is typically O(1) (Bercovici et al. 2001a; Bercovici &
Ricard 2005). The (1 −φ) in denominator of (eq. 11) may not be
necessary in the void limit, but the range of porosities in our study
make this term negligible anyways. The quantity k A represents the
rate of grain growth (Karato 1989) and η(φ) is a dimensionless
function of porosity (see eq. 7); the term proportional to k A rep-
resents the contribution of irreversible loss of interfacial area (via
grain growth) to the evolution of thermal energy. The exponent p in
the term proportional to k A is related to surface tension driven grain
growth which will be further elaborated on later in this section. The
viscous deformational work is given by Previous analyses that have incorporated a damage evolution
equation similar to the fineness evolution equation suggested the
association of this equation to inverse grain size as well (Tackley
2000b; Auth et al. 2003), and in both cases choose p = 1 in eq. (15). The fineness evolution equation (eq. 15) in the absence of damage
satisfies DA
Dt ≈−kA Ap ⇒
1
Ap−1 ≈(p −1)kAt. (17) (12) = (1 −φ)∇v : τ,
(12) (17) a fraction 1 −f of which is partitioned into dissipative heating. The
evolution of energy associated with work done on the interface and
interface generation is a fraction 1 −f of which is partitioned into dissipative heating. The
evolution of energy associated with work done on the interface and
interface generation is Since we want to associate A with inverse grain size, A = 1/g, and
the evolution of grain size undergoing grain growth is often found to
vary as (kAt)
1
2 , 1
3 (e.g. Karato 1989; Evans et al. 2001) we therefore,
choose p = 3. 2.2.1 Mass The association of the
term proportional to k A with surface tension driven grain growth
(and hence the curvature of grains) implies a direct relationship to
the size of the grains, hence we assume the k A healing term is only
in the equation governing the evolution of grain size (eq. 15) and not
in eq. (14). The term proportional to k A in (eq. 15) acts to decrease
the fineness (or increase grain size), and decreasing fineness leads
to an increase in viscosity and, therefore, heals the damaged zone
(i.e. plate boundary). We will consider cases where both the void-
and fineness-generating damage mechanisms operate by them self
as well as in tandem with each other. The partitioning fraction of
damage in the void-generating case is given by where f = f φ + f A must be less than one. The association of the
term proportional to k A with surface tension driven grain growth
(and hence the curvature of grains) implies a direct relationship to
the size of the grains, hence we assume the k A healing term is only
in the equation governing the evolution of grain size (eq. 15) and not
in eq. (14). The term proportional to k A in (eq. 15) acts to decrease
the fineness (or increase grain size), and decreasing fineness leads
to an increase in viscosity and, therefore, heals the damaged zone
(i.e. plate boundary). We will consider cases where both the void-
and fineness-generating damage mechanisms operate by them self
as well as in tandem with each other. The partitioning fraction of
damage in the void-generating case is given by fφ = f ∗
(Dφ/Dt)2
γ + (Dφ/Dt)2 ,
(16) fφ = f ∗
(Dφ/Dt)2
γ + (Dφ/Dt)2 , (16) 2.2.1 Mass Therefore, the pre-
factor k A plays the role of the growth rate, which is experimentally
found to be a function of temperature (Karato 1989). As we will
discuss in the next section, temperature variations in our model
lithosphere are not included which implies that the growth rate
k A in our model is constant. The lithosphere is likely strongest at
intermediate depths (30–70 km at 100 Myr) and, therefore, choosing
a grain growth healing rate associated with this depth would be
most appropriate in the thin-sheet formulation. The first term in
eq. (15) allows for the input of deformational work to go into the
reduction of grain size and increase the surface energy in the system. Grain size reduction in Earth minerals is experimentally found to
occur by dynamic recrystallization (Karato et al. 1980), but there
exists no complete development for the relationship between grain
size and these processes (Hall & Parmentier 2003). Our evolution
equation takes a similar form as previous studies, but we caution
that our grain size reduction term comes about from the partitioning
of deformational work between reversible and irreversible processes
and not from a model that is meant to specifically represent any one
experimentally determined grain size reduction mechanism. Recent
work by Austin & Evans (2007) has also suggested that grain size
reduction is determined by the rate of deformational work, and their
proposed evolution equation for grain size (which is very similar to
eq. 15) reproduces experimental results quite well. σ Dα
Dt = −P Dφ
Dt −B
Dφ
Dt
2
−σηkA Ap + f . (13) (13) Since changes in α can be manifested as either changes in porosity
or changes in inverse grain/void size we can decompose (eq. 13)
into one damage evolution equation for porosity and one for grain
size (Bercovici & Ricard 2005). The damage equation for porosity
is is σ A dη
dφ
Dφ
Dt
= −P
Dφ
Dt
−B
Dφ
Dt
2
+ fφ
(14) (14) and the evolution equation for fineness is DA
Dt = f A
ση −kA Ap,
(15) (15) where f = f φ + f A must be less than one. 2.2.1 Mass Choosing p = 1 leads to a logarithmic integration
where the grain growth rate increases with time, while choosing
p = 3, 4 allows for grain growth to be driven by surface tension
and curvature. This simple analysis will provide the guidance for
our choice of the healing exponent in eq. (15). Therefore, the pre-
factor k A plays the role of the growth rate, which is experimentally
found to be a function of temperature (Karato 1989). As we will
discuss in the next section, temperature variations in our model
lithosphere are not included which implies that the growth rate
k A in our model is constant. The lithosphere is likely strongest at
intermediate depths (30–70 km at 100 Myr) and, therefore, choosing
a grain growth healing rate associated with this depth would be
most appropriate in the thin-sheet formulation. The first term in
eq. (15) allows for the input of deformational work to go into the
reduction of grain size and increase the surface energy in the system. Grain size reduction in Earth minerals is experimentally found to
occur by dynamic recrystallization (Karato et al. 1980), but there
exists no complete development for the relationship between grain
size and these processes (Hall & Parmentier 2003). Our evolution
equation takes a similar form as previous studies, but we caution
that our grain size reduction term comes about from the partitioning
of deformational work between reversible and irreversible processes
and not from a model that is meant to specifically represent any one
experimentally determined grain size reduction mechanism. Recent
work by Austin & Evans (2007) has also suggested that grain size
reduction is determined by the rate of deformational work, and their
proposed evolution equation for grain size (which is very similar to
eq. 15) reproduces experimental results quite well. Since we want to associate A with inverse grain size, A = 1/g, and
the evolution of grain size undergoing grain growth is often found to
vary as (kAt)
1
2 , 1
3 (e.g. Karato 1989; Evans et al. 2001) we therefore,
choose p = 3. Choosing p = 1 leads to a logarithmic integration
where the grain growth rate increases with time, while choosing
p = 3, 4 allows for grain growth to be driven by surface tension
and curvature. This simple analysis will provide the guidance for
our choice of the healing exponent in eq. (15). C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS 2.3 Lithosphere model formulation We will employ the same
dimensional scales as used for the mantle, except that we will in-
clude an additional length scale Aref (m−1) to non-dimensionalize
the fineness (inverse grain size) in order to keep the macroscopic
length scale (D) separate from the microstructural one since they
differ by about nine orders of magnitude. Substituting the aforemen-
tioned scales into our governing equations (with the bar designating
a vertically averaged quantity subsequently dropped) results in the
following non-dimensional equations where the subscript L designates that the stresses are in the thin
layer and we have neglected the buoyancy force of the top layer. where the subscript L designates that the stresses are in the thin
layer and we have neglected the buoyancy force of the top layer. In the ˆz eq. (19) we will neglect the shear stress term since it goes
as O(h/L) (since ∂τ xz/∂x ∼huL/L, where uL is the horizontal
velocity in the lithosphere), which implies σzzL is constant. Taking
the vertical average of both equations (and assuming the lithospheric
thickness h is constant) results in 0 = ∂
∂x
σ ¯α + (1 −¯φ)
¯τxxL −¯P
−1
h
(1 −¯φ)τxzm|z=D
(20)
∂σzzL
∂z
= 0 ⇒σzzL|D+h
D
= 0 ⇒
σ ¯α + (1 −¯φ)
¯τzzL −¯P
= τzzm|z=D
(21) 0 = ∂
∂x
σ ¯α + (1 −¯φ)
¯τxxL −¯P
−1
h
(1 −¯φ)τxzm|z=D
(20) (20) am
bm
−bm
−am
∂uL
∂x
wL
h |z=1
=
1
h
x
0 (1 −φ)τxzm|z=Ddx′ + τxzo −ˆσ Aλ
τzzm|z=1 −ˆσ Aλ
+ μR
F
F
,
(29) 1
h
x
0 (1 −φ)τxzm|z=Ddx′ + τxzo −ˆσ Aλ
τzzm|z=1 −ˆσ Aλ
+ μR
F
F
,
(29) (21) (29) using the continuity of vertical and shear stress at the boundary
between the lithosphere and Newtonian layer. We define σzzL =
σ ¯α + (1−¯φ)(¯τzzL−¯P) tobethetotal vertical stress inthelithosphere,
and the subscript m refers to stresses in the Newtonian mantle. Eq. (21) results from our approximation that σzzL is constant in the
lithosphere, hence the vertically averaged σzzL is equal to σzzL at z =
D + h (and also at z = D). To close the system of equations we also
make the approximation that the average of a product is the product
of the averages (e.g. φτL = ¯φ ¯τL). 2.3 Lithosphere model formulation The thin layer that represents the lithosphere is assumed to have a
free-slip, impermeable surface on top and a no-slip interface with the
Newtonian layer. Since our lithosphere thickness is assumed small
we will employ the thin-sheet approximation to solve for the dy-
namics of this layer (England & McKenzie 1982; Wdowinski et al. 1989; Ribe 1992; Weinstein & Olson 1992; Lemery et al. 2000). In the thin-sheet approximation, vertical gradients in horizontal
velocity are assumed negligible in comparison to the horizontal The fineness generating damage equation presented in this paper
is different from its previous form (Bercovici & Ricard 2005) in
that it now explicitly contains a healing term. In the case where
fineness represents inverse grain size (the only case considered in C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection
1069 1069 Plate generation and two-phase damage theory in a model of mantle convection
1069 gradients. The thin-sheet formulation, therefore, involves determin-
ing the lithosphere’s vertically averaged horizontal velocity and the
vertical velocity at the interface with the Newtonian layer. We thus
examine the horizontal and vertical components of the momentum
equation in a vertically averaged sense. The x and z components of
the momentum equation in the lithosphere are: where the constant τxzo is determined using periodic boundary con-
ditions, and where the constant τxzo is determined using periodic boundary con-
ditions, and am = (1 −¯φ)
4
3μL + Kμo
¯φ
bm = (1 −¯φ)
2
3μL −Kμo
¯φ
λ =
¯η + (1 −¯φ) d¯η
d ¯φ
. (28) 0 = ∂
∂x [σα + (1 −φ)(τxxL −P)] + ∂
∂z [(1 −φ)τxzL]
(18)
0 = ∂
∂z [σα + (1 −φ)(τzzL −P)] + ∂
∂x [(1 −φ)τxzL],
(19) (18) λ =
¯η + (1 −¯φ) d¯η
d ¯φ
. (28) (28) 0 = ∂
∂z [σα + (1 −φ)(τzzL −P)] + ∂
∂x [(1 −φ)τxzL],
(19) (19) We next consider the non-dimensionalization of the equations gov-
erning the behaviour of the lithosphere. 2.3 Lithosphere model formulation 4), while the fineness evolution equa-
tion becomes (23) The vertically averaged evolution equations for damage are The vertically averaged evolution equations for damage are ¯P = −σ ¯A d¯η
d ¯φ −Kμo
¯φ
∇· ¯v + F,
(24)
where
∇· ¯v = ∇H · ¯v −wL
h
z=D, ¯v = (uL, 0, wL)
(25)
and
F = f ∗
(1 −¯φ)2∇· ¯v
γ + (1 −¯φ)2(∇· ¯v)2 ¯
(26) ¯P = −σ ¯A d¯η
d ¯φ −Kμo
¯φ
∇· ¯v + F,
(24) DA
Dt = f AμR
η ˆσ
A−m∗−ˆkA Ap,
(33)
where
ˆkA = kA Ap−1
ref D2
κ
,
(34)
∗= (1 −φ)
∇v : [∇v + [∇v]t −2
3(∇· v)]
. (35) DA
Dt = f AμR
η ˆσ
A−m∗−ˆkA Ap,
(33) (24) (24) (33) where where where
∇· ¯v = ∇H · ¯v −wL
h
z=D, ¯v = (uL, 0, wL)
(25) where
ˆkA = kA Ap−1
ref D2
κ
,
(34)
∗= (1 −φ)
∇v : [∇v + [∇v]t −2
3(∇· v)]
. (35) (25) (25) ˆkA = kA Ap−1
ref D2
κ
, (34) and F = f ∗
(1 −¯φ)2∇· ¯v
γ + (1 −¯φ)2(∇· ¯v)2 ¯
(26) ∗= (1 −φ)
∇v : [∇v + [∇v]t −2
3(∇· v)]
. (35) (26) (35) under the same assumptions that lead to eqs (20) and (21). Sub-
stituting in the expressions for ¯τxxL, ¯τzzL and ¯P eqs (22–24) into
the momentum eqs (20 and 21) we get the following system of
equations for ∂uL
∂x and wL
h |z=D: The general scheme for solving the model system proceeds along
the following steps. (1) Solve for the velocity and stress in the New-
tonian mantle (eqs 1–2) given the current density distribution sub-
ject to free-slip bottom and no-slip top boundary conditions. (2) The
flow calculated in (1) generates a stress at the lithosphere–mantle
interface that is used to calculate the velocity in the lithosphere via
(eq. 29). (3) The lithospheric velocity is then used to drive cavity
flow in the Newtonian mantle, and the velocity and stress in the
Newtonian mantle are the sum of solutions from (1) and (3). 2.3 Lithosphere model formulation The vertically averaged stress is
given by integrating eq. (6), and making use of the assumption that
the vertical velocity is zero at the top boundary; this results in using the continuity of vertical and shear stress at the boundary
between the lithosphere and Newtonian layer. We define σzzL =
σ ¯α + (1−¯φ)(¯τzzL−¯P) tobethetotal vertical stress inthelithosphere,
and the subscript m refers to stresses in the Newtonian mantle. Eq. (21) results from our approximation that σzzL is constant in the
lithosphere, hence the vertically averaged σzzL is equal to σzzL at z =
D + h (and also at z = D). To close the system of equations we also
make the approximation that the average of a product is the product
of the averages (e.g. φτL = ¯φ ¯τL). The vertically averaged stress is
given by integrating eq. (6), and making use of the assumption that
the vertical velocity is zero at the top boundary; this results in where am = (1 −φ)
4
3
μR
Am + KμR
φ
,
bm = (1 −φ)
2
3
μR
Am −KμR
φ
. (30) (30) We have defined the following non-dimensional quantities in the
above equations as q
ˆσ = σ Aref D2
μmκ
,
(31)
μR = μo
μm
,
(32) ˆσ = σ Aref D2
μmκ
,
(31) ˆσ = σ Aref D2
μmκ
, (31) ¯τxxL = ¯μL
4
3
∂¯uL
∂x + 2
3
wL
h |z=D
(22)
¯τzzL = ¯μL
−4
3
wL
h
z=D −2
3
∂¯uL
∂x
. (23) (22) μR = μo
μm
,
(32) μR = μo
μm
, (32) where μo is the reference lithospheric viscosity. The non-
dimensional version of the mass equation is exactly the same as
the dimensional case (eq. 2.5 Scaling analysis for fineness magnitude Without including a healing term in the damage eq. (15) it is obvious
that fineness would continue to increase (i.e. grain size continues
to decrease) without bound which is an unphysical scenario. Given
that grain growth acts to increase the average grain size, even in
the presence of deformation (Karato 1983, 1989) it is important to
consider its effects on steady state fineness. We first examine how
the magnitude of fineness depends upon the damage parameters
( f A, ˆkA and m), as well as consider the stability of the fineness
evolution eq. (15) to an infinitesimal perturbation. We seek the
maximum value of fineness at the boundaries where dA/dx is zero,
and advection does not affect the fineness magnitude. Expanding
the fineness field around its steady state value (Ao) uL
dA
dx = f AμR
ˆση
A−m ˙ε2 −ˆkA Ap. (36) (36) We assume functions with the proper symmetries for the velocity
and fineness in the vicinity of the plate boundary, and then substitute
them into eq. (15) to solve for the boundary widths. We thus assume uL = ±umax tanh(x/δ)
A = Ao exp(−x2/δ2),
(37) = ±umax tanh(x/δ)
A
(
2/δ2)
(37) ( / )
A = Ao exp(−x2/δ2),
(37) (37) A = Ao + ϵ A′(t),
(41) (41) where the ± in the velocity equation distinguishes the relations be-
tween convergent (−) and divergent (+) zones, uL is the surface
velocity and Ao is the steady-state maximum amplitude of fineness. The constant δ is the characteristic width for each boundary (conver-
gent and divergent) and is assumed to be equal for both the fineness
and velocity field (i.e. δ A = δv). Multiplying eq. (36) through by Am
and substituting the above functional forms (eq. 37) into eq. (36)
gives where A′ is the perturbation fineness and ϵ ≪1, and substituting
this into where A′ is the perturbation fineness and ϵ ≪1, and substituting
this into dA
dt = f A Am
τ 2
ˆσημR
−ˆkA Ap
(42) (42) we determine how the stability of steady state fineness is governed
by the damage parameters. To 0th order in ϵ [assuming that the
stress (τ) is constant] the steady-state fineness goes as we determine how the stability of steady state fineness is governed
by the damage parameters. 2.3 Lithosphere model formulation We focus our analysis on both
boundary widths and strain-rates for each numerical experiment
to characterize the degree and type of localization present. The
scaling analysis for δ above depends upon the steady-state fineness
amplitude (Ao), and Ao is clearly a function of the input damage
parameters. It is clear that eq. (40) provides a scaling law for both boundary width
(δ) and boundary strain-rate umax/δ. We focus our analysis on both
boundary widths and strain-rates for each numerical experiment
to characterize the degree and type of localization present. The
scaling analysis for δ above depends upon the steady-state fineness
amplitude (Ao), and Ao is clearly a function of the input damage
parameters. 2.4 Scaling analysis for boundary width/strain-rate In the study of plate generation, an important goal is the formation of
narrow zones of weakness (plate boundaries) which separate strong
plate-like interiors, especially when the analysis is confined to two
dimensions. Before proceeding with a large number of numerical
experiments it is useful to develop a scaling analysis that can guide
our exploration of parameter space as well as provide insight into
the different behaviours our numerical experiments produce. We are
interested in determining how the plate boundary widths depend
upon the free parameters in our model at steady state; to that end,
we look at the fineness evolution eq. (33) in the absence of time-
dependence: 2.3 Lithosphere model formulation (4) We am
bm
−bm
−am
∂uL
∂x
wL
h |z=D
=
1
h
x
0 (1 −¯φ)τxzm|z=Ddx′ + τxzo −σ ¯Aλ
τzzm|z=D −σ ¯Aλ
+
F
F
,
(27) (27) C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS W. Landuyt, D. Bercovici and Y. Ricard 1070 iterate on (2) and (3) until the L2 norm of the difference between
successive iterations reaches 10−6 or less, at which point the litho-
sphere and mantle are assumed to be in mechanical equilibrium. (E) Finally we update the temperature field in the mantle (eq. 3)
and the damage variables in the lithosphere (eqs 4 and 33) with the
equilibrium velocities determined at the end of (4). Convection in
the Newtonian mantle is solved numerically via a spectral method
that employs a Propagator Matrix method for solving Stokes flow
(Hager & O’Connell 1981) and finite difference for the temperature
equation. The cavity flow velocity field is calculated using a Prop-
agator Matrix method as well. The sensitivity of lithospheric and
convective dynamics to variations in damage parameters we will
consider in this study include varying f φ and f A, k A and m, which
are the fraction of deformational work partitioned into generating
voids and fineness, grain growth healing rate, and the viscosity
exponent for grain size sensitivity, respectively. of terms yields of terms yields
π
m + p
ˆkA Am+p
o
+ (∓)
√π
(m + 1)3/2 Am+1
o
umax
δ
−4
3μR
f A
ˆση
umax
δ
2
= 0,
(39) (39) with which we can estimate δ (or umax/δ, the boundary strain-rate)
in terms of our governing free parameters. The term proportional
to (umax/δ)0 in eq. (39) is associated with healing, the term propor-
tional to (umax/δ)1 is associated with advection [(−) is for divergent
and (+) is for convergent], and the term proportional to (umax/δ)2
is associated with deformational work. Solving for the boundary
widths for convergence (δc) and divergence (δd) gives δd,c =
±1 +
1 +
16
3√π
(m+1)3
(m+p)1/2 Ap−m−2
o
μR
fA
ˆση ˆkA
2 (m+1)3/2
(m+p)1/2 Ap−1
o
ˆkA
umax
. (40) (40) It is clear that eq. (40) provides a scaling law for both boundary width
(δ) and boundary strain-rate umax/δ. C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS 2.6 A note on boundary widths and the influence
of temperature Table 1. List of the values for the model parameters: f φ is the fraction
of deformational work that goes into void generating damage, f A is the
fraction of deformational work that goes into fineness generating damage,
m controls the sensitivity of viscosity to fineness, ˆkA is the healing rate
related to grain growth and γ modulates the amount of void generating
damage. Some important points can be made regarding the results of the
scaling analysis for plate boundary widths. Convergent boundaries
are predicted to be smaller than divergent boundaries for a given
plate velocity, and this difference arises predominately from the dif-
ference in sign of the velocity field (see eq. 37). Consideration of
data from global seismicity would imply the opposite of the above
prediction, specifically that divergent boundaries are narrower than
convergent boundaries (Dumoulin et al. 1998). The difference in
boundary widths between divergent and convergent zones seen on
Earth are likely to not arise solely due to the difference in sign of
the divergence field, but it also points out the importance of temper-
ature variations in the lithosphere. The characteristic temperature
of convergent and divergent boundaries in the lithosphere is very
different, and this necessarily plays an important role for the varia-
tions in boundary widths between convergence and divergence. The
effects of horizontal variations in temperature within the plate and
their subsequent influence on viscosity are not explicitly included
in this model due to the assumed isothermal structure of the plate. While we include the zeroth order influence of temperature depen-
dent viscosity in that μo > μm the existence of horizontal variations
in viscosity due to horizontal variations in temperature likely influ-
ences the difference between our scaling law predictions and Earth. Inspection of the scaling law in eq. (40) suggests that allowing for
temperature variations in the lithosphere would most readily affect
the viscosity (μo) and the healing rate (k A); an increase (decrease)
in temperature would decrease (increase) viscosity while increas-
ing (decreasing) healing rate. The divergent boundary width with
temperature variations would, therefore, decrease compared to the
isothermal case and vice versa for convergent boundaries. Given
that the scaling analysis is derived by considering local variations
we could just as easily assume that μo and k A are different for
convergent and divergent boundaries. 2.5 Scaling analysis for fineness magnitude To 0th order in ϵ [assuming that the
stress (τ) is constant] the steady-state fineness goes as Ao =
τ 2
ˆσημR
1
p−m f A
ˆkA
1
p−m
,
(43) ±2umax
δ2
Am+1
o
x tanh(x/δ) exp[−(m + 1)x2/δ2]
= f A
ˆση
μRu2
max
δ2
cosh−4(x/δ) −ˆkA Am+p
o
exp[−(m + p)x2/δ2]. (38) (43) and to first order in ϵ (38) dA′
dt
= (m −p)ˆkA Ap−1
o
A′. (44) (44) We next integrate eq. (38) around the plate boundary over a domain
much greater than the boundary width δ. We make the assumption
that the integral of eq. (38) from [−δ/2 −L, + δ/2 + L] is approx-
imately equal to the integral with the limits (−∞, +∞) to facilitate
evaluation of the above integral (i.e. δ ≪L). We also approximate
tanh (x/δ) by x/δ which doesn’t produce any significant error since
the Gaussian in the relevant term decays much faster than the linear
term grows. The resultant integration of eq. (38) and organization We next integrate eq. (38) around the plate boundary over a domain
much greater than the boundary width δ. We make the assumption
that the integral of eq. (38) from [−δ/2 −L, + δ/2 + L] is approx-
imately equal to the integral with the limits (−∞, +∞) to facilitate
evaluation of the above integral (i.e. δ ≪L). We also approximate
tanh (x/δ) by x/δ which doesn’t produce any significant error since
the Gaussian in the relevant term decays much faster than the linear
term grows. The resultant integration of eq. (38) and organization Two interesting points can be made. First, the steady-state fineness is
predicted to be a function of the ratio of f A and ˆkA, second, stability
of the steady-state to perturbations is determined solely by the sign
of m −p. The scaling above for the magnitude of the steady-state
fineness also provides a diagnostic to determine whether or not
fineness magnitudes are determined by the competition between
deformational work and healing. C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection 1071 2.6 A note on boundary widths and the influence
of temperature 2.6 A note on boundary widths and the influence
of temperature Since it is difficult
to glean information from numerical experiments that fail to con-
verge to a stable solution our goal is to find a range of parameters
that will produce numerically resolvable, steady-state behaviours,
while still remaining in parameter regimes that produce Earth-like
convective patterns. We will look at the following plate character-
istics: divergent and convergent boundary width (we will measure
this quantity by the full-width at half-max for the divergence field),
as well as the concentration of deformation and rigidity of plate
interiors as measured by the integrated diagnostic plateness [Pl =
(π −Pl∗)/(π −2), where Pl∗= (1/|u|avg)
|˙εxx|dx] as defined
by Weinstein & Olson (1992). The definition of plateness results
in a sinusoidal velocity field having a plateness of 0, and boxcar
velocity field having a plateness of 1. Looking at the individual the damage rheology is initiated the plate is assumed to have both
constant porosity and fineness profiles, with values of 1 per cent
and 1, respectively. The exponents (a and b) in the interfacial area
density function η(φ) (7) are chosen to be 0.5 throughout the results
section, and test cases with variations in a and b showed little to
no difference in the system behaviour from the cases with a = b =
0.5. We employ 128 gridpoints in the horizontal direction and 50
gridpoints in the vertical direction. The chosen numerical resolution
faithfully reproduced the results of test runs at higher resolutions
but at significantly reduced computational times. Since it is difficult
to glean information from numerical experiments that fail to con-
verge to a stable solution our goal is to find a range of parameters
that will produce numerically resolvable, steady-state behaviours,
while still remaining in parameter regimes that produce Earth-like
convective patterns. We will look at the following plate character-
istics: divergent and convergent boundary width (we will measure
this quantity by the full-width at half-max for the divergence field),
as well as the concentration of deformation and rigidity of plate
interiors as measured by the integrated diagnostic plateness [Pl =
(π −Pl∗)/(π −2), where Pl∗= (1/|u|avg)
|˙εxx|dx] as defined
by Weinstein & Olson (1992). The definition of plateness results
in a sinusoidal velocity field having a plateness of 0, and boxcar
velocity field having a plateness of 1. Looking at the individual C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS 2.6 A note on boundary widths and the influence
of temperature The scaling law, therefore,
seems capable of predicting boundary width variations seen on
Earth when the appropriate lithospheric temperature variations are
taken into consideration. We will discuss more later about the influ-
ence of variations in viscosity due to temperature and its inclusion
in subsequent models. g
f φ
f A(×10−4)
m
ˆkA
γ
0
0.05
1.2
1
0
0
0.25
1.2
0.75, 1, 2.5
0
0
0.5
1.2
2, 2.5, 5, 10, 25, 50
0
0
0.75
1.2
2.5, 3, 3.75, 5, 7.5, 15
0
0
1
1.2
4
0
0
0.05
1.4
5
0
0
0.25
1.4
5
0
0
0.5
1.4
10, 25, 50, 100
0
0
0.75
1.4
15
0
0
0.05
1.6
5
0
0
0.25
1.6
15, 37.5
0
0
0.5
1.6
25, 37.5, 50, 100
0
0
0.75
1.6
37.5
0
0
0.05
1.8
10
0
0
0.1
1.8
20
0
0
0.25
1.8
50
0
0
0.5
1.8
75, 80, 85, 100, 200
0
0
0.75
1.8
150
0
0
0.05
2
25
0
0
0.1
2
50
0
0
0.25
2
125
0
0
0.5
2
200, 250, 375
0
0
0.75
2
375
0
0
2.5
2
1250
0
0
25
2
12 500
0
0
50–1000
2
250 000
0
0
0.5
2.5
725, 750, 1000
0
0.5
0
0
0
102, 1, 10−2, 10−4
0.4
0.1
1.2, 2
2.5, 250
10−2
0.25
0.25
1.2, 2
2.5, 250
10−2, 1, 103
0.2
0.3
1.2, 2
2.5, 250
10−2
0.1
0.4
1.2, 2
2.5, 250
10−2 the damage rheology is initiated the plate is assumed to have both
constant porosity and fineness profiles, with values of 1 per cent
and 1, respectively. The exponents (a and b) in the interfacial area
density function η(φ) (7) are chosen to be 0.5 throughout the results
section, and test cases with variations in a and b showed little to
no difference in the system behaviour from the cases with a = b =
0.5. We employ 128 gridpoints in the horizontal direction and 50
gridpoints in the vertical direction. The chosen numerical resolution
faithfully reproduced the results of test runs at higher resolutions
but at significantly reduced computational times. 3 N U M E R I C A L R E S U LT S We next explore numerical solutions of our model in a sampling
of parameter space. All of our convection simulations have a sim-
ple Rayleigh–Bernard heating mode, and the Rayleigh number is
106 with an aspect ratio of two for each case. We choose to use
this simple bottom heating mode in order to take advantage of the
symmetries and steady-state behaviour of this convective system. In all cases we initiated our damage rheology calculations with
a Newtonian convection simulation that has reached steady-state. For each set of parameters investigated (see Table 1 for list of all
parameters), we attempted to find steady-state solutions to find ro-
bust measurements of the plate characteristics (boundary widths,
concentration of deformation and rigidity of plate interiors) that
we are most interested in understanding. We also choose the litho-
spheric plate viscosity to be four orders of magnitude greater than
the viscosity of the Newtonian mantle (i.e. μR = 104). With typ-
ical values of mantle scales (μm = 1021 Pa s,σ = 1 N m−1, κ =
10−6 m2 s−1, D ∼106 m and Aref ∼103 m−1 implying a grain size
of 1 mm) the non-dimensional surface tension, ˆσ is approximately
1, and we will assume that it is throughout the calculations. When W. Landuyt, D. Bercovici and Y. Ricard 1072 boundaries, but this variation produces no noticeable improvement
on localization for void-generating damage. This result corroborates
the work of (Bercovici & Ricard 2005) in that pure void-generating
damage is insufficient to allow for plate-like flow. It should be noted
that Bercovici & Ricard (2005) found that void-generating damage
was specifically unable to produce significant toroidal flow which is
not relevant to this study; however both their and our results suggest
that voids in the current two-phase framework do not allow for the
types of plate boundary behaviour seen on Earth. A possible way to
improve the plate-inducing capabilities of void-generating damage
would be to change the sensitivity of viscosity to porosity. This was
done in Bercovici & Ricard (2005) by specifying that the viscos-
ity undergoes a sharp drop when porosity goes above some critical
value, and Katz et al. (2006) employed a viscosity exponentially
dependent on porosity. 3.2 Fineness-generating damage The fineness generating cases are distinguished by the variations
in the parameters m, f A, ˆkA (see eq. 17). The healing exponent is
assumed constant at p = 3 throughout to reduce the number of
variables under consideration in this study. In the fineness evolution
eq. (33) the variables f A and μR multiply each other and can be
treated as a single variable. Most of the results we present in the
following sections are for 0 ≤f AμR ≤1, and since μR = 104
this implies 0 ≤f A ≤10−4. As we show in the following sections
the steady state results are not highly dependent upon the absolute
magnitude of f AμR. Therefore, we can map out the general plate
generating behaviours of our model at smaller values of f AμR, but
at reduced computational times compared to calculations at larger
f AμR. Our results also suggest that f A is not required to be large
in order for significant amounts of localization to be generated. -4.4
0.0
4.4
0.00
0.22
Surface Velocity
Porosity
t = 0.05
Pl ~ .1
(a)
(b)
(c) -4.4
0.0
4.4
0.00
0.22
Temperature
Surface Velocity
Porosity
t = 0.05
Pl ~ .1
x
0
1
2
(a)
(b)
(c)
Figure 2. Numerical results for case with only void generating damage. In
(a) we plot the porosity profile in the lithosphere, (b) we plot the surface
velocity profile in the lithosphere and (c) we plot the temperature field. The
parameter values are f φ = 0.5 and γ = 10−2, and at t = 0.05 the results for
surface velocity are in steady state. This example of void generating damage
is representative of all such cases where the model results are similar to the
case of a Newtonian rigid lid. The contour interval for the temperature field
is 0.1 (and will be the same contour interval for all plots of the temperature
field). The plateness (Pl) for the velocity field is 0.1. 3.1 Void-generating damage The void-generating damage cases are distinguished by the variation
in the parameter γ (and subsequently f φ), which primarily controls
thesensitivityof void-generatingdamagetothedeformational work. We examine cases for four different values of γ (102, 1, 10−2, 10−4)
with constant f ∗= 0.5, and show the results for γ = 10−2 in Fig. 2. Solutions for different γ display similar trends: plateness is very low
(∼1, hence deformation is spatially distributed) and similar to the
case where there is no damage (i.e. f A = f φ = 0), and porosity is
greater over ridges than in subduction zones (Fig. 2a). That voids are
generated over ridges and closed over subduction zones is indicative
of ridges and subduction zones undergoing tensional and compres-
sional environments, respectively. In the current void-generating
damage framework it seems that we are unable to generate narrow
plate boundaries and rigid plate interiors (Fig. 2b). Previous results
employing void-generating damage in simple-shear found that shear
localization occurred upon increasing γ and deformational work in
tandem (Bercovici & Ricard 2003), but in the current study the de-
formational work is approximately constant since we don’t vary the
Rayleigh number. Decreasing the magnitude of γ results in a slight
increase in the rate at which voids are generated around divergent 3 N U M E R I C A L R E S U LT S While these different viscosity formulations
had varying degrees of success for the given model, we will not
explore variations on the porosity sensitivity to viscosity since it is
unclear of the relevance of the previous formulations to our study
and a more appropriate form is unknown at this time. boundary widths will provide insight into how each deformational
zone responds to model parameters, while plateness will quantify
the narrowness of the boundaries as well as the rigidity of the plate
interiors. C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection Further
increases in f A forces the system to an episodic regime followed by a region -5.6
-4.2
-2.8
-1.4
0.0
0.000
0.167
0.333
0.500
+
+
+
+
++++
•
•
•
•
•
•
•• •• ••
+
+
+
+
++
++
•
•
•
•
•
•
•• •• ••
stable
plate like
episodic
epi spl
+
•
log ( fA )
δd
δc ,
kA = 2.5E5
kA = 2.5E2
stagnant
lid
sl Figure 5. The above plot shows the behaviours in boundary widths for
variations in f A with the healing rate ˆkA kept constant for viscosity exponent
m = 2 and two different healing rates. The grey inset is for ˆkA = 250 and
the other plot is for ˆkA = 2.5 × 105, but the axes are the same for both
plots. For both healing rates we find that at small values of f A (where small
depends upon healing rate) the system is in a stagnant lid regime. Further
increases in f A forces the system to an episodic regime followed by a region
characterized by stable plate behaviour. Increases in f A beyond the steady
state regions results in numerically unresolvable phenomena. Figure 3. Variations in surface velocity and fineness for variations in f A
with ˆkA = 2.5 and m = 1.2. erations (Auth et al. 2003; Bercovici & Ricard 2005). However, in
Bercovici & Ricard (2005) they looked at variations in f A/σ (with
the surface tension σ assumed small) and were possibly able to al-
low for more significant localization, though they primarily looked
at generation of toroidal motion without characterizing boundary
width variations. This study suggests that variations in the amount
of fineness generating damage available in a convecting system at
steady state produces the same amount of localization. Fig. 5 dis-
plays the results for variations in boundary widths for variations in
f A at two different healing rates. For a given healing rate at small
values of f A the system is in a stagnant lid regime, and increases
in f A cause a transition to time dependent behaviour characterized
by episodic plate-like behaviour. Upon further increasing f A the
system enters a stable plate-like regime. For a given healing rate,
there is little variation in localization in the stable plate regime. Plate generation and two-phase damage theory in a model of mantle convection 1073 0
25
50
0
1
2
-100
0
100
m = 1.2, kA = 2.5
fA = .05x10-4
fA = .25x10-4
fA = .5x10-4
fA = .75x10-4
Fineness
Surface Velocity
x
Figure 3. Variations in surface velocity and fineness for variations in f A
with ˆkA = 2.5 and m = 1.2. 0
25
50
0
1
2
-100
0
100
m = 1.2, kA = 2.5
fA = .05x10-4
fA = .25x10-4
fA = .5x10-4
fA = .75x10-4
Fineness
Surface Velocity
x
gure 3. Variations in surface velocity and fineness for variations in f
th ˆk
2 5 and m
1 2 -5.6
-4.2
-2.8
-1.4
0.0
0.000
0.167
0.333
0.500
+
+
+
+
++++
•
•
•
•
•
•
•• •• ••
+
+
+
+
++
++
•
•
•
•
•
•
•• •• ••
stable
plate like
episodic
epi spl
+
•
log ( fA )
δd
δc ,
kA = 2.5E5
kA = 2.5E2
stagnant
lid
sl
Fi
5 Th
b
l t h
th
b h
i
i
b
d
idth f -5.6
-4.2
-2.8
-1.4
0.0
0.000
0.167
0.333
0.500
+
+
+
+
++++
•
•
•
•
•
•
•• •• ••
+
+
+
+
++
++
•
•
•
•
•
•
•• •• ••
stable
plate like
episodic
epi spl
+
•
log ( fA )
δd
δc ,
kA = 2.5E5
kA = 2.5E2
stagnant
lid
sl
Figure 5. The above plot shows the behaviours in boundary widths for
variations in f A with the healing rate ˆkA kept constant for viscosity exponent
m = 2 and two different healing rates. The grey inset is for ˆkA = 250 and
the other plot is for ˆkA = 2.5 × 105, but the axes are the same for both
plots. For both healing rates we find that at small values of f A (where small
depends upon healing rate) the system is in a stagnant lid regime. Plate generation and two-phase damage theory in a model of mantle convection With that being said, increasing the fineness inducing damage char-
acterized by f A does allow for a viscosity drop that increases plate
mobility without decreasing boundary widths (and hence increasing
localization). 0.00
0.33
0.66
1.00
0.20
0.26
0.33
0.40 +
+
+
+
+
+
+
+
+
0.00
0.33
0.66
1.00
0.05
0.11
0.18
0.25
+
+
+
+
+
+
+
+
+
0.00
0.33
0.66
1.00
0.25
0.50
0.75
1.00
+
+
+
+
+
+
+
+
+
0.00
0.33
0.66
1.00
0
33
66
100
+
+
+
+
+
+
+
+
+
Plateness
umax
fA ( x10-4 )
(a)
(b)
(c)
(d)
(e)
(f)
m = 1.2 , kA = 2.5 m = 1.6 , kA = 37.5 m = 2.0 , kA = 250
fA ( x10-4 )
0
33
267
400
+
+
+
+
+
+
+
+
+
0
200
400
600
+
+
+
+
+
+
+
+
+
0.00
0.33
0.66
1.00
0.00
0.33
0.66
1.00 0.00
0.33
0.66
1.00
0.25
0.50
0.75
1.00
+
+
+
+
+
+
+
+
+
Plateness
(a)
( ) 0.00
0.33
0.66
1.00
0
33
66
100
+
+
+
+
+
+
+
+
+
umax
(b)
d (b) (d) 0.00
0.33
0.66
1.00
0.05
0.11
0.18
0.25
+
+
+
+
+
+
+
+
+
(c) (d)
f
0
200
400
600
+
+
+
+
+
+
+
+
+
0.00
0.33
0.66
1.00 (f) 0.00
0.33
0.66
1.00
0.20
0.26
0.33
0.40 +
+
+
+
+
+
+
+
+
fA ( x10-4 )
(e) (f)
fA ( x10-4 )
0
33
267
400
+
+
+
+
+
+
+
+
+
0.00
0.33
0.66
1.00 3.2.1 Variations in f A C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection 3.2.2 Variations in healing rate ˆkA m = 1.2 , kA = 2.5 m = 1.6 , kA = 37.5 m = 2.0 , kA = 250 We now consider how variations in healing rate ˆkA affect the be-
haviours of convergent and divergent boundary widths (Figs 6c and
e). Our results show that increasing the healing rate (at constant
damage fraction f A and viscosity exponent m) significantly re-
duces plate velocity (Fig. 6b), has little effect on plateness (Fig. 6a),
and has varied effects on convergent and divergent boundary width
(Figs 6c and e). Increasing the healing rate leads to an increased
plate viscosity which acts to decrease the plate velocity and bound-
ary strain-rate. Convergent boundary widths show unsystematic
behaviour to variations in healing rate except when ˆkA becomes
small and δc generally decreases mildly. The trends of δd for all
m values suggest that the effect of increasing ˆkA initially leads to
a reduced boundary width, but when ˆkA reaches a critical value
divergent boundary width begins to increase and eventually reach
an asymptote. The initial decrease in δd is consistent with our pre-
vious analysis that a decrease in the healing length scale umax/ˆkA
(dominated by the decrease in umax) will lead to a reduction in δd. Therefore, we see that an increase in healing can actually play a
role in localization of divergent boundaries by causing the reduced Figure 4. The range of plate-like behaviours observed (+, with connecting
dashed lines) and predicted (solid lines) from our study for the case of
variations in fineness inducing damage characterized by f A. (a) Plateness
versus f A, (b) Plate velocity versus f A, (c) Convergent boundary width
versus f A, (d) Convergent boundary strain-rate versus f A, (e) Divergent
boundary width versus f A and (f) Divergent zone strain-rate versus f A. Figure 4. The range of plate-like behaviours observed (+, with connecting
dashed lines) and predicted (solid lines) from our study for the case of
variations in fineness inducing damage characterized by f A. (a) Plateness
versus f A, (b) Plate velocity versus f A, (c) Convergent boundary width
versus f A, (d) Convergent boundary strain-rate versus f A, (e) Divergent
boundary width versus f A and (f) Divergent zone strain-rate versus f A. Figure 4. 3.2.1 Variations in f A We consider variations in f A (which represents the partition of
work towards fineness inducing damage) at constant values of ˆkA
(healing rate) and m (viscosity exponent for grain size sensitiv-
ity). Increases in f A result in increases in the plate mobility (de-
fined as the ratio of plate velocity to rms velocity of the mantle)
and boundary strain-rate (uL/δd,c), while the boundary widths and
plateness remain fairly constant and insensitive to changes in f A,
which is somewhat unexpected. These results imply that the fraction
of deformational work that goes into fineness-generating damage
is important for providing the reduction in strength (or increase
in strain-rate) necessary for plate boundary lubrication, but con-
tributes little to the localization of deformation. In Fig. 3, we show
the fineness and surface velocity fields for m = 1.2, ˆkA = 2.5 and
f A = (0.05–0.75)10−4. At f A = 0.05 × 10−4 the fineness field
shows a small perturbation above the baseline value (of 1) and the
associated boundary width is relatively small (see Figs 4c and e). As we increase f A the maximum values of fineness increase as
expected (Ao ∼( f A/ˆkA)1/(p−m)) and there is a slight broadening
of δc (see Fig. 4c). The convergent boundary width δc calculated
at f A = 0.05 × 10−4 is smaller than at larger f A in Fig. 3, and
we can easily see that as f A is reduced the variations in the fine-
ness field get smoothed out until they disappear at f A = 0. The (c) x
0
1
2 Figure 2. Numerical results for case with only void generating damage. In
(a) we plot the porosity profile in the lithosphere, (b) we plot the surface
velocity profile in the lithosphere and (c) we plot the temperature field. The
parameter values are f φ = 0.5 and γ = 10−2, and at t = 0.05 the results for
surface velocity are in steady state. This example of void generating damage
is representative of all such cases where the model results are similar to the
case of a Newtonian rigid lid. The contour interval for the temperature field
is 0.1 (and will be the same contour interval for all plots of the temperature
field). The plateness (Pl) for the velocity field is 0.1. C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS 3.2.2 Variations in healing rate ˆkA The range of plate-like behaviours observed (+, with connecting
dashed lines) and predicted (solid lines) from our study for the case of
variations in fineness inducing damage characterized by f A. (a) Plateness
versus f A, (b) Plate velocity versus f A, (c) Convergent boundary width
versus f A, (d) Convergent boundary strain-rate versus f A, (e) Divergent
boundary width versus f A and (f) Divergent zone strain-rate versus f A. divergent boundary widths in Fig. 4(e) display less systematic be-
haviour as a function of f A than the convergent boundary widths,
and in Fig. 3 appear to not change significantly. The increase in
strain-rate as f A is increased, therefore, comes about due to the
increase in uL at approximately constant δc,d (Figs 4d and f). Since
increasing the fraction of fineness inducing damage does nothing to
increase the rigidity of the plate (and δc,d ≈constant) the plateness
remains approximately unchanged. Increasing the fraction of defor-
mational work that goes into the generation of fineness damage at
constant ˆkA and m, therefore, does not seem to improve the plate
characteristics in any dramatic fashion contrary to previous consid- C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS W. Landuyt, D. Bercovici and Y. Ricard 1074 0
133
267
400
0.20
0.26
0.33
0.40++++++
+
+++
+
+++ +
+
++++
+
+ +
+
0
133
267
400
0.05
0.11
0.18
0.25
++++++
+
+++ +
+++ +
+
++++
++ +
+
0
133
267
400
0.25
0.50
0.75
1.00
++++++
+
+++ +
+++ +
+
++++
++ +
+
0
133
267
400
0
25
50
75+
+
+
++
+
+
+
+
+
+
+
+
+
+++
+
+
+
+
+
Plateness
umax
kA
kA
(a)
(b)
(c)
(d)
(e)
(f)
m = 1.2
m = 1.6
m = 1.4
m = 1.8
m = 2
0
33
267
400
0
200
400
600
++
+
+
++
+
+
+ +
+
++
+
+
++++
+ +
+
+
+
+
+
+
+
++
+
+
+ +
+
++
+
+
+++
+
+ +
+
+
+
0
133
267
400
0
133
267
400
Figure 6. 3.2.3 Variations in f A and ˆkA with their ratio constant It seems that the effect of increasing ˆkA
acts to distribute deformation by increasing the plate strength at old
lithosphere, while the value of f A does not contribute significantly
to weakening of the plate at subduction zones. These results again
highlight the conclusion that the combination of plate velocity and
healing may allow for narrow divergent boundaries, but increases
in fineness inducing damage characterized by f A still remain insuf-
ficient for generating narrow convergent boundaries. In Fig. 9 we (f) (e) kA
(f)
0
33
267
400
++
+
+
++
+
+
+ +
+
++
+
+
++++
+ +
+
+
+
0
133
267
400 0
133
267
400
0.20
0.26
0.33
0.40++++++
+
+++
+
+++ +
+
++++
+
+ +
+
kA
(e) m = 1.2
m = 1.6
m = 1.4
m = 1.8
m = 2 Figure 6. The range of plate-like behaviours observed (+, with connect-
ing dashed lines) and predicted (solid lines) from our study for the case of
variations in healing rate characterized by ˆkA with f A = 0.5 × 10−4. (a)
Plateness versus ˆkA, (b) Plate velocity versus ˆkA, (c) Convergent bound-
ary width versus ˆkA, (d) Convergent boundary strain-rate versus ˆkA, (e)
Divergent boundary width versus ˆkA and (f) Divergent zone strain-rate
versus ˆkA. grain size at a ridge to substantially heal within a short distance
from the ridge. When the healing rate reaches the critical value,
the velocity (and boundary strain-rate) approach their small-
magnitude asymptotes. At this point the lithosphere begins to ap-
proach the properties of a stagnant lid, and hence the boundary
widths increase (divergent) or remain the same (convergent). Our
expectation that increasing healing rate would lead to a modera-
tion of localization while allowing for an increase in plate strength
seems to be partially born out. While increasing healing rate does
strengthen the plate interiors and allows for steady-state convec-
tion, it will eventually choke off surface motion and cause a rigid
lid. 3.2.3 Variations in f A and ˆkA with their ratio constant (d) 0
133
267
400
0.05
0.11
0.18
0.25
++++++
+
+++ +
+++ +
+
++++
++ +
+
(c) (d)
0
200
400
600+
+
+
+
++
+
+
+ +
+
++
+
+
+++
+
+ +
+
+
+
0
133
267
400 We further consider the physics of advection in plate boundary
development by maintaining fixed relative sizes of damage and
healing. The previous sections showed that increasing f A resulted
in an increase in plate velocity and boundary strain-rate, while in-
creasing the healing rate (ˆkA) resulted in a decrease in plate velocity
and boundary strain-rate. Our results for constant f A/ˆkA at various
values of m (note that the ratio f A/ˆkA is different for each m value,
and is chosen to be the largest ratio that is numerically resolvable
(e.g. f A/ˆkA for m = 1.2 is 0.25 × 10−4 while f A/ˆkA for m = 2 is
0.002 × 10−4)) are shown in Figs 7 and 8. We see that plate ve-
locity remains fairly unchanged with variations in f A and ˆkA while
f A/ˆkA is kept constant (Fig. 8b), whereas we say umax grows with an
increasing ratio of f A to ˆkA in Figs 4 and 6. Our results show that di-
vergent boundary width undergoes a decline as f A and ˆkA increase
before eventually asymptoting to its minimum value (Fig. 8e). This
result again shows the phenomena discussed in the previous section
where a decrease in the healing length scale (umax/ˆkA) may allow
for localization of divergent boundaries. Contrary to the previous
section though the plate velocity remains constant as ˆkA goes up
and the model does not approach the stagnant lid case; therefore,
increasing f A simultaneously with the healing rate allows for di-
vergent boundaries to undergo further localization than when f A
remains small. The asymptotic value for δd is eventually controlled
by the competition between healing and deformational work when
both fineness inducing damage and healing rate dwarf the advective
component. We have already seen that convergent boundaries re-
spond somewhat negligibly to variations in f A, but here we see that
δc actually grows for increasing healing rate and fineness inducing
damage fraction (Fig. 8c). 3.2.2 Variations in healing rate ˆkA The range of plate-like behaviours observed (+, with connect-
ing dashed lines) and predicted (solid lines) from our study for the case of
variations in healing rate characterized by ˆkA with f A = 0.5 × 10−4. (a)
Plateness versus ˆkA, (b) Plate velocity versus ˆkA, (c) Convergent bound-
ary width versus ˆkA, (d) Convergent boundary strain-rate versus ˆkA, (e)
Divergent boundary width versus ˆkA and (f) Divergent zone strain-rate
versus ˆkA. 0
133
267
400
0.25
0.50
0.75
1.00
++++++
+
+++ +
+++ +
+
++++
++ +
+
Plateness
(a) 0
133
267
400
0
25
50
75+
+
+
++
+
+
+
+
+
+
+
+
+
+++
+
+
+
+
+
umax
(b)
(d) ture to Fig. 5, except that stagnant lid (stable plate-like) behaviour
would be at large (small) k A instead of small (large) f A. An intrigu-
ing and surprising result is that increasing healing rate (in a specific
range) may allow for localization at divergent plate boundaries via
a reduction in the healing length scale. Plate generation and two-phase damage theory in a model of mantle convection The range of plate-like behaviours observed (+, with connecting
dashed lines) and predicted (solid lines) from our study for the case of
variations in viscosity exponent m with f A = 0.5 × 10−4. (a) Plateness
versus m, (b) Plate velocity versus m, (c) Convergent boundary width versus
m, (d) Convergent boundary strain-rate versus m, (e) Divergent boundary
width versus m, and (f) Divergent zone strain-rate versus m. Figure 8. The range of plate-like behaviours observed (+, with connect-
ing dashed lines) and predicted (solid lines) from our study for the case of
simultaneous variations in f A and ˆkA. (a) Plateness versus f A, (b) Plate
velocity versus f A, (c) Convergent boundary width versus f A, (d) Conver-
gent boundary strain-rate versus f A, (e) Divergent boundary width versus
f A and (f) Divergent zone strain-rate versus f A. m (Fig. 10a), irrespective of the ratio f A to ˆkA. Increases in the
viscosity exponent, therefore, also increase the rigidity of the plate
while also decreasing the boundary widths. As m is increased, the
boundary zone strain-rate increases dramatically (Figs 10d and f),
primarily due to the decrease in boundary width, but strain-rate
is also influenced by the corresponding increase in plate velocity. Allowing for greater viscosity sensitivity to grain size by increasing
m, therefore, allows for significantly greater plate-like behaviours
by increasing plate velocity and plateness, while narrowing both
divergent and convergent plate boundaries. -5
-3
-1
0.000
0.133
0.267
0.400
+ +
+ ++
+
+ +
+
+ + ++
+
+
+
log ( fA )
fA μR / kA = .002
δd
δc ,
Figure 9. The variation in plate boundary width as a function of damage
fraction for 5 × 10−5 ≤f A ≤0.1. This case is for m = 2 and constant ratio
of f A to ˆkA equal to 0.002 × 10−4. We can see that the variations in plate
boundary width saturate well before f A approaches 0.1. -5
-3
-1
0.000
0.133
0.267
0.400
+ +
+ ++
+
+ +
+
+ + ++
+
+
+
log ( fA )
fA μR / kA = .002
δd
δc , In Fig. 11, we show the relevant plate characteristics for a case
where m = 2.5, f A = 0.5 × 10−4, and ˆkA = 750 at steady state
along with its time series. Plate generation and two-phase damage theory in a model of mantle convection (a) Plateness versus f A, (b) Plate
velocity versus f A, (c) Convergent boundary width versus f A, (d) Conver-
gent boundary strain-rate versus f A, (e) Divergent boundary width versus
f A and (f) Divergent zone strain-rate versus f A. 1.0
1.4
1.8
2.2
0.20
0.26
0.33
0.40
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1.0
1.4
1.8
2.2
0.05
0.11
0.18
0.25
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1.0
1.4
1.8
2.2
0.25
0.50
0.75
1.00
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1.0
1.4
1.8
2.2
0
33
66
100
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Plateness
umax
m
m
(a)
(b)
(c)
(d)
(e)
(f)
fA = 0.5 x10-4 , kA = 25, 50, 100, 200, 250
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1.0
1.4
1.8
2.2
1.0
1.4
1.8
2.2
0
33
267
400
0
200
400
600
Figure 10. The range of plate-like behaviours observed (+, with connectin
dashed lines) and predicted (solid lines) from our study for the case o
variations in viscosity exponent m with f A = 0.5 × 10−4. (a) Platenes
versus m, (b) Plate velocity versus m, (c) Convergent boundary width versu
m, (d) Convergent boundary strain-rate versus m, (e) Divergent boundar
width versus m, and (f) Divergent zone strain-rate versus m. 3.2.4 Variations in m We finally consider how variations in viscosity exponent m affect
the plate-like behaviour of our model at constant values of f A and
ˆkA. The results shown in Fig. 10 are for various combinations of
f A and ˆkA, but all show that an increase in m results in a decrease
in boundary width for both δd and δc (Figs 10c and e), which is
expected from our scaling analysis and previous observations (Auth
et al. 2003; Bercovici & Ricard 2005). Our calculations also show
that the plateness of the flow increases approximately linearly with Plate generation and two-phase damage theory in a model of mantle convection The time series values of plateness, Nus-
selt number and maximum fineness show the transition from our
initial state, through the period of transient oscillations to an even-
tual steady state. The velocity field is very plate-like, with narrowly
deforming boundaries and a rigid plate interior. The value of fine-
ness at the subduction zone is approximately five, while the mini-
mum value of fineness in the plate interior is about 0.05 for a range
of fineness of about two orders of magnitude. Given our reference
grain size of 1 mm, this would correspond to a range of grain sizes
of about 200 μ m to 20 mm. While this range of values for grain size
may be possible within the Earth, we would expect the grain size in
a shear zone and plate interior to be somewhat reduced compared
to our calculated values (Evans et al. 2001). In our efforts to find
damage parameters that are able to produce plate like flows though
it is encouraging that successful results don’t require unreasonable
values of material parameters, such as grain size. Figure 9. The variation in plate boundary width as a function of damage
fraction for 5 × 10−5 ≤f A ≤0.1. This case is for m = 2 and constant ratio
of f A to ˆkA equal to 0.002 × 10−4. We can see that the variations in plate
boundary width saturate well before f A approaches 0.1. see the effect of increasing f A to significantly larger values (and
correspondingly ˆkA) demonstrates that boundary widths still remain
saturated. Therefore, our analysis of behaviours at smaller f A dis-
plays the variations brought about maintaining constant advection
by fixing the ratio of damage to healing. 3.2.3 Variations in f A and ˆkA with their ratio constant The transition in convective behaviours would be similar in na- m = 2
0
10
-30
0
30
0
1
2
fA = .05x10-4 , kA = 25
fA = .25x10-4 , kA = 125
fA = .75x10-4 , kA = 375
0
50
-100
0
100
0
1
2
fA = .15x10-4 , kA = 0.6
fA = .5x10-4 , kA = 2
fA = .75x10-4 , kA = 3
m = 1.2
Fineness
Surface Velocity
x
x
Figure 7. Variations in velocity and fineness for different m, f A and ˆkA values, but keeping f A/ˆkA = constant. C⃝2008 The Authors, GJI, 174, 1065–1080 m = 2
0
10
-30
0
30
0
1
2
fA = .05x10-4 , kA = 25
fA = .25x10-4 , kA = 125
fA = .75x10-4 , kA = 375
x 0
50
-100
0
100
0
1
2
fA = .15x10-4 , kA = 0.6
fA = .5x10-4 , kA = 2
fA = .75x10-4 , kA = 3
m = 1.2
Fineness
Surface Velocity
x Figure 7. Variations in velocity and fineness for different m, f A and ˆkA values, but keeping f A/ˆkA = constant. C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection 1.0
1.4
1.8
2.2
0.20
0.26
0.33
0.40
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1.0
1.4
1.8
2.2
0.05
0.11
0.18
0.25
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1.0
1.4
1.8
2.2
0.25
0.50
0.75
1.00
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1.0
1.4
1.8
2.2
0
33
66
100
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Plateness
umax
m
m
(a)
(b)
(c)
(d)
(e)
(f)
fA = 0.5 x10-4 , kA = 25, 50, 100, 200, 250
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1.0
1.4
1.8
2.2
1.0
1.4
1.8
2.2
0
33
267
400
0
200
400
600 0.00
0.33
0.66
1.00
0.05
0.11
0.18
0.25
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0.00
0.33
0.66
1.00
0.25
0.50
0.75
1.00
+ +
+
+
+
+
+
+
+
++
+
+
+
++
+
+
+
Plateness
(a)
(c)
( ) 0.00
0.33
0.66
1.00
0
33
66
100
+ +
+
+
+
+
+
+
+
++
+
+
+
++
+
+
+
umax
(b)
(d) 1.0
1.4
1.8
2.2
0
33
66
100
+
+
+
+
+
+
+
+
+
+
+
+
+
+
umax
(b)
(d)
600 0.00
0.33
0.66
1.00
0.05
0.11
0.18
0.25
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0.00
0.33
0.66
1.00
0.25
(c)
( ) (d) (d) m
(d)
(f)
kA = 25 50 100 200 250
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1.0
1.4
1.8
2.2
1.0
1.4
1.8
2.2
0
33
267
400
0
200
400
600 (d)
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1.0
1.4
1.8
2.2
0
200
400
600 (f) (f) (e) 1.0
1.4
1.8
2.2
0.20
0.26
0.33
0.40
+
+
+
+
+
+
+
+
+
+
+
+
+
+
m
(e) 0.00
0.33
0.66
1.00
0.20
0.26
0.33
0.40
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
(e)
fA ( x10-4 ) m fA = 0.5 x10-4 , kA = 25, 50, 100, 200, 250 Figure 10. Plate generation and two-phase damage theory in a model of mantle convection 1075 0.00
0.33
0.66
1.00
0.20
0.26
0.33
0.40
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0.00
0.33
0.66
1.00
0.05
0.11
0.18
0.25
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0.00
0.33
0.66
1.00
0.25
0.50
0.75
1.00
+ +
+
+
+
+
+
+
+
++
+
+
+
++
+
+
+
0.00
0.33
0.66
1.00
0
33
66
100
+ +
+
+
+
+
+
+
+
++
+
+
+
++
+
+
+
Plateness
umax
(a)
(b)
(c)
(d)
(e)
(f)
fA ( x10-4 )
fA ( x10-4 )
+ +
+
+
+
+
+
+
+
++
+
+
+
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0
33
267
400
0
200
400
600
0.00
0.33
0.66
1.00
0.00
0.33
0.66
1.00 0.00
0.33
0.66
1.00
0.20
0.26
0.33
0.40
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0.00
0.33
0.66
1.00
0.05
0.11
0.18
0.25
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0.00
0.33
0.66
1.00
0.25
0.50
0.75
1.00
+ +
+
+
+
+
+
+
+
++
+
+
+
++
+
+
+
0.00
0.33
0.66
1.00
0
33
66
100
+ +
+
+
+
+
+
+
+
++
+
+
+
++
+
+
+
Plateness
umax
(a)
(b)
(c)
(d)
(e)
(f)
fA ( x10-4 )
fA ( x10-4 )
+ +
+
+
+
+
+
+
+
++
+
+
+
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0
33
267
400
0
200
400
600
0.00
0.33
0.66
1.00
0.00
0.33
0.66
1.00
Figure 8. The range of plate-like behaviours observed (+, with connect-
ng dashed lines) and predicted (solid lines) from our study for the case of
simultaneous variations in f A and ˆkA. 3.3 Combined void- and fineness-generating damage In this final section we consider the combined effects of both
void- and fineness-generating damage mechanisms on plate-like C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS 1076
W. Landuyt, D. Bercovici and Y. Ricard
Temperature
0.0
0.5
1.0
0.0000
0.0257
0.0514
0.0771
time
Pl
-20
0
20
Surface Velocity
10
12
Nu
0.0
9.2
Fineness
0
15
Max A
0.000
0.035
t = 0.0514
Porosity
0.000
0.026
Max φ
Figure 11. Results for parameter set m = 2.5, f A = 0.5 × 10−4 and ˆkA = 750. -4.2
0.0
4.2
0
2
0.000
0.026
Temperatur e
Surface Velocity
Finenes s
Porosity
t = 0.0025
fA = 0
fφ = 0.5
-12
0
12
0
12
0.000
0.037
t = 0.00353
-81
0
81
0
16
0.000
0.023
t = 0.0131
fA = 0.25
fφ = 0.25
fA = 0.5
fφ = 0
Figure 12. Results for case when both void and fineness generating damage are generated by deformational work. In these plots ˆkA = 2.5 × 106, m = 2 and
γ = 10−2. behaviour in the model We have so far found that void generat-
subsequent to frames shown The reason for the ultra localization 1076 W. Landuyt, D. Bercovici and Y. Ricard 0.0
0.5
1.0
0.0000
0.0257
0.0514
0.0771
time
Pl
10
12
Nu
0
15
Max A
0.000
0.026
Max φ Temperature
0.0
0.5
1.0
0.0000
0.0257
0.0514
0.0771
time
Pl
-20
0
20
Surface Velocity
10
12
Nu
0.0
9.2
Fineness
0
15
Max A
0.000
0.035
t = 0.0514
Porosity
0.000
0.026
Max φ
Figure 11. Results for parameter set m = 2.5, f A = 0.5 × 10−4 and ˆkA = 750. Figure 11. Results for parameter set m = 2.5, f A = 0.5 × 10−4 and ˆkA = 750. -12
0
12
0
12
0.000
0.037
t = 0.00353
fA = 0.25
fφ = 0.25 -4.2
0.0
4.2
0
2
0.000
0.026
Temperatur e
Surface Velocity
Finenes s
Porosity
t = 0.0025
fA = 0
fφ = 0.5
-12
0
12
0
12
0.000
0.037
t = 0.00353
-81
0
81
0
16
0.000
0.023
t = 0.0131
fA = 0.25
fφ = 0.25
fA = 0.5
fφ = 0
Figure 12. Results for case when both void and fineness generating damage are generated by deformational work. 3.3 Combined void- and fineness-generating damage In these plots ˆkA = 2.5 × 106, m = 2 and
γ = 10−2. Temperatur e
Surface Velocity Figure 12. Results for case when both void and fineness generating damage are generated by deformational work. In these plots ˆkA = 2.5 × 106, m = 2 and
γ = 10−2. Figure 12. Results for case when both void and fineness generating damage are generated by deformational work. In these p
γ = 10−2. behaviour in the model. We have so far found that void generat-
ing is insufficient by itself to produce plate like flow in the cur-
rent two-phase framework, which is in line with previous work
(Bercovici & Ricard 2005). On the other hand, fineness generat-
ing damage has shown to be successful at producing plate like
flow under certain circumstances in this study as well as previously
(Bercovici & Ricard 2005). Fig. 12 shows results for a case with
both void and fineness generating damage, the void- and fineness-
generating end member cases are presented alongside for com-
parison. Previously we found that employing a damage rheology
with only void generating damage and γ = 10−2 produced very
unplate-like flows (see Fig. 12). However, when these same pa-
rameters are employed with moderate fineness generating damage
( fφ = f A = 0.25, ˆkA = 2.5 × 106, m = 2) the result is local-
ized plate boundaries and significantly more plate-like flow. The
combined case in fact becomes too localized to numerically resolve subsequent to frames shown. The reason for the ultra localization
in this case is due to the f A/η(φ) term in eq. (15). The void-
generating damage allows for the porosity field to undergo larger
variations than when f φ = 0, and hence η(φ) is reduced in the
convergent and divergent zones. The reduction in η(φ) results in
an increase in the effective amount of fineness generating dam-
age, f A/η(φ). Physically, the reduction in η(φ) (or interfacial area
density) allows for the continued input of damage to have a more
significant impact than if the system was already heavily damaged,
since inputting deformational work into a material with little dam-
age results in a greater viscosity drop than if the material was already
significantly deformed. While our results for boundary width vari-
ations in f A (with f φ = 0 at constant ˆkA and m) (Fig. C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS 4.1 Earth-like parameters and our model we eventually force the system to be similar to the case when
f φ = 0. φ
The results of allowing combined void- and fineness-generating
damage in our convective model is summarized in Fig. 13. For
some cases with combined forms of damage the system became
numerically unresolvable (i.e. solution became too localized before
reaching steady state), hence we choose to measure the maximum
strain-rate when the convergent boundary width was less than three
gridpoints. For each choice of m, k A and γ the plate velocity is
generally the same, so increasing strain-rate [(u/δ)max] is indica-
tive of a reduction in boundary width and hence localization. The
results show that an increase in plate-like behaviour, specifically
localization, is achieved by allowing for both forms of damage
when the fineness generating parameters by themselves are associ-
ated with stable plate-like behaviour (ˆkA = 2.5 × 105, f = 0.05
and ˆkA = 2.5 × 106, f = 0.5 in Fig. 13). In contrast, the com-
bination of void and fineness generating damage, when the fine-
ness parameters are associated with episodic or stagnant lid be-
haviour (ˆkA = 2.5 × 106, f = 0.05 in Fig. 13), does not result
in an increase in plate localization or mobilization. Combining the
two types of damage mechanisms can result in more significant
localization, but this seems to occur only when the fineness pa-
rameters permit stable plate-like solutions. Addition of void gen-
erating damage does not enhance plate like behaviour unless the
fineness parameters allow for it. As previously discussed, increas-
ing the parameter γ acts to force the system back to the case when
f φ = 0 and decreasing γ causes the convective system to local-
ize at a faster rate. These results suggest that an interesting inter-
play between void and fineness generating damage may exist that
could facilitate shear localization within the Earth. From a mineral
physics perspective microcracks distributed in a poly-crystalline
material may act to pin grain boundaries, therefore, inhibiting grain
growth. If grain growth is stunted due to the presence of pores (by
f φ ̸= 0) then grain size will remain smaller and viscosity will cor-
respondingly remain small. Results from looking at natural samples
has shown that grain size reduction can lead to the formation of
brittle faults and shear localization (Jin et al. 1998). 4.1 Earth-like parameters and our model Plate generation necessitates breaking the strongest part of the litho-
sphere; we would therefore, like to know if the range of parameters
in our thin-sheet model is associated with the strongest portion of the
lithosphere [i.e. brittle–ductile transition (Kohlstedt et al. 1995)]. One way of estimating this is through the grain growth rate k A in
eq. (15). From mineral physics we know that the grain growth rate in
olivine and other minerals is highly temperature dependent (Karato
1989). The growth rate has an Arrhenius type functional form that
can be solved for temperature such that Figure 13. The effect of combined void and fineness generating damage
on plate-like behaviour for different values of total damage and fineness
healing. For this plot m = 2, f = 0.05, 0.5, ˆkA = 2.5 × 105, 2.5 × 106 and
γ = 10−2. The quantity f is defined to be the sum of f A and f φ. kA = ko exp
−H
RT
⇒T =
H
R ln
ko
kA
,
(45) (45) where ko is the reference grain growth rate, H is the activation
enthalpy, and R is the gas constant. Given the values of ˆkA we deter-
mined to be capable of producing plate-like flows we can then get an
estimate of the lithospheric temperature controlling the rheology in
our model. Using values of k o = 10−8 m2 s−1 and H = 2 × 105 J (mol
K)−1 (Karato 1989) and ˆkA = 250 –1000(2.3–9.0 × 10−21m2 s−1 in
dimensional form) for m = 2 and f AμR = 0.5 from our calculations
we arrive at a temperature range of T = 800–900 K. This range of
values for temperature will put us in the brittle–ductile regime for
most of the oceanic lithosphere. Our thin-sheet damage rheology
is, therefore, associated with the strongest region of the lithosphere,
which is what is desired in a thin-sheet model. The kinetics of grain
growth at such low temperatures might be significantly different
than eq. (45) predicts, but experimental data on single-phase grain
growth likely predict much faster grain growth than actually occurs
in the mantle due to the inhibition of grain growth by secondary
phases (Evans et al. 2001). Therefore, our temperature estimate
might be a lower limit of the temperature or depth represented by
our vertically averaged rheology. 4.1 Earth-like parameters and our model Brittle fail-
ure may lead to significant grain size reduction as well, though
the reduction process may be more related to generation of fault
gouge than grain size reduction via dynamic recrystallization or
similar processes. The interaction of brittle and ductile deformation
mechanisms remains a poorly understood rheological phenomena
from both an experimental and theoretical perspective (Kohlstedt
et al 1995) 4 D I S C U S S I O N A N D C O N C LU S I O N 0.0
0.5
1.0
0
1000
2000
fA / f
| . max |
kA = 2.5E6
kA = 2.5E5
f = 0.5
f = 0.05
f = 0.05 3.3 Combined void- and fineness-generating damage 4) didn’t
show any significant localization it seems that a larger effective f A
(with f φ > 0) is capable of generating much greater plate-like flow. When γ is increased to 103 (with the same fineness parameters) C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS 1077 Plate generation and two-phase damage theory in a model of mantle convection 0.0
0.5
1.0
0
1000
2000
fA / f
| . max |
kA = 2.5E6
kA = 2.5E5
f = 0.5
f = 0.05
f = 0.05
Figure 13. The effect of combined void and fineness generating damage
on plate-like behaviour for different values of total damage and fineness
healing. For this plot m = 2, f = 0.05, 0.5, ˆkA = 2.5 × 105, 2.5 × 106 and
γ = 10−2. The quantity f is defined to be the sum of f A and f φ. C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS R E F E R E N C E S Austin, N. & Evans, B., 2007. Paleowattmeters: a scaling relation for dy-
namically recrystallized grain size, Geology, 35(4), 343–346. Auth, C., Bercovici, D. & Christensen, U., 2003. Two-dimensional convec-
tion with a self-lubricating, simple-damage rheology, Geophys. J. Int.,
154, 783–800. Bercovici, D., 1993. A simple model of plate generation from mantle flow,
Geophys. J. Int., 114, 635–650. Bercovici, D., 1995. A source-sink model of the generation of plate tectonics
from non-Newtonian mantle flow, J. geophys. Res., 100, 2013–2030. Bercovici, D., 1998. Generation of plate tectonics from lithosphere–mantle
flow and void-volatile self-lubrication, Earth planet. Sci. Lett., 154, 139–
151. Bercovici, D., 2003. The generation of plate tectonics from mantle convec-
tion, Earth planet. Sci. Lett., 205, 107–121. Bercovici, D. & Ricard, Y., 2003. Energetics of a two-phase model of litho-
spheric damage, shear localization and plate-boundary formation, Geo-
phys. J. Int., 152, 581–596. Bercovici, D. & Ricard, Y., 2005. Tectonic plate generation and two-phase
damage: void growth versus grain size reduction, J. geophys. Res., 110,
1–18. While our model does include the zeroth order effect of temper-
ature on the viscosity of the lithosphere (i.e. lithospheric viscosity
is much greater than Newtonian mantle viscosity), the effect of a
laterally varying temperature on the lithospheric dynamics is an im-
portant consideration to look into for the future. A laterally variable
temperature in the plate would likely allow for the formation of even
narrower divergent plate boundaries. The area of ridge formation is
significantly warmer than assumed in the model, and including this
effect would lead to a lower viscosity as well as increase the amount
of healing in the grain size evolution equation. The viscosity of
the region of old lithosphere would be similar to the zeroth order
viscosity assumed in our model and, therefore, would likely be little
affected by allowing for lateral temperature variations. Bercovici, D., Ricard, Y. & Richards, M., 2000. The relation between mantle
dynamics and plate tectonics: a primer, in History and Dynamics of Global
Plate Motions, Geophys. Monogr. Ser., Vol. 121, pp. 5–46, eds Richards,
M.A., Gordon, R. & van der Hilst, R., Am. Geophys. Union, Washington,
DC. Bercovici, D., Ricard, Y. & Schubert, G., 2001a. A two-phase model of
compaction and damage, 1. General theory, J. geophys. Res., 106(B5),
8887–8906. Bercovici, D., Ricard, Y. & Schubert, G., 2001b. A two-phase model of
compaction and damage, 3. Applications to shear localization and plate
boundary formation, J. 4.2 Final thoughts This behaviour may also be important for the forma-
tion of narrow rifts in that plate thinning under tension may allow
for significant healing outside of the failure region thereby forming
a localized rift zone. Increases in the viscosity exponent m lead to
the formation of narrow plate boundaries (both convergent and di-
vergent) as well as increases in the rigidity of the plate interior, and
the values of m explored fall within the range of values expected for
mantle rheologies (Karato & Wu 1993). These results suggest that
the mechanism of localization at convergent and divergent bound-
aries need not originate from the same process. Our model does not
incorporate the generation of melts which will strongly influence
the strength of ridges as well (Tackley 2000a,b), and melt forma-
tion obviously leads to different localization mechanism than what
affects subduction zones. While grain size sensitive creep (coupled
with crack formation) may be needed to form narrow convergent
boundaries, the generation of narrow divergent boundaries may
only need some small damage input coupled with strong healing
near the ridge. Allowing for both forms of damage causes a feed-
back mechanism which allows for the most significant localization
of plate boundaries in our study. The feedback mechanism begins
with void-generation allowing for greater variability in porosity, the
range of η(φ) (or interfacial area density) increases, which leads
to greater damage input into increasing fineness (hence reduction
in boundary viscosity), which focuses the variations in the poros-
ity field, hence completing the cycle that facilitates the formation
of narrow plate boundaries. While many studies have considered
the effect of cracking and grain size reduction on plate generation
or convection separately (Hall & Parmentier 2003; Bercovici &
Ricard 2005), our study suggests that it is the interaction of these
two forms of damage which allows for the formation of plate tec-
tonics on Earth. Further investigation into the coupling between
void- and fineness-generating damage in a depth-dependent prob-
lem, hence allowing us to follow the plate boundary formation
process in a vertical section of the lithosphere, is a necessary next
step. dependent rheologies would enable us to explore the possibilities of
fault formation and progression in a more complete manner. For ex-
ample, localization may be initiated by grain size reduction at mid to
deep lithospheric depths leading to failure at shallower depths via
void generation completing the formation of a lithospheric fault. AC K N OW L E D G M E N T S The authors benefited from discussions with Shun-ichiro Karato and
Garrett Leahy, as well as the feedback in the Yale mantle/core semi-
nar. The suggestions from two anonymous reviewers also helped to
clarify many different aspects of this paper. Support was provided
by the National Science Foundation (NSF, grant EAR-0105269) and
the Centre National de la Recherche Scientifique (CNRS). 4.2 Final thoughts These considerations are an important step for future analyses, but
the current thin-sheet model does allow for a fairly comprehensive
understanding of how two-phase damage theory could facilitate the
formation of tectonic plates on Earth. 4.2 Final thoughts Increases in the viscosity exponent m lead to
the formation of narrow plate boundaries (both convergent and di-
vergent) as well as increases in the rigidity of the plate interior, and
the values of m explored fall within the range of values expected for
mantle rheologies (Karato & Wu 1993). These results suggest that
the mechanism of localization at convergent and divergent bound-
aries need not originate from the same process. Our model does not
incorporate the generation of melts which will strongly influence
the strength of ridges as well (Tackley 2000a,b), and melt forma-
tion obviously leads to different localization mechanism than what
affects subduction zones. While grain size sensitive creep (coupled
with crack formation) may be needed to form narrow convergent
boundaries, the generation of narrow divergent boundaries may
only need some small damage input coupled with strong healing
near the ridge. Allowing for both forms of damage causes a feed-
back mechanism which allows for the most significant localization
of plate boundaries in our study. The feedback mechanism begins
with void-generation allowing for greater variability in porosity, the
range of η(φ) (or interfacial area density) increases, which leads
to greater damage input into increasing fineness (hence reduction
in boundary viscosity), which focuses the variations in the poros-
ity field, hence completing the cycle that facilitates the formation
of narrow plate boundaries. While many studies have considered
the effect of cracking and grain size reduction on plate generation
or convection separately (Hall & Parmentier 2003; Bercovici &
Ricard 2005), our study suggests that it is the interaction of these
two forms of damage which allows for the formation of plate tec-
tonics on Earth. Further investigation into the coupling between
void- and fineness-generating damage in a depth-dependent prob-
lem, hence allowing us to follow the plate boundary formation
process in a vertical section of the lithosphere, is a necessary next
step. characteristics we find. We have found that increasing fineness in-
ducing damage f A (for fixed healing rate k A and viscosity expo-
nent m) does not do much to improve the plate characteristics in
our model, though allowing for void-generating damage in tandem
with fineness-generating damage dramatically changes this result. Increases in the healing term k A can lead to an important reduction
in divergent boundary width due to the reduction in the healing
length scale. 4.2 Final thoughts The goal of this study is to test our hypothesis that two-phase dam-
age theory will allow for shear localization leading to the develop-
ment of plate tectonics on Earth. We have employed a 2-D model
of mantle convection in order to test the plate generating capa-
bilities of two-phase damage theory. The primary characteristics
of plate tectonics on Earth that we have attempted to address in
this study include the formation of narrow boundaries at both con-
vergent and divergent zones, rigid plate interiors, and temporally
persistent plate-like velocity profiles. We have attempted to under-
stand how the different factors (i.e. advection, deformational work,
and healing) affect the rheological variables (fineness and porosity)
to produce the aforementioned plate-like features. We have explored how each form of damage, void and fineness
generating (or grain size reducing), by itself and in tandem, can
facilitate plate-like behaviour in a convectively driven system. Our
results for void and fineness generating damage by themselves have
shown some similar consequences to separate problems as previ-
ous work (Bercovici & Ricard 2005), namely that void generating
damage was unsuccessful while fineness generating damage was
quite successful at inducing plateness in this study and toroidal
motion in Bercovici & Ricard (2005). An important part of this
study has been to understand how the range of different param-
eters that govern grain size (fineness) evolution affect the plate C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS W. Landuyt, D. Bercovici and Y. Ricard 1078 characteristics we find. We have found that increasing fineness in-
ducing damage f A (for fixed healing rate k A and viscosity expo-
nent m) does not do much to improve the plate characteristics in
our model, though allowing for void-generating damage in tandem
with fineness-generating damage dramatically changes this result. Increases in the healing term k A can lead to an important reduction
in divergent boundary width due to the reduction in the healing
length scale. This behaviour may also be important for the forma-
tion of narrow rifts in that plate thinning under tension may allow
for significant healing outside of the failure region thereby forming
a localized rift zone. A P P E N D I X : S C A L I N G A NA LY S I S :
P R E D I C T I O N S V E R S U S R E S U LT S Karato, S., 1983. Grain-size distribution and rheology of the upper mantle,
Tectonophysics, 104, 155–176. Given the extensive numerical data generated, we test how well the
scaling analyses developed in the previous sections fit the data. In
Fig. A1, we show the results of plotting Ao (the maximum steady
state value of fineness, see eq. 37) at both ridges and subduction
zones versus eq. (43) for each numerical experiment. The fit of the
data to the prediction is quite good, and indicates that our assumption
that stress (deformational work) is constant in a given environment
(i.e. divergent vs. convergent) is reasonably accurate. The slopes of
the predicted fineness magnitudes are actually different for ridges
and subduction zones owing to the fact that the second invariant of
stress between these two different regions is different. These results
suggest that the steady-state amplitude of fineness at subduction
zones and ridges is governed by the competition between deforma-
tional work and healing, and advection plays no role in determining
the amplitude. Karato, S., 1989. Grain growth kinetics in olivine aggregates, Tectono-
physics, 168, 255–273. Karato, S. & Wu, P., 1993. Rheology of the upper mantle: a synthesis,
Science, 260, 771–778. Karato, S., Toriumi, M. & Fujii, T., 1980. Dynamic recrystallization of
olivine single crystals during high temperature creep, Geophys. Res. Lett.,
7, 649–652. Katz, R., Spiegelman, M. & Holtzman, B., 2006. The dynamics of melt
and shear localization in partially molten aggregates, Nature, 442, 676–
679. King, S., 2001. Subduction zones: observations and geodynamic models,
Phys. Earth planet. Int., 127, 9–24. Kohlstedt, D., Evans, B. & Mackwell, S., 1995. Strength of the lithosphere:
Constraints imposed by laboratory experiments, J. geophys. Res., 100,
17 587–17 602. Lemery, C., Ricard, Y. & Sommeria, J., 2000. A model for the emergence of
thermal plumes in Rayleigh-Benard convection at infinite Prandtl number,
J. Fluid Mech., 414, 225–250. The results of comparing our numerical data for δc/umax and
δd/umax with our scaling analysis is presented in Fig. A2(a). In
Fig. A2(b) we plot ratio of the numerical result for δc,d/umax to the
predicted value for each point in parameter space, since we expect McKenzie, D., 1984. The generation and compaction of partially molten
rock, J. Petrol., 25, 713–765. Moresi, L. & Solomatov, V., 1998. Mantle convection with a brittle litho-
sphere: thoughts on the global tectonic style of the Earth and Venus,
Geophys. J. Int., 133, 669–682. A P P E N D I X : S C A L I N G A NA LY S I S :
P R E D I C T I O N S V E R S U S R E S U LT S 0.0
15.8
31.5
47.3
0.0
16.5
33.1
49.6
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+++
+
+
+
+
+ +
+
+
+
+
+
+
+
+++++++
++ +
++++
+
++
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+++
+
+
+
+
++
+
+
+
+
+
+
+
+++++++
+++
+++++++
Ridge
Subduction Zone
Calculated Fineness
Predicted Fineness
Figure 14. The values of fineness plotted on the ordinate come from the
numerical experiments, while the values of fineness plotted on the abscissa
are from the scaling analyses eq. (43). The results from the numerical exper-
iments and the scaling analyses are for the maximum values of steady-state
fineness (Ao) in both subduction zones and ridges. 0.0
15.8
31.5
47.3
0.0
16.5
33.1
49.6
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+++
+
+
+
+
+ +
+
+
+
+
+
+
+
+++++++
++ +
++++
+
++
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+++
+
+
+
+
++
+
+
+
+
+
+
+
+++++++
+++
+++++++
Ridge
Subduction Zone
Calculated Fineness
Predicted Fineness Ogawa,
M.,
2003. Plate-like
regime
of
a
numerically
modeled
thermal convection in a fluid with temperature-, pressure-, and
stress-history-dependent
viscosity,
J. geophys. Res.,
108,
2067,
doi:10.1029/2000JB000069. Ribe, N., 1992. The dynamics of thin shells with variable viscosity and
the origin of toroidal flow in the mantle, Geophys. J. Int., 110, 537–
552. Ricard, Y. & Bercovici, D., 2003. Two-phase damage theory and crustal rock
failure: the theoretical ‘void’ limit, and the prediction of experimental
data, Geophys. J. Int., 155, 1057–1064. Figure 14. The values of fineness plotted on the ordinate come from the
numerical experiments, while the values of fineness plotted on the abscissa
are from the scaling analyses eq. (43). The results from the numerical exper-
iments and the scaling analyses are for the maximum values of steady-state
fineness (Ao) in both subduction zones and ridges. Ricard, Y. & Froidevaux, C., 1986. Stretching instabilities and lithospheric
boudinage, J. geophys. Res., 91, 8314–8324. Ricard, Y., Bercovici, D. & Schubert, G., 2001. Plate generation and two-phase damage theory in a model of mantle convection Plate generation and two-phase damage theory in a model of mantle convection 1079 Plate generation and two-phase damage theory in a model of mantle convection Gurnis, M., Zhong, S. & Toth, J., 2000. On the competing roles of fault
reactivation and brittle failure in generating plate tectonics from mantle
convection, in History and Dynamics of Global Plate Motions, Geophys. Monogr. Ser., Vol. 121, pp. 73–94, eds Richards, M.A., Gordon, R. & van
der Hilst, R., Am. Geophys. Union, Washington, DC. weakening and asthenosphere, Geochem. Geophys. Geosystems (G3), 1,
2000GC000043. Wdowinski, S., O’Connell, R. & England, P., 1989. A continuum model
of continental deformation above subduction zones: Application to the
Andes and the Aegean, J. geophys. Res., 94, 10 331–10 346. Weinstein, S., 1996. Thermal convection in a cylindrical annulus with a
non-Newtonian outer surface, PAGEOPH, 146, 551–572. Hager, B. & O’Connell, R., 1981. A simple global model of plate dynamics
and mantle convection, J. geophys. Res., 86, 4843–4867. Hall, C. & Parmentier, E., 2003. Influence of grain size evolution on convec-
tive stability, Geochem. Geophys. Geosystems (G3), 1, 2003GC000308. Weinstein, S. & Olson, P., 1992. Thermal convection with non-Newtonian
plates, Geophys. J. Int., 111, 515–530. Jin, D., Karato, S. & Obata, M., 1998. Mechanisms of shear localization in
the continental lithosphere: Inference from the deformation microstruc-
tures of peridotites from the Ivrea zone, northwestern Italy, J. Struct. Geol., 20, 195–209. R E F E R E N C E S geophys. Res., 106(B5), 8925–8940. Cawood, P.A., Kroner, A. & Pisarevsky, S., 2006. Precambrian plate tecton-
ics: criteria and evidence, GSA Today, 16(7), 4–11. Christensen, U. & Harder, H., 1991. Three-dimensional convection with
variable viscosity, Geophys. J. Int., 104, 213–226. While a thin-sheet formulation for lithospheric dynamics cap-
tures a significant component of lithospheric behaviour, allowing
for depth-dependent rheologies might allow for a wider range of
plate generating scenarios. As we noted previously the likelihood
of void-generating damage manifesting itself at mid to deep litho-
spheric depths is small, but it is entirely possible that localization in
the lithosphere may vary as a function of depth. Allowing for depth- Dumoulin, C., Bercovici, D. & Wessel, P., 1998. A continuous plate-tectonic
model using geophysical data to estimate plate margin widths, with a
seismicity based example, Geophys. J. Int., 133, 379–389. England, P. & McKenzie, D., 1982. A thin viscous sheet model for conti-
nental deformation, Geophys. J. R. astr. Soc., 70, 295–321. Evans, B., Renner, J. & Hirth, G., 2001. A few remarks on the kinetics of
static grain growth in rocks, Int. J. Earth Sci., 90, 88–103. C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection A P P E N D I X : S C A L I N G A NA LY S I S :
P R E D I C T I O N S V E R S U S R E S U LT S A two-phase model of
compaction and damage, 2, Applications to compaction, deformation,
and the role of interfacial surface tension, J. geophys, Res., 106(B5),
8907–8924. 0.00
0.08
0.16
0.25
0.00
0.02
0.04
0.06
+ +
++++
++++
+
+
++++++
+
+
+++ + +
+
++
+ +
+++
+
+
++++
++++
+
++++
+ +
+
+
+
+
+++ +
+++ +
+
+
+++++ +
+
+
+++ +
+
+
+ +
+
+
+++
+
+ +++
++++
+
+
++
+
+
+
+
+
0
18
35
53
0.000
0.333
0.667
1.000
+++++++++++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++++++++++
Divergent
Convergent
numerical run #
(a)
(b)
Figure 15. (a) Ordinate values are boundary widths over umax (or one
over the strain-rate) that we determined from numerical experiments plotted
against the values on the abscissa which are from the scaling analyses. (b)
Ordinate values are the ratio of inverse strain-rate (δc,d/umax) for the scaling
analysis to the numerical experiments plotted against the run number. This
graph displays the constant scale that differs between the scaling predictions
and numerical experiments. 0.00
0.08
0.16
0.25
0.00
0.02
0.04
0.06
+ +
++++
++++
+
+
++++++
+
+
+++ + +
+
++
+ +
+++
+
+
++++
++++
+
++++
+ +
+
+
+
+
+++ +
+++ +
+
+
+++++ +
+
+
+++ +
+
+
+ +
+
+
+++
+
+ +++
++++
+
+
++
+
+
+
+
+
0
18
35
53
0.000
0.333
0.667
1.000
+++++++++++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++++++++++
Divergent
Convergent
numerical run #
(a)
(b) 0.00
0.08
0.16
0.25
0.00
0.02
0.04
0.06
+ +
++++
++++
+
+
++++++
+
+
+++ + +
+
++
+ +
+++
+
+
++++
++++
+
++++
+ +
+
+
+
+
+++ +
+++ +
+
+
+++++ +
+
+
+++ +
+
+
+ +
+
+
+++
+
+ +++
++++
+
+
++
+
+
+
+
+
Divergent
Convergent
(a) Schubert, G., Turcotte, D. & Olson, P., 2001. Mantle Convection in the Earth
and Planets, Cambridge Univ. Press, Cambridge, UK. Spiegelman, M., 1993a. Flow in deformable porous media, part 1, Simple
analysis, J. Fluid Mech., 247, 17–38. Spiegelman, M., 1993b. Physics of melt extraction: theory, implications and
applications, Phil. Trans. R. Soc. London, Ser. A, 342, 23–41. applications, Phil. Trans. R. Soc. C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS A P P E N D I X : S C A L I N G A NA LY S I S :
P R E D I C T I O N S V E R S U S R E S U LT S London, Ser. A, 342, 23–4 Tackley, P., 1998. Self-consistent generation of tectonic plates in three-
dimensional mantle convection, Earth planet. Sci. Lett., 157, 9–22. Figure 15. (a) Ordinate values are boundary widths over umax (or one
over the strain-rate) that we determined from numerical experiments plotted
against the values on the abscissa which are from the scaling analyses. (b)
Ordinate values are the ratio of inverse strain-rate (δc,d/umax) for the scaling
analysis to the numerical experiments plotted against the run number. This
graph displays the constant scale that differs between the scaling predictions
and numerical experiments. Figure 15. (a) Ordinate values are boundary widths over umax (or one
over the strain-rate) that we determined from numerical experiments plotted
against the values on the abscissa which are from the scaling analyses. (b)
Ordinate values are the ratio of inverse strain-rate (δc,d/umax) for the scaling
analysis to the numerical experiments plotted against the run number. This
graph displays the constant scale that differs between the scaling predictions
and numerical experiments. Tackley, P., 2000a. Self-consistent generation of tectonic plates in
time-dependent, three-dimensional mantle convection simulations, 1. Pseudoplastic yielding, Geochem. Geophys. Geosystems (G3), 1,
2000GC000036. Tackley, P., 2000b. Self-consistent generation of tectonic plates in time-
dependent, three-dimensional mantle convection simulations, 2. Strain Tackley, P., 2000b. Self-consistent generation of tectonic plates in time-
dependent, three-dimensional mantle convection simulations, 2. Strain Tackley, P., 2000b. Self-consistent generation of tectonic plates in time-
dependent, three-dimensional mantle convection simulations, 2. Strain C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS C⃝2008 The Authors, GJI, 174, 1065–1080
Journal compilation C⃝2008 RAS W. Landuyt, D. Bercovici and Y. Ricard 1080 between the numerical experiments and analytic prediction (where
the analytic prediction has been multiplied by the constant deter-
mined in Fig. A2b). The regression line fits all of the data points to
within approximately 15 per cent which we will assume allows us
to assign some validity to the analytic expression we derived. our scaling analysis to only fit the numerical data up to some multi-
plicative constant (e.g. a geometrical factor of order 1 not included
in a scaling analysis). The fit for convergent boundaries is especially
good, but both boundaries exhibit the general features suggested by
the scaling in eq. (40) as demonstrated by the linear relationship
|
https://openalex.org/W2940533028
|
http://ijres.iaescore.com/index.php/IJRES/article/download/13520/pdf
|
English
| null |
ARM Controller and EEG based Drowsiness Tracking and Controlling during Driving
|
International Journal of Reconfigurable and Embedded Systems/International Journal of Reconfigurable & Embedded Systems (IJRES)
| 2,018
|
cc-by-sa
| 2,770
|
International Journal of Reconfigurable and Embedded Systems (IJRES)
Vol. 6, No. 3, November 2017, pp. 127~132
ISSN: 2089-4864, DOI: 10.11591/ijres.v6.i3.pp127-132 International Journal of Reconfigurable and Embedded Systems (IJRES)
Vol. 6, No. 3, November 2017, pp. 127~132
ISSN: 2089-4864, DOI: 10.11591/ijres.v6.i3.pp127-132 127 127 127 B. Naresh, Email: narib.naresh@gmail.com ARM Controller and EEG Based Drowsiness Tracking and
Controlling during Driving B. Naresh, S. Rambabu, D. Khalandar Basha
Department of Electronics and Communication Engineering, Institute of Aeronautical Engineering, India ABSTRACT This paper discussed about EEG-Based Drowsiness Tracking during
Distracted Driving based on Brain computer interfaces (BCI). BCIs are
systems that can bypass conventional channels of communication (i.e.,
muscles and thoughts) to provide direct communication and control between
the human brain and physical devices by translating different patterns of
brain activity commands through controller device in real time. With these
signals from brain in mat lab signals spectrum analyzed and estimates driver
concentration and meditation conditions. If there is any nearest vehicles to
this vehicle a voice alert given to driver for alert. And driver going to sleep
gives voice alert for driver using voice chip. And give the information about
traffic signal indication using RFID. The patterns of interaction between
these neurons are represented as thoughts and emotional states. According to
the human feelings, this pattern will be changing which in turn produce
different electrical waves. A muscle contraction will also generate a unique
electrical signal. All these electrical waves will be sensed by the brain wave
sensor and it will convert the data into packets and transmit through
Bluetooth medium. Level analyzer unit (LAU) is used to receive the raw data
from brain wave sensor and it is used to extract and process the signal using
Mat lab platform. The nearest vehicles information is information is taken
through ultrasonic sensors and gives voice alert. And traffic signals condition
is detected through RF technology. Article history:
Received July 05, 2017
Revised Sep 06, 2017
Accepted Sep 20, 2017 ARM controller
Bluetooth
Brain wave sensor
DC motor
Ultrasonic sensor Copyright © 2017 Institute of Advanced Engineering and Science.
All rights reserved. Corresponding Author: B. Naresh,
Department of Electronics and Communication Engineering,
Institute of Aeronautical Engineering,
Dundigal, Telangana, India. Email: narib.naresh@gmail.com B. Naresh,
Department of Electronics and Communication Engineering,
Institute of Aeronautical Engineering,
Dundigal, Telangana, India. Email: narib.naresh@gmail.com B. Naresh,
Department of Electronics and Communication Engineering,
Institute of Aeronautical Engineering,
Dundigal, Telangana, India. Email: narib.naresh@gmail.com B. Naresh,
Department of Electronics and Communication Engineering,
Institute of Aeronautical Engineering,
Dundigal, Telangana, India.
Email: narib.naresh@gmail.com 1.
INTRODUCTION Driving is a complex task, requiring full concentration and a calm attitude. Stressed and strong
emotions, whether they result from the driving task itself or unrelated matters, can affect a driver’s abilities. For example, research has shown that angry drivers are more likely to take risks such as speeding, rapidly
switching lanes, tailgating and jumping red lights. EEG signal can be one of the features of BCI for detecting
driver’s state of mind. The main objective of this paper is to study the different mental state recognition methods based on
EEG signals. Later work can be done on analyzing different mental states of a driver in different traffic
situation and based on the study, predicting whether driver is mentally fit or not. In recent past, it has been
observed that drivers with mental fatigue lead to accident. In this proposol, we proposed a robust real-time
embedded platform to monitor the loss of attention of the driver during day and night driving conditions. The
alertness level can be measured by using different parameters, such as electro encephalogram (EEG) signals,
visual features, blood samples, speech and others. The EEG- based method has been reported to be highly
authentic for estimating the state of drowsiness. In a new method for multi fractal analysis of EEG signals Journal homepage: http://iaescore.com/journals/index.php/IJRES/index ISSN: 2089-4864 128 named generalized Higuchi fractal dimension spectrum (GHFDS) was proposed and applied in mental
arithmetic task recognition from EEG signals. named generalized Higuchi fractal dimension spectrum (GHFDS) was proposed and applied in mental
arithmetic task recognition from EEG signals. 2.1.2. Working Principle The principle of operation is quite simple. It consists of two dry sensors are detect and filters the
EEG signals. The sensor tip detects electrical signals from the forehead of the brain. At the same time, the
sensor picks up ambient noise generated by human muscle, computers, light bulbs, electrical sockets and
other electrical devices. The sensor and ear clip picks up the information from grounds and reference, which
allows think gear chip to filter out the electrical noise. The device measures the raw signal, power spectrum
(alpha, beta, delta, gamma, theta), attention level, mediation level and blink detection. For every clock
frequency of 512 Hz, it receives the raw data from EEG. Other measured values are made every second. Therefore, raw EEG data is a main source of information on EEG signals using Mind Wave MW001. 2. PROPOSED BLOCK DIAGRAM The above block diagram shows the control of vehicle through ARM Controller, it identifies the
drowsiness while driving and also it detects the obstacles near to the vehicle. Figure 1 shows BCI- attention
tracking. It consists of two sections, i.e. (a) design and implementation and (b) system hardware using ARM
Processor. Figure 1. BCI- attention tracking (a) design and implementation and (b) system hardware using
ARM processor Figure 1. BCI- attention tracking (a) design and implementation and (b) system hardware using
ARM processor 2.1. Design and Implementation Electroencephalography (EEG) is the measurement of electrical activity in the living brain. In this
project we used a brainwave sensor MW001 to analyze the EEG signals. This design discuss about
processing and recording the raw EEG signal from the sensor MW001 in the MATLAB environment and
through Serial transmission control commands will be passed to the voice chip. This MW001 sensor is not
used for medical purpose, but it is used to measure neurons feedback. The Brain control interface is a direct
communication pathway between the brain and an external device. 2.2. System Hardware LPC2148 Processor 2.2. System Hardware LPC2148 Processor
In this paper it uses LPC2148 processor, is belongs to one of the family of ARM7. This processor
employs the pipeline architecture, hence data processing and memory operations are executing parallel, it
reduces the execution time. y
In this paper it uses LPC2148 processor, is belongs to one of the family of ARM7. This processor
employs the pipeline architecture, hence data processing and memory operations are executing parallel, it
reduces the execution time. It consists of two instruction sets. It consists of two instruction sets.
Standard 32-bit ARMv5TE set
16-bit THUMB set 2.2.1. Thinkgear Asic Module
It is used to interface the dry electrode, One EEG channel with three contacts namely EEG, REF, and
GND.
It is used to interface the dry electrode, One EEG channel with three contacts namely EEG, REF, and
GND.
Improper fit detected through “Poor Signal Quality” warning from ASIC to reset if off the head for four
consecutive seconds, or if it is receiving a poor signal for seven consecutive seconds.
Improper fit detected through “Poor Signal Quality” warning from ASIC to reset if off the head for four
consecutive seconds, or if it is receiving a poor signal for seven consecutive seconds.
It is applicable for low power consumption devices.
Max power consumption 15mA @ 3.3 V
Raw EEG data output at 512 bits per second. 2.2.2. DC Motor A motor consists of a rotor and a permanent magnetic field stator. The magnetic field is maintained
using either permanent magnets or electromagnetic windings. Motors are the devices that provide the actual
speed and torque in a drive system. This family includes AC motor types (single and multiphase motors,
universal, servo motors, induction, synchronous, and gear motor) and DC motors (brush less, servo motor,
and gear motor) as well as linear, stepper and air motors, and motor contactors and starters. 3.
SYSTEM SOFTWARE Using MATLAB’s GUIDE, the processes of laying out a GUI (i.e., its buttons, pop-up menus, etc.)
and programming the operation of the GUI are divided conveniently into two easily managed and relatively
independent tasks. 2.2.3. Ultrasonic Sensor Ultra Sonic Sensor Our ultrasonic range finder is capable of allowing the user to determine his or
her distance from an object or wall. It is used to identify the obstacle, nearer to vehicle while driving. 2.1.4. The Communications Protocol 2.1.4. The Communications Protocol It consists of 7 basic steps, which are presented in following steps
Load Think Gear library into MATLAB It consists of 7 basic steps, which are presented in following steps. Load Think Gear library into MATLAB Get a connection ID handle to Think Gear Attempt to connect the connection ID handle to serial port "COMx" Waiting to establish the connection Read packets from the connection In the first step are functions with parameters in the following order In the first step are functions with parameters in the following order Libisloaded („Think gear‟)–returns true if the Think Gear library is loaded, and false otherwise. Libisloaded („Think gear‟)–returns true if the Think Gear library Load library ('Think gear.dll','think gear.h')–loads the functions defined in the header file and found
library. Now, the function calllib () can call a function in the ThinkGear library. Calllib ('Thinkgear'TG_GetDriverVersion')–returns the version of loaded library. In the next step, the
function calllib ('Thinkgear', 'TG_GetNewConnectionId') gets a new connection ID handle to ThinkGear. The value -1 is returned if too many connections have been created. Select Connection ID, number of the
serial port, Baud rate and type of data from the TG connect. The number of the serial port is given during
the pairing of the device. The device can connect on modes 1200, 2400, 4800, 9600, 57600 and 115200
bits per second (bps). Here, we use the 9600 bps rate and stream 5V RAW mode, because these
parameters have the minimum of transmission errors. IJRES ISSN: 2089-4864 2.1.3. Matlab Based Design Theory The MATLAB allows including thinkgear.dll. This environment has broad support in toolbox,
which makes it ideal for a scientific research. This paper presents how recording and processing the raw EEG
signal in MATLAB environment using Mind Wave sensor. a system of digital rules for message exchange
between MATLAB environment and Mind Wave MW001 device is processing through communications
protocol. This section also presents the main parameters of think gear library. IJRES Vol. 6, No. 3, November 2017 : 127 – 132 129 IJRES 3.1. Role of Matlab in BCI The MATLAB allows including thinkgear.dll. This environment has broad support in toolbox,
which makes it ideal for a scientific research. This paper presents how recording and processing the raw EEG
signal in MATLAB environment using Mind Wave sensor. The Communication Protocol shows a system of ARM Controller and EEG Based Drowsiness Tracking and Controlling during… (B. Naresh) 130 ISSN: 2089-4864 digital rules for message exchange between MATLAB environment and Mind Wave MW001 device. This
section also presents the main parameters of thinkgear library. digital rules for message exchange between MATLAB environment and Mind Wave MW001 device. This
section also presents the main parameters of thinkgear library. The Communication Protocol, shows a system of digital rules for message exchange between
MATLAB environment and Mind Wave MW001 device. This section also presents the main parameters of
thinkgear library. The connection is established through command: calllib ('Thinkgear ','TG_ Connect', Id,
ComPortName,TG_BAUD_115200,TG_ STR EAM _5VRAW). In the next step, we must attempt to read a
Packet of data from the connection. We use the TG_Read Packets () function with ID parameter and number
of packet to read. The command calllib ('Thinkgear','TG_ReadPackets', Id, 1). Figure 2 shows Brian signal
representation in MATLAB. Figure 2. Brian signal representation in MATLAB Figure 2. Brian signal representation in MATLAB The above graph representation includes the attention value and blink strength. Based on this signals
device control file will be opened and the device will get controlled based on the brain signals. IJRES Vol. 6, No. 3, November 2017 : 127 – 132 [1]
Chin-Teng Lin, Fu-Chang Lin, Shi-An Chen, Shao-Wei Lu, Te-Chi Chen, Li-Wei Ko, “EEG-based Brain-computer
Interface for Smart Living Environmental Auto-adjustment”, Journal of Medical and Biological Engineering, Year:
2010, Vol. 30, Page: 237-245. 5.
CONCLUSION The main goal of this paper is to design and implement a human thought controlled electrical
switching system using BCI technology for the physically challenged people. Non invasive BCI method is
used for capturing the brain signals with the help of brain wave sensor. Different cognitive state of the user
like attention level, meditation level, and eye-blink can be used to measure the human thought. The signals
are classified according their respective frequencies ranging from 0.5 Hz to 30 Hz. By setting the threshold
value for an event, the interrupt is generated. This Interrupt is sent to the microcontroller unit for operating
the appliances. 4.
ANALYSIS OF HARDWARE SECTION
ARM Controller read the value of raw ARM Controller read the value of raw EEG signal with the maximum frequency of 512 Hz. Sampling frequency is set on 512 Hz, and we control time delays in sampling. The value of the signal and
time are written to the array data.The data which are stored in array will be compared with the threshold
points given by the user. In this Paper, the Matlab section waits for three consecutive blink in order to send
the hardware activation signal. Then based on the blink level signal, the cursor will be places upon any
section to get the device control. Further the person has to raise the attention to switch the device. Figure 3
shows Brain wave signal identification in Matlab. Figure 4 shows Drowsiness identification through Matlab IJRES Vol. 6, No. 3, November 2017 : 127 – 132 131 ISSN: 2089-4864 IJRES IJRES Figure 3. Brain wave signal identification in Matlab
Figure 4. Drowsiness identification through Matlab Figure 3. Brain wave signal identification in Matlab
Figure 4. Drowsiness identification through Matlab Figure 3. Brain wave signal identification in Matlab [2]
Wolpaw, J.R.,”Brain-computer interface research comes of age: traditional assumptions meet emerging realities.”
Journal of motor behavior, Year: 2010, Vol. 42, Page: 351-353. [4]
T. Kameswara Rao, M. Rajyalakshmi, Dr. T. V. Prasad, “An Exploration on Brain Computer Interface and Its
Recent Trends”, International Journal of Advanced Research in Artificial Intelligence, Year: 2012, Vol. 1, Page:
17-22. ARM Controller and EEG Based Drowsiness Tracking and Controlling during… (B. Naresh) ]
Anupama.H.S, N.K.Cauvery, Lingaraju. G.M, Brain computer interface and its types - a study , Internat
Journal of Advances in Engineering & Technology, Year: 2012, Vol. 8, Page: 117-119. f
,
,
,
g
]
Anupama.H.S, N.K.Cauvery, Lingaraju. G.M, “Brain computer interface and its types - a study”, Internation IJRES Vol. 6, No. 3, November 2017 : 127 – 132 REFERENCES [1]
Chin-Teng Lin, Fu-Chang Lin, Shi-An Chen, Shao-Wei Lu, Te-Chi Chen, Li-Wei Ko, “EEG-based Brain-computer
Interface for Smart Living Environmental Auto-adjustment”, Journal of Medical and Biological Engineering, Year:
2010, Vol. 30, Page: 237-245. g
[2]
Wolpaw, J.R.,”Brain-computer interface research comes of age: traditional assumptions meet emerging realities.”
Journal of motor behavior, Year: 2010, Vol. 42, Page: 351-353. f
,
,
,
g
[3]
Anupama.H.S, N.K.Cauvery, Lingaraju. G.M, “Brain computer interface and its type
Journal of Advances in Engineering & Technology, Year: 2012, Vol. 8, Page: 117-119. [4]
T. Kameswara Rao, M. Rajyalakshmi, Dr. T. V. Prasad, “An Exploration on Brain Computer Interface and Its
Recent Trends”, International Journal of Advanced Research in Artificial Intelligence, Year: 2012, Vol. 1, Page:
17-22.
132 ISSN: 2089-4864 132 ISSN: 2089-4864 BIOGRAPHIES OF AUTHORS BIOGRAPHIES OF AUTHORS BIOGRAPHIES OF AUTHORS
B.Naresh working as Associate professor in Institute of aeronautical engineering,
Hyderabd, Telangana. Area of interest is Embedded Systems and VLSI System Design. S.Rambabu working as Assistant professor in Institute of aeronautical engineering,
Hyderabd, Telangana.Area of interest is VLSI. D.Khalander Basha working as Associate professor in Institute of aeronautical
engineering, Hyderabd, Telangana. Area of interest is Image processing. B.Naresh working as Associate professor in Institute of aeronautical engineering,
Hyderabd, Telangana. Area of interest is Embedded Systems and VLSI System Design. S.Rambabu working as Assistant professor in Institute of aeronautical engineering,
Hyderabd, Telangana.Area of interest is VLSI. S.Rambabu working as Assistant professor in Institute of aeronautical engineering,
Hyderabd, Telangana.Area of interest is VLSI. D.Khalander Basha working as Associate professor in Institute of aeronautical
engineering, Hyderabd, Telangana. Area of interest is Image processing. D.Khalander Basha working as Associate professor in Institute of aeronautical
engineering, Hyderabd, Telangana. Area of interest is Image processing. IJRES Vol. 6, No. 3, November 2017 : 127 – 132
|
https://openalex.org/W2596124770
|
https://www.nature.com/articles/srep44696.pdf
|
English
| null |
Experimental investigation on the coupled effect of effective stress and gas slippage on the permeability of shale
|
Scientific reports
| 2,017
|
cc-by
| 7,157
|
Diansen Yang1, Wei Wang1,2, Weizhong Chen1,3, Shugang Wang3 & Xiaoqiong Wang4 Diansen Yang1, Wei Wang1,2, Weizhong Chen1,3, Shugang Wang3 & Xiaoqiong Wang4 Permeability is one of the most important parameters to evaluate gas production in shale reservoirs. Because shale permeability is extremely low, gas is often used in the laboratory to measure
permeability. However, the measured apparent gas permeability is higher than the intrinsic
permeability due to the gas slippage effect, which could be even more dominant for materials with
nanopores. Increasing gas pressure during tests reduces gas slippage effect, but it also decreases
the effective stress which in turn influences the permeability. The coupled effect of gas slippage and
effective stress on shale permeability remains unclear. Here we perform laboratory experiments
on Longmaxi shale specimens to explore the coupled effect. We use the pressure transient method
to measure permeability under different stress and pressure conditions. Our results reveal that the
apparent measured permeability is controlled by these two competing effects. With increasing gas
pressure, there exists a pressure threshold at which the dominant effect on permeability switches from
gas slippage to effective stress. Based on the Klinkenberg model, we propose a new conceptual model
that incorporates both competing effects. Combining microstructure analysis, we further discuss the
roles of stress, gas pressure and water contents on gas permeability of shale. With recent development of novel technologies, unconventional gas extraction has made great success in the
North America. Shale gas has been considered as an attractive low-carbon solution for the transition period to a
future power by renewable energy1. As an important supplement to conventional energy resources, shale gas has
obtained an increasing attention in the past years. Due to its extremely low permeability, shale gas is still difficult to
be recovered, particularly in deep formations. For example, the average burial depth of shale gas in the US is about
800–2000 m, while the average depth in China is over 3500 m where the stress is significantly high (>50 MPa)2. In order to better evaluate the production of shale gas, an accurate permeability of shale under in situ stress condi-
tions is required. As the mean pore size of shale is often at the nanoscale, the permeability of shale is substantially
low, typically less than 10−19 m2, which is difficult to be measured in the laboratory. Inert gases such as helium,
azote, nitrogen, are often used to measure the low permeability. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 16 August 2016
accepted: 13 February 2017
Published: 17 March 2017 Experimental investigation on
the coupled effect of effective
stress and gas slippage on the
permeability of shale received: 16 August 2016
accepted: 13 February 2017
Published: 17 March 2017 Methods Pressure transient method and experimental setup. Pressure transient method or known as pressure
pulse decay13,20,21 is often used to effectively measure low permeability of tight rocks (<100 nD (10−19 m2)), as it
is less time consuming. Recently, pressure transient measurements on ultra-tight rocks have been interpreted by
accounting for the effects of gas-slippage22, transitional flow, and Knudsen diffusion flow23. Its principle was first
proposed by Brace et al13. It consists of a sample connected with two reservoirs that have an equilibrated initial
pressure P0 in the whole system. An increment pressure ΔP0 is suddenly imposed into one of the two reservoirs
and the pressure evolution in two reservoirs over time is recorded as demonstrated in Fig. 1a. Analyzing the var-
iation of gas pressures, permeability can be then determined either using the method developed by Brace13,21 or
back analysis method20.i y
Based on the principle of pressure transient method, a specific setup of gas permeability measurements has
been designed and developed in the Institute of Rock and Soil Mechanics of Wuhan, CAS. In this system, a triax-
ial core holder, capable of accepting membrane-sheathed cylindrical samples (2.5 cm diameter) and of applying
independent loading in the radial and axial directions, is connected to two gas reservoirs. In order to shorten the
testing time, small reservoirs are designed and the volume of upstream reservoir is about 8.2 cm3 and the volume
of downstream reservoir is about 6.6 cm3. Axial and confining (radial) stresses up to 56 MPa are independently
applied using two ISCO 260D pumps with control up to ± 0.007 MPa. Gas pressure is controlled by a ISCO 500D
pump. Different transducers (temperature, pressure, and displacement transducers) are connected to a computer
to automatically record the experimental data. All parts of this apparatus are located in a temperature controlled
cabinet where the temperature can be maintained constant with a deviation of +/− 0.1 °C. The whole system is
shown in Fig. 1b. Tests on aluminum samples have been carried out to calibrate the system and check possible
leakage and the gas permeability accuracy of the system is up to 10−23 m2. Sample description. In this study, shale samples were recovered from the Lower Silurian Longmaxi (LM)
shale formation located in Qianjiang, southeast of Chongqing. Longmaxi shale formation is comprised of dark
gray to black graptolite shale, carbonaceous shale, siliceous shale, silty shale, and argillaceous siltstone24. Diansen Yang1, Wei Wang1,2, Weizhong Chen1,3, Shugang Wang3 & Xiaoqiong Wang4 Gas can shorten the testing time and can also
avoid the hydration reaction of shale with high content of clay minerals that are sensitive to water. However, gas
slippage effect could be very large in nanopores and thus could affect gas permeability measurement. Numerous
investigations have been performed to study gas slippage phenomenon which was initially noted in the 1940s3–8. Due to gas slippage, the steady-state flow rate through small capillaries is higher, which results in an overestima-
tion of permeability. To estimate the gas slippage effect on the permeability, Klinkenberg proposed an empirical
equation written as =
+
K
K
b P
(1
/
)
(1)
a
int
m =
+
K
K
b P
(1
/
)
a
int
m (1) where Ka is the apparent permeability, Kint is the intrinsic permeability, Pm is the average gas pressure in pores, and b
is the slope of Klinkenberg straight line related to the mean free path of gas molecules. Many researchers have where Ka is the apparent permeability, Kint is the intrinsic permeability, Pm is the average gas pressure in pores, and b
is the slope of Klinkenberg straight line related to the mean free path of gas molecules. Many researchers have 1State Key Laboratory of Geomechanics and Geotechnical Engineering, Institute of Rock and Soil Mechanics, Chinese
Academy of Sciences, Wuhan, Hubei 430071, China. 2University of Chinese Academy of Science, Beijing, 100049, China. 3Geotechnical and Structural Engineering Research Center, Shandong University, Jinan, Shandong, China. 4Unconventional
Natural Gas Institute, China University of Petroleum, Beijing 102249, China. Correspondence and requests for materials
should be addressed to D.S.Y. (email: dsyang@whrsm.ac.cn) or S.G.W. (email: sdgeowsg@gmail.com) SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 1 www.nature.com/scientificreports/ Figure 1. Illustration of the pressure transient method for gas permeability measurement (a) principle of the
method; (b) the experimental setup). Figure 1. Illustration of the pressure transient method for gas permeability measurement (a) principle of the
method; (b) the experimental setup). investigated this phenomenon from the flow dynamics of gases in micro-channels with high Knudsen numbers
(Kn), defined as the ratio of the molecular mean free path to the characteristic length of the flow path9,10. Methods
such as the direct simulation Monte Carlo approach and the lattice Boltzmann method have been used to estimate
the gas slippage effect3. A few new expanded models have been proposed to correct Klinkenberg’s equation4,6. Diansen Yang1, Wei Wang1,2, Weizhong Chen1,3, Shugang Wang3 & Xiaoqiong Wang4 In
the experimental tests, Klinkenberg effect is often characterized using different gas pressures to measure perme-
ability11. High gas pressure is often applied to reduce the gas slippage effect because high gas pressure can lead to
a smaller mean free path of gas molecules and thus minimize Klinkenberg effect. However, high gas pressure also
reduces the effective stress and thus increases permeability. The dependence of permeability on the stress has been
well observed in the laboratory. Dong et al. in 2010 found that the permeability of shale was more sensitive to the
effective confining stress than sandstone12. Based on numerous experimental data, stress dependent permeability
could be described by either an exponential relationship13,14 or a power law15–18. Katsuki et al. in 2013 discussed
poroelastic effects on the stress-dependent permeability of a stiff shale and found that pore pressure changed gas
permeability of shale significantly19. However, there have been only a few studies to date aiming at understanding
the coupled effect of gas slippage and effective stress principle on the permeability of shale. This paper will present
an experimental investigation on this topic and discuss various factors influencing the gas permeability of shale. SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 Methods The
organic content of shale is larger than 0.5%, with a mean TOC of 2.54%. LM shales are mainly composed of clay,
quartz and calcite, while in contrast pyrite and feldspar are minor. The grain density of shale sample is about
2.72 g/cm3 and the total porosity is 0.25–3.25% and the initial water content is less than 4%. Two cylindrical
samples with a diameter of 25.0 mm and a length of 40.4 mm were drilled from the same LM shale block and
they were undamaged and the measured longitudinal wave velocity (Vp) at the initial state was nearly the same
(2267 m/s for the sample No.1 and 2254 m/s for the sample No.2). The dry density of the sample is 2.32 g/cm3 and
the connected porosity measured by Autopore IV9510 is 2.50%. The pore size distribution of LM shale has been
characterized by the mercury intrusion porosimetry (MIP) method (Fig. 2a) and by the gas adsorption method SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 2 www.nature.com/scientificreports/ Figure 2. Pore size distribution of Longmaxi shale using (a) the MIP method and (b) the gas adsorption
method. Figure 2. Pore size distribution of Longmaxi shale using (a) the MIP method and (b) the gas adsorption
method. (Fig. 2b). The results show that the pore size of LM shale is complex and it covers a range from several nanometers
to hundred nanometers and the average pore size is 3.0 nm. Experimental procedure. In order to evaluate the coupled effect of effective stress and gas slippage on gas
permeability of shale, a series of gas permeability tests have been performed on the shale samples subjected to dif-
ferent confining stresses and gas pressures. Sample No.1 was at unsaturated state and the water content was 1.24%,
which was measured at the end of testing by drying it at 105 °C for 24 hours. Before testing, sample No.2 with an
initial water content of 1.50% was dried. To estimate the effect of effective stress on gas permeability, a cycle of
loading and unloading is performed on the sample No.1 with a constant confining stress at 15 MPa. The path of
applied axial stress follows: 15 MPa → 30 MPa → 45 MPa → 50 MPa → 55 MPa → 45 MPa → 30 MPa → 15 MPa. The maximum deviatoric stress is about 40 MPa, under which little damage could occur in the shale samples. Methods At
different stress levels, the initial gas pressure was maintained at 3 MPa, and the increment gas pressure for the
pressure pulse varied between 0.2–0.4 MPa to measure gas permeability. At the end of the mechanical unloading,
a hydrostatic stress of 16 MPa was applied on the sample No.1 and a series of gas permeability measurements
were carried out with different gas pressures (1 MPa, 2 MPa, 3 MPa, 4 MPa) to study the gas slippage effect. Three
levels of hydrostatic stress (12 MPa, 14 MPa, 16 MPa) were applied on the dry sample No.2 and under these stress
conditions, different gas pressures were selected to measure the gas permeability of the sample. The histories of
mechanical loading and gas pressure performed on the samples (No.1 and No.2) are illustrated in Fig. 3a and b,
respectively. Finally, 14 pulse tests have been conducted for sample No.1 and 8 pulse tests for sample No.2 and
they are shown in Fig. 3. The duration of the tests has lasted about 60 days. In this study, nitrogen was chosen to
measure gas permeability and the temperature of the whole system was kept at 30 °C. SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 Results
i Results
Determination of the apparent gas permeability. The apparent gas permeability Ka was firstly evalu-
ated using Brace’s equation13,25, Determination of the apparent gas permeability. The apparent gas permeability Ka was firstly evalu-
ated using Brace’s equation13,25, αηβ
α
=
+
= −
∆
∆
k
L
S
V
V
P t
P
1
1/
1/
,
ln
( ) /t
(2)
a
up
down
0 (2) where Vup and Vdown (m3) are the volumes in the upstream reservoir and downstream reservoir; Pup is the gas
pressure in the upstream reservoir (Pa) and Pf is the final equilibrium pressure of the system (Pa); η (Pa.s) is the SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 3 www.nature.com/scientificreports/ Figure 3. Mechanical loading path and gas pressure evolution during gas permeability tests (a) wet sample
No.1, (b) dry sample No.2). Figure 3. Mechanical loading path and gas pressure evolution during gas permeability tests (a) wet sample
No.1, (b) dry sample No.2). Nitrogen viscosity at the temperature of 30 °C and the mean pore pressure; β is the isothermal compressibility
coefficient of the pore fluid (Pa−1) and it is gas pressure dependent; α is the decay exponent; L and S are the length
and cross-sectional area of the sample, and t is the testing time (s).hl Nitrogen viscosity at the temperature of 30 °C and the mean pore pressure; β is the isothermal compressibility
coefficient of the pore fluid (Pa−1) and it is gas pressure dependent; α is the decay exponent; L and S are the length
and cross-sectional area of the sample, and t is the testing time (s).hl p
g
Brace’s formula was derived assuming that Darcy law is valid and gas is ideal gas. The fluid volume in the pores
of the rock sample is ignored. However, this will not induce a large error for tight rock with very low porosity (e.g.,
granite, shale). To estimate the error, a series of permeabilities, chosen by changing the value determined from the
formula (2) with an increment of several 10−22 m2, were used to calculate the theoretical pressure in the upstream
and downstream reservoirs following Brace’s method as the following. ∆
∆
∆
=
+
. +
. =
−
. +
. =
+
. Results
i +
α
α
−
−
P
V
V
V
e
P
V
V
V
e
V
V
V
P
P
,
P
P
,
P
P
P
(3)
t
down
t
up
f
0
up
up
down
f
0
down
up
down
f
0
0
up
up
down ∆
∆
∆
=
+
. +
. =
−
. +
. =
+
. +
α
α
−
−
P
V
V
V
e
P
V
V
V
e
V
V
V
P
P
,
P
P
,
P
P
P
(3)
t
down
t
up
f
0
up
up
down
f
0
down
up
down
f
0
0
up
up
down (3) Numerical results were then compared with experimental results (e.g., Fig. 4a and b). These two figures illus-
trate the evolution of the gas pressure in the upstream and downstream reservoirs during the pulse tests of the
sample No.1 (3rd stage) and No.2 (4th stage), respectively. The results show that the numerical results matches well
against the experimental results with an appropriate Ka.h The obtained apparent gas permeability Ka and the gas permeability determined directly from Brace’s formula
Ka
B of the shale samples at different stages are listed in the Table 1 and 2. Table 1 presents the gas permeability
evolution Ka of sample No.1 at different stages, and Table 2 lists permeabilities measured for sample No.2. The
results show that the difference between the permeability determined from the formula (a) and the appropriate
obtained during the numerical comparison is less than 5%. As the shale samples have a very low porosity (<2%),
we believe that the Brace method is reliable in determining gas permeability in this work. Permeability versus deviatoric stress with the same gas pressure. As shown in Table 1, the appar-
ent gas permeability decreases during the loading process and increases during the unloading process, but the SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 4 www.nature.com/scientificreports/ Figure 4. Experimental and numerical data of gas pressure evolutions in upstream and downstream
reservoirs during pressure transient tests for determination of gas permeability (a) 2nd stage of the wet sample,
Ka = 4.3 × 10−21m2, (b) 8th of the dry sample, Ka = 39.5 × 10−21m2). Figure 4. Results
i Experimental and numerical data of gas pressure evolutions in upstream and downstream
reservoirs during pressure transient tests for determination of gas permeability (a) 2nd stage of the wet
Ka = 4.3 × 10−21m2, (b) 8th of the dry sample, Ka = 39.5 × 10−21m2). Stage in Fig. 3a
1st
2nd
3rd
4th
5th
6th
7th
8th
9th
10th
11th
12th
13th
14th
Confining stress (MPa)
15
15
15
15
15
15
15
15
15
16
16
16
16
16
Axial stress (MPa)
15
30
45
50
55
45
30
15
15
16
16
16
16
16
Gas pressure (MPa)
3
3
3
3
3
3
3
3
4
4
2
3
4
1
Ka (x10−21 m2)
17.6
4.3
3.9
3.7
3
3.1
3.5
3.7
3.6
3.6
4.1
3.7
3.6
4.8
Ka
B (x10−21 m2)
17.1
4.4
3.9
3.6
3.1
3.2
3.5
3.6
3.5
3.5
4.1
3.6
3.5
4.7
Table 1. Apparent permeability of shale sample No.1 at different stages. Stage in Fig. 3a
1st
2nd
3rd
4th
5th
6th
7th
8th
9th
10th
11th
12th
13th
14th
Confining stress (MPa)
15
15
15
15
15
15
15
15
15
16
16
16
16
16
Axial stress (MPa)
15
30
45
50
55
45
30
15
15
16
16
16
16
16
Gas pressure (MPa)
3
3
3
3
3
3
3
3
4
4
2
3
4
1
Ka (x10−21 m2)
17.6
4.3
3.9
3.7
3
3.1
3.5
3.7
3.6
3.6
4.1
3.7
3.6
4.8
Ka
B (x10−21 m2)
17.1
4.4
3.9
3.6
3.1
3.2
3.5
3.6
3.5
3.5
4.1
3.6
3.5
4.7
Table 1. Apparent permeability of shale sample No.1 at different stages. Table 1. Apparent permeability of shale sample No.1 at different stages. Stage in Fig. 3b
1st
2nd
3rd
4th
5th
6th
7th
8th
Hydrostatic stress
(MPa)
16
16
12
12
12
16
14
14
Gas pressure (MPa)
1
4
1.15
2
3
4
1.14
2
Ka (x10−21 m2)
57
37
51
44
43
37
49.5
39.5
Ka
B (x10−21 m2)
56.8
37.2
50.9
44.2
43.1
36.9
49.8
39.7
Table 2. Apparent permeability of shale sample No.2 under the constant hydrostatic stress for different gas
pressures. Stage in Fig. Results
i 3b
1st
2nd
3rd
4th
5th
6th
7th
8th
Hydrostatic stress
(MPa)
16
16
12
12
12
16
14
14
Gas pressure (MPa)
1
4
1.15
2
3
4
1.14
2
Ka (x10−21 m2)
57
37
51
44
43
37
49.5
39.5
Ka
B (x10−21 m2)
56.8
37.2
50.9
44.2
43.1
36.9
49.8
39.7 Table 2. Apparent permeability of shale sample No.2 under the constant hydrostatic stress for different gas
pressures. Table 2. Apparent permeability of shale sample No.2 under the constant hydrostatic stress for different gas
pressures. permeability is not fully recoverable. The reduction of permeability can be explained by the compression of the
shale due to the increase of stress which will reduce the effective porosity. At the first stage of loading, during
which the deviatoric stress increases to 15 MPa, permeability change (from 17.6 × 10−21 m2 to 4.3 × 10−21 m2)
is much larger compared with other stages during which the variation is less than 1 × 10−21 m2. This could be
due to the fact that the first stage is the hydrostatic loading while the following stages are all deviatoric loading. Permeability change is consistent with the deformation evolution of the sample. The axial strain obtained during
the hydrostatic loading is larger than that during the deviatoric loading for the same magnitude variation of SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 5 www.nature.com/scientificreports/ Figure 5. Permeability versus gas pressure at different isotropic stress (a) wet sample No.1, (b) dry sample
No.2). Figure 5. Permeability versus gas pressure at different isotropic stress (a) wet sample No.1, (b) dry sample
No.2). stresses. Permeability decline is consistent with the compression of the sample during mechanical loading. The
irreversible permeability loss is believed to be related to the irreversible strain, which is 0.18% in the axial direc-
tion at the end of the cycle of loading and unloading. Permeability versus gas pressure under constant hydrostatic stress. The results in Table 1 and 2
demonstrate that measured permeability of the samples (No.1 and No.2) decreases with the increase of gas pres-
sure under different constant hydrostatic stresses (Fig. 5) below a certain pressure value. This permeability reduc-
tion is often explained by the effect of gas slippage which is weakened when the gas pressure increases. Sample
No.2 was subjected to an isostatic stress of 16 MPa, 12 MPa, 14 MPa, successively. Results
i For the same level of gas pres-
sure (about 1 MPa), permeability is smaller at the isostatic of 14 MPa than at 12 MPa. This should be related to
the dependence of permeability on the stress as presented above. The higher stress induces a large compression
of shale and thus reduces the effective porosity and results in a decrease in permeability. Permeability of sample
No.2 is much larger than of sample No.1 under similar conditions, which can be explained by the role of water
saturation on gas permeability. Scientific Reports | 7:44696 | DOI: 10.1038/srep44696 Discussions Our experimental results show that measured apparent permeability is influenced by both confining stress and
gas slippage effect. The effective stress principle by Terzaghi is widely used to characterize the influence of pore
pressure on the material. Biot’s effective stress σ′ can be defined by σ
σ
′ =
−
′b p, where σ′ is the Cauchy stress
tensor, p is pore pressure and b’ is Biot’s coefficient. In linear elastic poromechanics26,27, it is calculated by
b’ = 1 − K/Ks, where Ks is the bulk modulus of solid grain, and K is the apparent elastic bulk modulus under
drained conditions. For soft material such as sand and soils, K«Ks, and then the Terzaghi effective stress is recov-
ered since b’ = 1. For hard material, b’ is often less than 1. Bemer et al.28 identified Cox shale Biot’s coefficient and
obtained an average value equal to 0.52. Homand et al. found that Biot’s coefficient decreased from 0.95 to 0.55
when the axial stress increased from 8 to 24 MPa20. Cariou et al.29 characterized Biot’s coefficient of partially sat-
urated Cox shale and identified the apparent Biot’s coefficient as 1. In this study, Biot’s coefficient is evaluated by
analyzing the difference in axial strain induced by confining stress and by gas pressure. For the wet sample No.1,
the axial strain is increased to 0.49% when the hydrostatic stress increases to 15 MPa. It is about −0.017% when
the gas pressure increases to 2.9 MPa. For the dry sample No.2, the axial strain variation is about 0.009%/MPa
during gas pressure increasing period and 0.024%/MPa during hydrostatic stress loading period. The different
contribution of hydrostatic stress and gas pressure on the axial strain shows that Biot’s coefficient of LM shale is Scientific Reports | 7:44696 | DOI: 10.1038/srep44696 6 www.nature.com/scientificreports/ less than 1.These displacement measurements confirm that different gas pressures will induce a non-negligible
strain. The augment of effective stress, which is controlled by confining stress and gas pressure, will compact the
pore volume and result in a decrease in permeability. Permeability decreases from 44×10−21 m2 to 39.5×10−21 m2
when the hydrostatic stress increases from 12 MPa to 14 MPa. Under the same hydrostatic stress, increasing gas
pressure will expand the pores and result in an increase in permeability, which is the opposite effect compared
with the influence of gas slippage. The relationship between permeability and gas pressure is nonlinear (Fig. Discussions 5)
and the Klinkenberg empirical formula cannot be directly used to characterize the intrinsic permeability of the
Longmaxi shale used in this work. We posit this would hold for other material sensible to the pore pressure
change as well. Therefore, the influence of the effective stress on permeability cannot be ignored, and it is sug-
gested to be considered to expand the Klinkenberg’s model.f p
g
Increasing gas pressure reduces effective stress, and also shortens the mean free path of gas molecules and thus
mits the effect of the gas slippage phenomenon. The mean free path of the gas is defined as πδ
λ =
K T
p
2
(4)
B
2 πδ
λ =
K T
p
2
B
2 (4) where KB is the Boltzmann constant (KB = 1.3805 × 10−23J/K), δ is the collision diameter of the gas molecule (for
nitrogen, δ =
. ×
−m
3 5
10 10
), T is temperature (K), and P is the pressure (Pa). When gas pressure is 1 MPa,
λ
. is 7 69 nm and it becomes 1.92 nm when gas pressure increases to 4 MPa. The mean free path of the gas is com-
parable to the average pore size of Longmaxi shale. As presented in the sample description section, the average
pore size of LM shale is about 3.0 nm. The ratio of mean free path of the gas (λ) to the average pore diameter of
material (d) is named as the Knudsen number
λ
=
K
d
/
n
. Different Knudsen number means different gas flow
regimes in material3: continuum flow for Kn < 0.001, slip flow (0.001 < Kn < 0.1); transition flow (0.1 < Kn < 10)
and free-molecule flow (Kn > 10). As shown in the MIP curve (Fig. 2), the pore size distribution of Longmaxi
shale is wide thus a variety of flow types might occur in Longmaxi shale, similar to those observed by
Dadmohammadi et al.22,23. As pores become smaller, gas slippage phenomenon will be more significant and
Darcy’s law will be invalid. This phenomenon is often evaluated by Klinkenberg’s equation (Eq. 1). This equation
establishes a linear relationship between the apparent permeability and the inverse of average pressure (1/pm). However, our experimental results (Fig. 5) cannot be explained by Klinkenberg’s linear equation. Discussions With increasing
gas pressure, we also observe a pressure threshold at which the dominant effect on the net permeability changes
from gas slippage to effective stress.i g
pp gf
Based on Klinkenberg’s work, Ashrafi et al. proposed a new expanded equation4, =
+
−
K
K
b
P
a
P
1
(5)
a
int
m
m
2 (5) This model describes a quadratic dependency between apparent gas permeability and mean pressure. The
constants, a and b are dependent on the fluid properties and pore geometry, b is similar to the Klinkenberg con-
stant and a is the slip effect with a non-constant velocity distribution and could be a representation of a dynamic
or secondary slippage factor. This new equation can describe the nonlinear relationship between the apparent
permeability and 1/p. However, this equation cannot completely explain the apparent permeability evolution. The obtained data in this work were used to fitting Klinkenberg and Ashrafi’s models and the results were shown
in Fig. 5 which shows permeability change is different from the theoretical predictions from Klinkenberg and
Ashrafi’s model. Specifically, when gas pressure increases to a certain threshold, net permeability increases again,
and this is dominated by the effective stress principle. Moreover, both Klinkenberg’s equation and Ashrafi’s model
consider that the pore size is constant while gas pressure increases. This is certainly not the case for geomaterial
subjected to mechanical loading. Therefore, one needs to consider the coupled effect of effective stress and gas
slippage in order to obtain an accurate estimation of permeability.htf pp g
p
y
The exponential relationship30 is often used to express the dependence of the permeability on the effective
stress and it is adopted as follows γ
σ
=
−
∆′
K
K
exp(
(
))
(6)
0 (6) where K is the permeability of the material subjected to the effective stress change (m2), K0 is the initial permea-
bility of the material (m2), γ is a material property (Pa−1), σ
∆′ = ∆σ −
′b p
(
) is the effective stress change. This
model can represent the increase of permeability due to the reduction in effective stress. Discussions To expand the
Klinkenberg’s model to incorporate both gas slippage and effective stress effects, we propose a new conceptual
model expressed as, where K is the permeability of the material subjected to the effective stress change (m2), K0 is the initial permea-
bility of the material (m2), γ is a material property (Pa−1), σ
∆′ = ∆σ −
′b p
(
) is the effective stress change. This
model can represent the increase of permeability due to the reduction in effective stress. To expand the
Klinkenberg’s model to incorporate both gas slippage and effective stress effects, we propose a new conceptual
model expressed as, γ
σ
=
+
⋅
−
∆′
K
K
b
P
exp
1
(
(
))
(7)
a
int
m (7) This model is capable of replicating the coupled and competing effect of both gas slippage effect and effective
stress effect. It allows the dominant effect on the net permeability to switch from gas slippage to effective stress
when increasing gas pressure. More tests will be performed in our future research activities to identify these
parameters and better constraint this conceptual model.h This model is capable of replicating the coupled and competing effect of both gas slippage effect and effective
stress effect. It allows the dominant effect on the net permeability to switch from gas slippage to effective stress
when increasing gas pressure. More tests will be performed in our future research activities to identify these
parameters and better constraint this conceptual model.h p
p
The measured gas permeability of the wet sample No.1 is lower than that of the dry sample No.2. Given that
the porosity of LM shale is 2.5% and the water content is 1.46%, the degree of water saturation of sample No.1 is Scientific Reports | 7:44696 | DOI: 10.1038/srep44696 7 www.nature.com/scientificreports/ estimated to be 83%. The dependence of gas permeability on water content has been observed in the past on Cox
shale25. The experimental results of Yang et al.25 show that there exists a quasi-linear relationship between log(Ka)
and the saturation. The obtained results in this work confirm that permeability of shale strongly depends on water
content and it should be considered during gas production design, because gas and water often co-exist in shale
formations. References
bl F., Walsh, J. B. & Frangos, W. T. Permeability of grani 14. David, C., Wong, T. F., Zhu, W. & Zhang, J. Laboratory measurement of compaction induced permeability change in porous rocks:
implication for the generation and maintenance of pore pressure excess in the crust Pure Apply Geophys 143 425 456 (1994) 14. David, C., Wong, T. F., Zhu, W. & Zhang, J. Laboratory measurement of compaction induced permeability change in porous r
implication for the generation and maintenance of pore pressure excess in the crust. Pure Apply Geophys. 143, 425–456 (1994) 5. Kwon, O., Kronenberg, A. & Gangi, A. Permeability of Wilcox shale and its effective pressure law, J. Geophys. Res. 106(B9)
19339–19353 (2001). 16. Shi, T. & Wang, C. Y. Pore pressure generation in sedimentary basins: overloading versus aquathermal. J. Geophys. Res. 91
2153–2162 (1986). (
)
17. Wang, S., Elsworth, D. & Liu, J. Permeability evolution in fractured coal: the roles of fracture geometry and water-content. Int. J. Coal
Geol. 87(1), 13–25 (2011). 8. Wang, S., Elsworth, D. & Liu, J. A mechanistic model for permeability evolution in fractured sorbing media. J. Geophys. Res
117(B06) (2012).ff 9. Katsuki, D. & Gutierrez, M. Poroelastic effects on the stress-dependent permeability of a stiff shale. Poromechanics V.1705–1714. doi
10.1061/9780784412992.202 (2013). 0. Homand, F., Shao, J., Giraud, A., Auvray, C. & Hoxha, D. Pétrofabrique et propriétés mécaniques des argilites. Comptes Rendu
Géosciences. 338(12–13), 882–891 (2006).f 21. Rathnaweera, T. D., Ranjith, R. G. & Perera, S. A. Experimental investigation of geochemical and mineralogical effects o
sequestration on flow characteristics of reservoir rock in deep saline aquifers. Sci. Rep. doi: 10.1038/srep19362 (2016). ql
p
q
p
p
(
)
22. Dadmohammadi, Y., Misra, S., Sondergeld, C. H. & Rai, C. S. Improved Petrophysical Interpretation of Laboratory Pressure-Step-Decay
Measurements on Ultra-Tight Rock Samples. In Unconventional Resources Technology Conference. San Antonio, Texas. (2016a).f ql
p
q
p
p
22. Dadmohammadi, Y., Misra, S., Sondergeld, C. H. & Rai, C. S. Improved Petrophysical Interpretation of Laboratory Pressure-Step-De
M
t
Ult
Ti ht R
k S
l
I U
ti
l R
T h
l
C
f
S
A t
i
T
(2016 ) ql
p
q
p
p
22. Dadmohammadi, Y., Misra, S., Sondergeld, C. H. & Rai, C. S. Improved Petrophysical Interpretation of Laboratory Pressure-Step-Decay g
p
gy
f
23. Dadmohammadi, Y., Misra, S., Sondergeld, C. H. & Rai, C. S. Discussions It is necessary to further characterize the gas permeability of unsaturated shale in the future, although
it would be challenging in the laboratory to control the saturation of the sample to as the porosity of Longmaxi
shale (2.50%) is much less than that of Cox shale (18%). (
)
(
)
In this study, nitrogen is used to measure gas permeability and it is a slightly sorptive gas which could induce
swelling of shale. Nuttall et al. studied the adsorption of Devonian black shales and found CO2 have an adsorption
capacity approximately 5 times greater than that of CH4
31. Battistuta et al.32 and Wang et al.17 studied the sorp-
tive effect of different gas (CO2, CH4 and N2) on dry coal and found that CO2 sorption on coal induces a bigger
swelling effect on the coal matrix than CH4 and N2. Wu and Zhang33 found that the swelling of matrix decreases
gas permeability. In this study, the adsorption effect on permeability was checked by repeated gas permeability
tests under the same condition at different stages as shown in Fig. 3a,b and Tables 1, 2 and the results show that
permeability does not change even though the gas tests have lasted two weeks. The reason could be due to the fact
that N2 has a relatively weak adsorption capacity. Thus, the swelling of shale due to gas adsorption can be ignored
to characterize gas permeability in this study. References
bl Simultaneous Estimation of Intrinsic Permeability, Effective Porosity,
Pore Volume Compressibility, and Klinkenberg-Slip Factor of Ultra-Tight Rock Samples Based on Laboratory Pressure-Step-Decay
Method. SPE-180266-MS, SPE Low Perm Symposium, Denver, Colorado, USA, Society of Petroleum Engineers (2016, May, 5–6). g
gy
f
23. Dadmohammadi, Y., Misra, S., Sondergeld, C. H. & Rai, C. S. Simultaneous Estimation of Intrinsic Permeability, Effective Por ,
,
,
,
g
,
R ,
y,f
y,
Pore Volume Compressibility, and Klinkenberg-Slip Factor of Ultra-Tight Rock Samples Based on Laboratory Pressure-Step-Decay
Method SPE-180266-MS, SPE Low Perm Symposium, Denver, Colorado, USA, Society of Petroleum Engineers (2016, May, 5–6) f
Pore Volume Compressibility, and Klinkenberg-Slip Factor of Ultra-Tight Rock Samples Based on Laboratory Pressure-Step-Decay
M h d SPE 180266 MS SPE L
P
S
i
D
C l
d
USA S
i
f P
l
E
i
(2016 M
5 6) ,
y p
y f
g
(
,
y,
)
24. Cao, C. H., Lv, Z. G., Li, L. W. & Du, L. Geochemical characteristics and implications of shale gas from the Longmaxi Formation,
Sichuan Basin, China. J. Nat Gas Geosci. doi: 10.1016/j.jnggs.2016.05.001 (2016). J
j j gg
25. Yang, D. S., Billotte, J. & Su, K. Characterization of the hydrome
permeability under deviatoric stress. Eng. Geol. 114, 116–122 (2010). j j gg
5. Yang, D. S., Billotte, J. & Su, K. Characterization of the hydromechanical behavior of argillaceous rocks with effective ga j j gg
25. Yang, D. S., Billotte, J. & Su, K. Characterization of the hydromechanical behavior of argillaceous ro
permeability under deviatoric stress. Eng. Geol. 114, 116–122 (2010). D. S., Billotte, J. & Su, K. Characterization of the hydromechanical behavior of argillaceous rocks with effective gas
ability under deviatoric stress. Eng. Geol. 114, 116–122 (2010). 5. Yang, D. S., Billotte, J. & Su, K. Characterization of the hydromechanical behavior of argillaceous rocks with effective ga
permeability under deviatoric stress. Eng. Geol. 114, 116–122 (2010). g
J
y
permeability under deviatoric stress. Eng. Geol. 114, 116–122 (2010). p
y
g
(
)
26. Biot, M. A. General theory of 3-dimensional consolidation. J. Appl. Phys. 12, 155–164 (1941). y
g
. A. General theory of 3-dimensional consolidation. J. Appl. Phys. 1 26. Biot, M. A. General theory of 3-dimensional consolidation. J. A y
pp
y
27. Coussy, O. Poromechanics. John Wiley and Sons, Chichester, England (2004). 28. Bemer, E., Longuemare, P. & Vincké, O. References
bl 1. Gensterblum, Y. et al. Gas storage capacity and transport in shale gas reservoirs – A review. Part A: Transport processes. J. Uncon. Oil Gas Res. 12, 87–122 (2015).h 2. Li, Y. B., Li, Y., Wang, B. Q., Chen, Z. E. & Nie, D. The status quo review and suggested policies for shale gas development in China
Renew. Sust. Energ. Rev. 59, 420–428 (2016). 3. Arabjamaloei, R. & Ruth, D. W. Lattice Boltzmann based simulation of gas flow regimes in low permeability porous media:
Klinkenberg’s region and beyond. J. Nat. Gas Sci. Eng. 31, 405–416 (2016).i g
g
y
g
4. Ashrafi-Moghadam, A. & Chalaturnyk, R. Expansion of the Klinkenberg’s slippage equation to low permeability porous media. Int. J. Coal Geol. 123, 2–9 (2014).l 5. Darabi, H., Ettehad, A., Javadpour, F. & Sepehrnoori, K. Gas flow in ultra-tight shale strata. J. Fluid Mech. 710, 641–658 (2012).l 5. Darabi, H., Ettehad, A., Javadpour, F. & Sepehrnoori, K. Gas flow in ultra tight shale strata. J. Fluid Mech. 710, 641 658 (2012). 6. Fathi, E., Tinni, A. & Yucel-Akkutlu, I. Correction to Klinkenberg slip theory for gas flow in nano-capillaries. Int. J. Coal Geol. 103
51–59 (2012).h 7. Klinkenberg, L. J. The permeability of porous media to liquids and gases. API Drilling and Production Practice. 200–213 (1941 h
8. Tanikawa, W. & Shimamoto, T. Comparison of Klinkenberg-corrected gas permeability and water permeability in sedimentary
rocks. Int. J. Rock Mech. Min. Sci. Geomech. Abstr. 46, 229–238 (2009).l J
(
)
9. Harley, J. C., Huang, Y., Bau, H. H. & Zemel, J. N. Gas flow in micro-channels. J. Fluid Mech. 284, 257–274 (1995). y
gl
10. Shan, X., Yuan, X. F. & Chen, H. Kinetic theory representation of hydrodynamics: a way beyond the Naiver Stokes equation. J. Fluid
Mech. 550, 413–441 (2006).f 1. Ren, X. X., Li, A. F., Wang, Y. Z. & Chen, M. Q. Gas permeability experimental study of low permeability core considering effect o
gas slippage, Nat. Gas Geosci. 26(4), 733–736 (2015). g
pp g
12. Dong, J. J. et al. Stress-dependence of the permeability and porosity of sandstone and shale from TCDP Hole-A. Int. J. Rock. Mech. Min.Sci. 47(7), 1141–1157 (2010). 3. Brace, W. F., Walsh, J. B. & Frangos, W. T. Permeability of granite under high pressure, J. Geophys. Res. 73, 2225–2236 (1968). d
h
h
b
f
d
d
b l
h 13. Brace, W. Acknowledgementsh g
This work is supported by the Chinese Fundamental Research (973) Program (2015CB057906), the National
Natural Science Foundation of China (Grant Nos 51379200, 41572290, 51225902, 41672281), the Chinese
government plan on the Recruitment of Global Young Talents and Youth Innovation Promotion Association
CAS. These supports are gratefully acknowledged. Author Contributions D.S.Y. and S.G.W. designed the research and analyzed the data, all authors performed the research, D.S.Y. and
S.G.W. wrote the paper, W.W. and X.Q.W. prepared the samples and carried out the tests, D.S.Y. and W.Z.C. analyzed the results. All authors discussed and approved the manuscript. The authors thank the reviewers for
their constructive comments and suggestions to improve the paper. www.nature.com/scientificreports/ carbon dioxide on dry Selar Cornish coal. Int. J. Coal Geol. 34, 39 48 (2010). 3. Wu, T. H. & Zhang, D. X. Impact of Adsorption on Gas Transport in Nanopores. Sci. Rep. doi: 10.1038/srep23629 (2016). References
bl Poroelastic parameters of Meuse/Haute Marne argillites: effect of loading and satur
states. Appl. Clay Sci. 26, 359–366 (2004). 28. Bemer, E., Longuemare, P. & Vincké, O. Po
states. Appl. Clay Sci. 26, 359–366 (2004). pp
y
9. Cariou, S., Duan, Z., Davy, C., Skoczylas, F. & Dormieux, L. Poromechanics of partially saturated Cox argillite. Appl. Clay Sci. 56
36–47 (2012). 30. Seidle, J. P., Jeansonne, M. W. & Erickson, D. J. Application of Matchstick Geometry to Stress Dependent Permeability in Coals. SPE-
24361, SPE Rocky Mountain Regional Meeting, Casper, Wyoming, USA, Society of Petroleum Engineers (1992, May 18–21). 30. Seidle, J. P., Jeansonne, M. W. & Erickson, D. J. Application of Matchstick Geometry to Stress Dependent Permeability in Coals. SPE-
24361, SPE Rocky Mountain Regional Meeting, Casper, Wyoming, USA, Society of Petroleum Engineers (1992, May 18–21). 31. Nuttall, B. C., Eble, C. F., Drahovzal, J. A. & Bustin, R. M. Analysis of Devonian black shales in Kentucky for potential carbon dioxide
sequestration and enhanced natural gas production. Report Kentucky Geological Survey/ University of Kentucky (DE-FC26-
02NT41442). http://www.osti.gov/scitech/servlets/purl/842849 (2005). 31. Nuttall, B. C., Eble, C. F., Drahovzal, J. A. & Bustin, R. M. Analysis of Devonian black shales in Kentucky for potential carbon dioxide
sequestration and enhanced natural gas production. Report Kentucky Geological Survey/ University of Kentucky (DE-FC26-
02NT41442). http://www.osti.gov/scitech/servlets/purl/842849 (2005). Scientific Reports | 7:44696 | DOI: 10.1038/srep44696 8 www.nature.com/scientificreports/ Scientific Reports | 7:44696 | DOI: 10.1038/srep44696 Additional Informationh Competing Interests: The authors declare no competing financial interests. How to cite this article: Yang, D. et al. Experimental investigation on the coupled effect of effective stress an
gas slippage on the permeability of shale. Sci. Rep. 7, 44696; doi: 10.1038/srep44696 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2017 Scientific Reports | 7:44696 | DOI: 10.1038/srep44696 9
|
W3090567294.txt
|
https://zenodo.org/records/4649631/files/17%2020572%20CE%204aug%202jul19%20N.pdf
|
en
|
Novel recommendation for enhancing optical properties of CP-WLEDs by Ba2Si5N8Eu2+ phosphor
|
International Journal of Power Electronics and Drive Systems/International Journal of Electrical and Computer Engineering
| 2,021
|
cc-by
| 2,478
|
International Journal of Electrical and Computer Engineering (IJECE)
Vol. 11, No. 2, April 2021, pp. 1063~1067
ISSN: 2088-8708, DOI: 10.11591/ijece.v11i2.pp1063-1067
1063
Novel recommendation for enhancing optical properties of
CP-WLEDs by Ba2Si5N8Eu2+ phosphor
Van-Duc Phan1, Minh Tran2, Q. S. Vu3
1Faculty
of Automobile Technology, Van Lang University, Ho Chi Minh City, Vietnam
2Optoelectronics Research Group, Faculty of Electrical and Electronics Engineering, Ton Duc Thang University,
Ho Chi Minh City, Vietnam
3School of Engineering-Technology and Arts, Hong Bang International University, Ho Chi Minh City, Vietnam
Article Info
ABSTRACT
Article history:
In this paper, the Ba2Si5N8Eu2+ phosphor is proposed as the novel
recommendation for enhancing the optical properties in terms of D-CCT,
CRI, CQS, and LO of the CP-WLEDs. Firstly, we conducted the physical
model of the CP-WLEDs in the LightTools software with the main
parameters like the real LEDs. Furthermore, the scattering process in LEDs
compound of the CP-WLEDs is simulated and investigated by the Matlab
software. Then the influence of the Ba2Si5N8Eu2+ concentration on the D-CCT,
CRI, CQS, and LO of the CP-WLEDs is investigated. Finally, the research
results showed that the Ba2Si5N8Eu2+ concentration has a considerable effect on
the D-CCT, CRI, CQS, and LO of the CP-WLEDs. From the results, we can
state that the Ba2Si5N8Eu2+ phosphor can be considered as the novel
recommendation for enhancing the optical properties of the CP-WLEDs.
Received Jul 2, 2019
Revised Aug 4, 2020
Accepted Sep 8, 2020
Keywords:
CP-WLEDs
CQS
CRI
D-CCT
LO
Optical properties
Red phosphor
This is an open access article under the CC BY-SA license.
Corresponding Author:
Minh Tran
Optoelectronics Research Group
Faculty of Electrical and Electronics Engineering
Ton Duc Thang University
Ho Chi Minh City, Vietnam
Email: tranhoangquangminh@tdtu.edu.vn
1.
INTRODUCTION
In the lighting revolution trend for improving civilian everyday life in over the world, the InGaNbased white-light-emitting diodes (LEDs) can be considered as the primary solution in comparison with the
conventional lighting method based on the excellent advantages such as environment-friendly, energy
efficiency, compactness, long lifetime, and designable features [1-3]. Nowadays, phosphor-converted LEDs
(pcLED) combines a blue LEDs chip, and the yellow emitting phosphor is the novel solution for lighting in civil
and industrial purposes [4, 5]. In the last few years, authors in many research focused on enhancing the lighting
properties of the white LEDs. Authors in [6, 7] proposed and investigated the effect of the phosphor layer
thickness and concentration on the optical properties of white LEDs and concluded that the lower phosphor
concentration and higher phosphor thickness led to the higher luminous efficacy. Furthermore, the authors in
[8] stated that the spatial color distribution (SPD) of white LEDs is significantly affected by the phosphor
layer parameter such as thickness, concentration, and size. As studied in [9-12], the blue light and yellow
light have a similar radiation pattern that can be lead to improving the SPD by varying the phosphor layer
location of the white LEDs. Besides, the green and red phosphor by adding to the phosphor layer can be
considered as a novel solution for enhancing the optical properties of the white LEDs as in [13-15].
Journal homepage: http://ijece.iaescore.com
1064
ISSN: 2088-8708
In this paper, the Ba2Si5N8Eu2+ is proposed as the novel recommendation for enhancing the optical
properties in terms of D-CCT, CRI, CQS, and LO of the CP-WLEDs. Firstly, we conducted the physical
model of the CP-WLEDs in the LightTools software with the main parameters like the real LEDs.
Furthermore, the scattering process in the CP-WLEDs is simulated and investigated by the Matlab software.
Then the influence of the Ba2Si5N8Eu2+ phosphor concentration on the D-CCT, CRI, CQS, and LO of the
CP-WLEDs is investigated. Finally, the research results showed that the Ba2Si5N8Eu2+ concentration has a
considerable effect on the D-CCT, CRI, CQS, and LO of the CP-WLEDs. From the results, we can state that
the Ba2Si5N8Eu2+ phosphor can be considered as the novel recommendation for enhancing the optical
properties of the CP-WLEDs. The rest of this paper can be formulated as follows. The physical model of
CPW-LEDs, optical properties of the red phosphor is proposed in the second section. The third section
presented the numerical results and some discussions. The last section concludes the research.
2.
PHYSICAL CPW-LEDS MODEL AND OPTICAL PROPERTIES OF THE RED PHOSPHOR
We use the LightTools software to simulate the real-world model of CPW-LEDs Figure 1(a) with
the main parameters, as shown in Figure 1(b) [13-15]. Among these (oxy) nitride phosphors, Ba2Si5N8Eu2+ has
high quantum efficiency and very low thermal quenching to improve the color rendering index Figure 2 [16-21].
(a)
(b)
Figure 1. (a) The white LEDs, (b) physical model
Figure 2. Optical properties of the Ba2Si5N8Eu2+ phosphor
Based on Mie theory [22-30], the scattering coefficients can be calculated as
sca ( ) N (r )Csca ( , r )dr
Int J Elec & Comp Eng, Vol. 11, No. 2, April 2021 : 1063 - 1067
(1)
Int J Elec & Comp Eng
ISSN: 2088-8708
abs ( ) N (r )Cabs ( , r )dr
1065
(2)
1
g ( ) 2 p( , , r ) f (r ) cos d cos dr
(3)
sca sca (1 g )
(4)
f (r ) f dif (r ) f phos (r )
(5)
N (r ) N dif (r ) N phos (r ) K N .[ f dif ( r ) f phos ( r )]
(6)
c K N M (r )dr
(7)
1
4
M (r ) r 3 [ dif f dif (r ) phos f phos (r )]
3
(8)
3.
NEMERICAL RESULTS AND DISCUSSIONS
Based on the Mie Theory, the scattering coefficients (SC) versus Ba2Si5N8Eu2+. concentration are
shown in Figure 3 with the red, blue, and yellow lights. From Figure 3, we can see that the (SC) of the yellow
light is better than others, and the SC of the red and blue lights are the same. The efficiency of the yellow
light in the phosphor layer can cause the enhancing the optical properties of the CPW-LEDs. In another way,
the scattering amplitude (SA) is investigated in Figure 4 with red, blue, and yellow lights. As shown in
Figure 4, the SA of the blue light is better than others to verify the enhancing the optical properties when we
added the red phosphor in the phosphor layer.
Figure 3. SC
Figure 4. The scattering amplitude
The function of D-CCT of the phosphor concentration is illustrated in Figure 5. In this figure, we
varied the phosphor concentration from 0 to 16% and investigated the D-CCT of the CPW-LEDs. As shown
in Figure 5, it can be observed that the D-CCT increases massively from 1500 K to 8500 K while we vary the
concentration of Ba2Si5N8Eu2+. Furthermore, the effect of the Ba2Si5N8Eu2+ phosphor concentration on
the CRI and CQS of the CPW-LEDs is proposed in Figures 6 and 7, respectively. The CRI and CQS increase
when the concentration of the Ba2Si5N8Eu2+ phosphor increases from 0% to 10% and then have a huge
decrease with rising of the phosphor concentration from 10% to 16%. The maximum values of CRI and CQS are
85 and 79, which can be obtained with 10% concentration of the Ba2Si5N8Eu2+ phosphor. Finally, the LO versus
the phosphor concentration is drawn in Figure 8. The LO has a massive fall with the rising of the
Ba2Si5N8Eu2+ phosphor. From the results, we can state that the concentration of Ba2Si5N8Eu2+ has a
considerable effect on the optical properties (D-CCT, CRI, CQS, and LO).
Novel recommendation for enhancing optical properties of CP-WLEDs … (Van-Duc Phan)
1066
ISSN: 2088-8708
Figure 5. D-CCT
Figure 6. CQS
Figure 7. CRI
Figure 8. The lumen output LO
4.
CONCLUSION
In this paper, Ba2Si5N8Eu2+ is proposed as the novel recommendation for enhancing the optical
properties in terms of D-CCT, CRI, CQS, and LO of the CP-WLEDs. Firstly, we conducted the physical
model of the CP-WLEDs in the LightTools software with the main parameters like the real LEDs.
Furthermore, the scattering process in the CP-WLEDs is simulated and investigated by the Matlab software.
Then the influence of Ba2Si5N8Eu2+ concentration on the D-CCT, CRI, CQS, and LO of the CP-WLEDs is
investigated. Finally, the research results showed that Ba2Si5N8Eu2+ concentration has a considerable effect
on the D-CCT, CRI, CQS, and LO of the CP-WLEDs. From the results, we can state that Ba2Si5N8Eu2+ can
be considered as the novel recommendation for enhancing the optical properties of the CP-WLEDs.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
Sheng Liu, Xiaobing Luo, “Design of LED Packaging Applications,” LED Packaging for Lighting Applications,
pp. 215-315, 2011.
Gibney, Elizabeth, “Nobel for Blue LED That Revolutionized Lighting,” Nature, vol. 514, no. 7521, pp. 152-153, 2014.
Winkler, Holger, Quang Trinh, Peter Bodrogi, and Tran Quoc Khanh, “LED Lighting: Technology and
Perception,” Weinheim: Wiley-VCH, 2015.
Luo, Xiaobing, Run Hu, Sheng Liu, and Kai Wang, “Heat and Fluid Flow in High-Power LED Packaging and
Applications,” Progress in Energy and Combustion Science, vol. 56, pp. 1-32, 2016.
Hu, Run, Xiaobing Luo, and Sheng Liu, “Effect of the Amount of Phosphor Silicone Gel on Optical Property of
White Light-Emitting Diodes Packaging,” 2011 12th International Conference on Electronic Packaging
Technology and High Density Packaging, Shanghai, 2011, pp. 1-4.
Tran, Nguyen T., and Frank G. Shi, “Studies of Phosphor Concentration and Thickness for Phosphor-Based White
Light-Emitting-Diodes,” Journal of Lightwave Technology, vol. 26, no. 21, pp. 3556-3559, 2008.
Shuai, Y., Y. He, N. T. Tran, F. G. Shi, “Angular CCT uniformity of phosphor-converted white LEDs: effects of
phosphor materials and packaging structures,” IEEE Photonics Technology Letters, vol. 23, no. 3, pp. 137-139, 2011.
Sommer, Christian, Franz-Peter Wenzl, Paul Hartmann, Peter Pachler, Marko Schweighart, Stefan Tasch, and
Günther Leising, “Tailoring of the Color Conversion Elements in Phosphor-Converted High-Power LEDs by
Optical Simulations,” IEEE Photonics Technology Letters, vol. 20, no. 9, pp. 739-741, 2008.
Int J Elec & Comp Eng, Vol. 11, No. 2, April 2021 : 1063 - 1067
Int J Elec & Comp Eng
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
ISSN: 2088-8708
1067
Li, Shuiming, Kai Wang, Fei Chen, Shuang Zhao, Zhili Zhao, and Sheng Liu, “Angular Color Uniformity
Enhancement of Phosphor Converted White LEDs Integrated with Compact Modified Freeform TIR Components,”
2012 13th International Conference on Electronic Packaging Technology & High Density Packaging, Guilin,
2012, pp. 1487-1490.
Liu, Zongyuan, Sheng Liu, Kai Wang, and Xiaobing Luo, “Analysis of Factors Affecting Color Distribution of
White LEDs,” 2008 International Conference on Electronic Packaging Technology & High Density Packaging,
Shanghai, 2008, pp. 1-8.
Liu, Zongyuan, Sheng Liu, Kai Wang, and Xiaobing Luo, “Optical Analysis of Color Distribution in White LEDs
with Various Packaging Methods,” IEEE Photonics Technology Letters, vol. 20, no. 24, pp. 2027-2029, 2008.
Liu, Zongyuan, Sheng Liu, Kai Wang, and Xiaobing Luo, “Effects of Phosphor’s Location on LED Packaging
Performance,” 2008 International Conference on Electronic Packaging Technology & High Density Packaging,
Shanghai, 2008, pp. 1-7.
Minh, Tran Hoang Quang, Nguyen Huu Khanh Nhan, Nguyen Doan Quoc Anh, and Hsiao-Yi Lee, “Red-Emitting
α-SrO•3B2O3:Sm2 Phosphor: an Innovative Application for Increasing Color Quality and Luminous Flux of
Remote Phosphor White LEDs,” Journal of the Chinese Institute of Engineers, vol. 40, no. 4, pp. 313-317, 2017.
Anh, Nguyen Doan Quoc, Hsiao-Yi Lee, Tran Thanh Phuong, Nguyen Huu Khanh Nhan, Tran Hoang Quang
Minh, and Truong Huu Ly, “Y2O3:Eu3 Phosphor: a Novel Solution for an Increase in Color Rendering Index of
Multi-Chip White LED Packages,” Journal of the Chinese Institute of Engineers, vol. 40, no. 3, pp. 228-234, 2017.
Anh, Nguyen Doan Quoc, and Hsiao-Yi Lee, “Improving the Angular Color Uniformity and the Lumen Output for
Multi-Chip White LED Lamps by Green Ce0.67Tb0.33MgAl11O19:Ce,Tb Phosphor,” Journal of the Chinese
Institute of Engineers, vol. 39, no. 7, pp. 871-875, 2016.
Chen, Changcheng, and Erqing Xie, “Synthesis and Luminescence Properties of Red-Emitting M2Si5N8:Eu2Based (M=Ca, Sr, Ba) Phosphors by a Simple Nitrate Reduction,” Science China Physics, Mechanics and Astronomy,
vol. 57, no. 3, pp. 433-436, 2014.
Duan, C. J., W. M. Otten, A. C. A. Delsing, and H. T. Hintzen, “Preparation and Photoluminescence Properties of Mn2Activated M2Si5N8 (M=Ca, Sr, Ba) Phosphors,” Journal of Solid State Chemistry, vol. 181, no. 4, pp. 751-757, 2008.
Li, Hui-Li, Rong-Jun Xie, Naoto Hirosaki, Takashi Takeda, and Guo-Hong Zhou, “Synthesis and Luminescence
Properties of Orange-Red-Emitting M2Si5N8:Eu2 (M=Ca, Sr, Ba) Light-Emitting Diode Conversion Phosphors by
a Simple Nitridation of MSi2,” International Journal of Applied Ceramic Technology, vol. 6, no. 4, pp. 459-464, 2009.
Li, Y. Q., J. E. J. Van Steen, J. W. H. Van Krevel, G. Botty, A. C. A. Delsing, F. J. Disalvo, G. De With, and H. T.
Hintzen, “Luminescence Properties of Red-Emitting M2Si5N8:Eu2 (M: Ca, Sr, Ba) LED Conversion Phosphors,”
Journal of Alloys and Compinds, vol. 417, no. 1-2, pp. 273-279, 2006.
Zeuner, Martin, Peter J. Schmidt, and Wolfgang Schnick, “One-Pot Synthesis of Single-Source Precursors for
Nanocrystalline LED Phosphors M2Si5N8:Eu2 (M: Sr, Ba),” Chemistry Materials, vol. 21, no. 12, pp. 2467-2573, 2009.
Fukuda, Yumi, Aoi Okada, and Ariane Keiko Albessard, “Luminescence Properties of Eu2+-Doped Red-Emitting
Sr-Containing Sialon Phosphor,” Applied Physics Express, vol. 5, no. 6, pp. 1-14, 2012.
“Beyond Mie's Theory II-The Generalized Mie Theory,” Optical Properties of Nanoparticle Systems, pp. 317-339, 2011.
Frisvad, Jeppe Revall, Niels Jørgen Christensen, and Henrik Wann Jensen, “Predicting the Appearance of Materials
Using Lorenz-Mie Theory,” The Mie Theory Springer Series in Optical Sciences, vol. 169, pp. 101-133, 2012.
Mackowski, Daniel, “The Extension of Mie Theory to Multiple Spheres,” The Mie Theory, vol. 169, pp. 223-256, 2012.
Wriedt, Thomas, “Mie Theory: A Review,” The Mie Theory Springer Series in Optical Sciences, pp. 53-71, 2012.
Luo, X., and Hu, R., “Chip packaging: Encapsulation of nitride LEDs,” Nitride Semiconductor Light-Emitting
Diodes (LEDs), pp. 441-481, 2014.
Phu Tran Tin, Minh Tran, Van-Duc Phan, Hoang-Nam Nguyen, Thanh Trang Tran, "K2SiF6:Mn4+ phosphor:
recommendation for improving LO and D-CCT of the 7700K RPWLEDs," International Journal of Electrical and
Computer Engineering (IJECE), vol. 9, no. 5, pp. 3975-3980, 2019.
Phu Tran Tin, Anh Vu Le, Minh Tran, Nguyen Huu Khanh Nhan, Tran Thanh Trang, "Improving CCT-D and LO
of the 6600K ICP-WLEDs by K2SiF6:Mn4+ Phosphor," International Journal of Power Electronics and Drive
Systems (IJPEDS), vol. 10, no. 2, pp. 1059-1063, 2019.
Phu Tran Tin, Nguyen Huu Khanh Nhan, Tran Hoang Quang Minh, Thi Phuong Thao Nguyen, "Red-Emitting
Ba2Si5N8Eu2+ Conversion Phosphor: A New Selection for Enhancing the Optical Performance of the In-cup
Packaging MCW-LEDs," Cogent Engineering, vol. 5, no. 1, pp. 1-11, 2018.
Q. S. Vu, Tang Tin Dao, Minh Tran, "Ca[Mg3SiN4]Ce3+ phosphor: effect of particle concentration on lighting
properties of the 7000K IPW-LEDs," International Journal of Power Electronics and Drive Systems (IJPEDS),
vol. 11, no. 2, pp. 566-570, 2020.
Novel recommendation for enhancing optical properties of CP-WLEDs … (Van-Duc Phan)
|
|
https://openalex.org/W3199098725
|
https://zenodo.org/records/5535233/files/CheckList_article_66867.pdf
|
English
| null |
Validating the presence of Spanish Flag, Gonioplectrus hispanus (Cuvier, 1828) (Perciformes, Serranidae), from the south-western Gulf of Mexico
|
Check list
| 2,021
|
cc-by
| 3,030
|
Keywords Chernas, Epinephelinidae, grouper, range extension, species distribution Academic editor: Hudson Tercio Pinheiro | Received 2 April 2021 | Accepted 28 August 2021 | Published 21 September 2021 Citation: Del Moral-Flores LF, López-Segovia E, Escartín-Alpizar VR, Jiménez-Badillo ML (2021) Validating the presence of Spanish Flag,
Gonioplectrus hispanus (Cuvier, 1828) (Perciformes, Serranidae), from the south-western Gulf of Mexico. Check List 17 (5): 1299–1303. https://
doi.org/10.15560/17.5.1299 Check List 17 (5): 1299–1303
https://doi.org/10.15560/17.5.1299
NOTES ON GEOGRAPHIC DISTRIBUTION Check List 17 (5): 1299–1303
https://doi.org/10.15560/17.5.1299
NOTES ON GEOGRAPHIC DISTRIBUTION Abstract We report the presence of Gonioplectrus hispanus (Cuvier, 1828) in Mexican waters of the Gulf of Mexico. Four
specimens (198–224 mm standard length) were caught at two locations south of Veracruz. These records increases
the number of species in the family Serranidae for Mexico to 113. We include morphometry and meristic data of our
specimens. Check List 17 (5): 1299–1303
https://doi.org/10.15560/17.5.1299 Check List 17 (5): 1299–1303
https://doi.org/10.15560/17.5.1299 NOTES ON GEOGRAPHIC DISTRIBUTION Validating the presence of Spanish Flag, Gonioplectrus hispanus
(Cuvier, 1828) (Perciformes, Serranidae), from the south-western
Gulf of Mexico Luis Fernando Del Moral-Flores1, Eduardo López-Segovia1,2, Viridiana R. Escartín-Alpizar1, 2,
María de Lourdes Jiménez-Badillo3 1 Laboratorio de Zoología, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Mexico City, Mexico •
LFDMF: delmoralfer@gmail.com
https://orcid.org/0000-0002-7804-2716 • VREA: viridianaescartin11d@gmail.com,
https://orcid.org/
0000-00018524-3850 2 Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Mexico City, Mexico • ELS: eduardosegovia@
comunidad.unam.mx
https://orcid.org/0000-0001-8065-1521 3 Instituto de Ciencias Marinas y Pesquerías, Universidad Veracruzana, Veracruz, Mexico • MLJB: ljimenez@uv.mx
https://orcid.org/0000-
0002-0324-433X * Corresponding author © The authors. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited. 2 Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Mexico City, Mexico • ELS: eduardosegovia@
comunidad.unam.mx
https://orcid.org/0000-0001-8065-1521 1 Laboratorio de Zoología, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Mexico City, Mexico •
LFDMF: delmoralfer@gmail.com
https://orcid.org/0000-0002-7804-2716 • VREA: viridianaescartin11d@gmail.com,
https://orcid.org/
0000-00018524-3850 Introduction in coastal, tropical and subtropical ecosystems, includ
ing mesophotic and rariphotic environments (Bullock
and Smith 1991; Baldwin et al. 2018). One of the genera
within the family is Gonioplectrus Gill, 1862, which is
monotypic, with Gonioplectrus hispanus (Cuvier, 1828),
Spanish Flag, restricted to Western Atlantic (Parenti and
Randall 2020). The family Serranidae Swainson, 1839 is a monophy
letic group and includes six subfamilies, 72 genera, and
between 579 and 583 species (Fricke et al. 2020; Parenti
and Randall 2020). This family has significant economic
importance, as it supports commercial fisheries and sport
fishing activities (Bullock and Smith 1991). In Mexico, it
supports one of the main fisheries in the Western Atlan
tic (Colás-Marrufo et al. 1998), and it is also important Gonioplectrus hispanus has a wide distribution from 1300 Check List 17 (5) North Carolina (USA) to south-eastern Brazil. How
ever, the records of this species are scarce because it
inhabits rocky areas and deep coral from 60 to 365 m
(McEachran and Fechhelm 2005). The north-western
distribution of this species includes the Gulf of Mex
ico, Texas, and other parts of the United States (Briggs,
1958; Briggs et al. 1964; Moore 1975; Smith 1976; Bull
ock and Smith 1991; Hoese and Moore 1998). There are
no confirmed records based on deposited specimens in
reference collections from Gulf of Mexico and the Mexi
can Caribbean, although it is mentioned in the faunistic
lists of Mexican Atlantic coasts (Lara-Domínguez et al. 1993; Baqueiro and Mendez 1994), including the coast
at Los Tuxtlas (Schaldach et al. 1997) and as larvae on
Campeche Bank (Flores-Coto et al. 2009). The only evi
dence of its presence in Mexican Atlantic coasts is a pho
tograph of a single specimen in the Instituto Nacional de
Pesca (1976). The geographic coordinates of the collec
tion site of this specimen are unknown. With the new
records presented here, we confirm the presence of G. hispanus in the south-western Gulf of Mexico. determination was made with specialized keys by Bull
ock and Smith (1991), McEachran and Fechhelman
(2005), and Heemstra et al. (2002). Electronic calipers
(0.01 mm) were used for morphological measurements,
and the meristic data were counted according to Hubbs
and Lagler (1958) with modifications for the group
(Betancur-R. et al. 2001; Oliveira and Barbosa 2010;
Sampaio et al. 2017). Gonioplectrus hispanus (Cuvier, 1828)
Figure 2; Table 1 Common names. Spanish Flag; mero payaso, payasín
(Spanish). New records. MEXICO – Veracruz • Gulf of Mexico;
ca. 11.8 km from Punta Puntilla, municipality of Ángel
R. Cabada; 18°46′56.7″N, 095°09′40.2″W; 280 m depth;
12.XI.2020; A. Campos-Pérez and E. Campos-Vicente
leg.; fishing line; 2 specimens, CIFI-1715 (192 and 208
mm SL) • same locality, 300 m depth; 09.I.2021; A. Cam
pos-Pérez leg.; fishing line; 1 specimen, CIFI-1801 (224
mm SL) • Gulf of Mexico; ca. 9 km from Playa Her
mosa, municipality of Ángel R. Cabada; 18°44′28.8″N,
095°04′52.0″W; 180 m depth; 24.VI.2021; A. Campos-
Pérez and E. Campos-Vicente leg.; fishing line; 1 speci
men, CIFI-1820 (208 mm SL). Results Family Serranidae Swainson 1839
Genus Gonioplectrus Gill, 1862 Family Serranidae Swainson 1839
Genus Gonioplectrus Gill, 1862 Methods Four specimens of Gonioplectrus hispanus were caught
accidentally by the artisanal fishery, using a bottom-
longline made with nylon monofilament and 60 to 150
hooks (size 6) at depths of 180–300 m (Fig. 1). The speci
mens were deposited in the Ichthyological Collection of
the Facultad de Estudios Superiores Iztacala (CIFI), Uni
versidad Nacional Autónoma de México. The taxonomic Figure 1. Sampling station of Gonioplectrus hispanus (black cross), ca. 11.8 km from Punta Puntilla, municipality of Ángel R. Cabada, Vera
cruz, south-western Gulf of Mexico (18°46′56.7″N, 095°09′40.2″W). Previous records of G. hispanus in the western Atlantic (red dots). Figure 1 Sampling station of Gonioplectrus hispanus (black cross) ca 11 8 km from Punta Puntilla municipality of Ángel R Cabada Vera Figure 1. Sampling station of Gonioplectrus hispanus (black cross), ca. 11.8 km from Punta Puntilla, municipality of Ángel R. Cabada, Vera
cruz, south-western Gulf of Mexico (18°46′56.7″N, 095°09′40.2″W). Previous records of G. hispanus in the western Atlantic (red dots). Del Moral-Flores et al. | Gonioplectrus hispanus from the south-western Gulf of Mexico 1301 Figure 2. Gonioplectrus hispanus (CIFI-1715, 208 mm SL) collected in the south-western Gulf of Mexico, showing typical coloration for the
species. The species was determined prior to the preservation of the specimen. Scale bar = 25 mm. Figure 2. Gonioplectrus hispanus (CIFI-1715, 208 mm SL) collected in the south-western Gulf of Mexico, showing typical coloration for the
species. The species was determined prior to the preservation of the specimen. Scale bar = 25 mm. Table 1. Morphometric and meristic data of specimens of Gonio
plectrus hispanus from the south-western Gulf of Mexico. Identification. Gonioplectrus hispanus is recognised by
its a serrated preopercle with a large spine on the ventral
edge directed forward. The opercle has three spines, and
the second spine is most elongated. The dorsal fin has
8 spines and 11–13 rays, and the anal fin has 3 spines
and 7 rays. The lateral line has 47–49 scales. The bases
of the dorsal, anal, and pectoral fins are covered with
scales. Gill rakers number 21–23. In addition, in vivo,
G. hispanus shows a striking pink coloration, with seven
bright yellow stripes on the sides from the head to the
base of the tail fin. A diagonal yellow stripe runs from
the snout through the eye and upper opercle to the back
of the body. There are yellow patches in the interorbital
region and cheek. Methods The pelvic fins are violet, and the anal
fin has a crimson spot from the second anal spine as far
as the first anal ray. There is a conspicuous, white, ven
tral blotch, which is much larger than the eye-diameter. Morphometrics (mm)
CIFI 1715
CIFI 1715
CIFI 1801
CIFI 1820
Total length
251
230
278
271
Standard length
208
192
224
222
Head length
88.66
82.21
96.06
93.82
Upper jaw length
39.47
37.06
42.25
43.97
Orbit diameter
15.78
15.35
16.03
16.24
Postorbital length
48.54
45.47
56.19
52.47
Interorbital width
14.70
12.62
16.13
15.84
Snout length
22.42
21.01
25.81
26.95
Suborbital length
9.53
8.55
11.20
11.48
Body depth
81.91
74.97
96.90
87.30
Caudal peduncule length
33.07
28.96
31.29
28.65
Caudal peduncule depth
32.58
30.70
33.78
34.92
Caudal fin base length
40.32
36.95
38.26
36.42
Dorsal fin base length
94.08
87.82
97.99
98.45
Anal fin base length
34.79
31.08
37.55
36.39
Predorsal length
84.15
81.70
93.45
96.12
Preanal length
144
134
156
15
Prepectoral length
84.11
74.2
87.96
85.13
Pectoral fin length
52.60
51.01
64.61
59.72
Prepelvic length
89.18
79.97
96.93
89.92
Pelvic fin length
42.61
41.66
46.10
46.51
Meristics
Dorsal fin spines
VIII
VIII
VIII
VII
Dorsal fin rays
11
13
13
13
Anal fin spines
III
III
III
III
Anal fin rays
7
7
7
7
Pectoral fin rays
16
15
16
17
Caudal fin rays
16
17
17
17
Pored scales lateral line
49
48
48
50
Lateral line scales
85
81
79
83
Scales around the caudal peduncle
47
45
45
48
Upper gill rakers
6
7
6
5
Lower gill rakers
15
15
15
15 Authors’ Contributions Hoese HD, Moore RH (1998) Fishes of the Gulf of Mexico, Texas,
Louisiana, and adjacent waters. Texas A&M University Press,
College Station, USA, 416 pp. Conceptualization: LFDM. Data curation: VREA. For
mal analysis: LFDM, ELS, MLJB, VREA. Visualiza
tion: ELS, VREA. Writing – original draft: LFDM, ELS. Writing – review and editing: LFDM, MLJB, VREA. Hubbs CL, Lagler KF (1958) Fishes of the Great Lakes region. Univer
sity of Michigan Press, Ann Arbor, USA, 213 pp. Instituto Nacional de Pesca (1976) Catálogo de peces marinos mexi
S
t
í d i d
t i
i
I
tit t
N
i
l d Instituto Nacional de Pesca (1976) Catálogo de peces marinos mexi
canos. Secretaría de industria y comercio, Instituto Nacional de
Pesca. Mexico City, Mexico, 462 pp.i Discussion https://doi.org/10.25268/bimc.invemar.2019.48.1.759 arque Nacional Natural Corales de Profundidad, usando buceo Colás-Marrufo T, Brulé T, Déniel C (1998) Análisis preliminar de
las capturas de meros realizados a través de unidades de la flota
mayor en el sureste del Golfo de México. Proceedings of the Gulf
and Caribbean Fisheries Institute 50: 780–803. Its occurrence in Punta Puntilla is probably due to
the dispersal of larvae, which influences the presence of
adults on coral reefs. Kendall and Fahay (1979) captured
larvae of G. hispanus in North Carolina, which represents
the northernmost record. However, in Mexico, there is a
gap in the knowledge of deep communities compared to
other countries where this species occurs (Simon et al. 2016; Baldwin et al. 2018; Chasqui-Velasco and González-
Corredor 2019) due to poor sampling efforts and the diffi
culty sampling mesophotic and rariphotic environments. Our new data of G. hispanus are an important contri
bution to understanding Mexico’s mesophotic and rari
photic ichthyofauna, and confirm the presence of this
species in the southwestern Gulf of Mexico. Flores–Coto C, Espinosa–Fuentes ML, Zavala García F, Sanvicente
Añorve L (2009) Ictioplancton del sur del Golfo de México: un
compendio. Hidrobiológica 19 (1): 49–76. Fricke R, Eschmeyer WN, Fong JD (2020) Species by family/sub
family. Version 2020. http://researcharchive.calacademy.org/
research/ichthyology/catalog/SpeciesByFamily.asp. Accessed on:
2021-1-28. Garcia-Júnior J, Nóbrega MF, Lins-Oliveira JE (2015) Coastal fishes
of Rio Grande do Norte, north-eastern Brazil, with new records. Check List 11 (3): 1659. https://doi.org/10.15560/11.3.1659 Check List 11 (3): 1659. https://doi.org/10.15560/11.3.1659 González-Gándara C (2020) Composition, distribution and relative
abundance of fishes linked to fringing reefs of southern Veracruz,
Gulf of Mexico. GSC Biological and Pharmaceutical Sciences 12
(2): 217–228. https://doi.org/10.30574/gscbps.2020.12.2.0268 (2): 217–228. https://doi.org/10.30574/gscbps.2020.12.2.0268 Guitart DJ (1977) Sinopsis de los peces marinos de Cuba. Tomo III. Academia de Ciencias de Cuba, Instituto de Oceanología, Ha
vana, Cuba, 325–608. Heemstra PC, Anderson WD, Lobel PS (2002) Serranidae. Groupers
(seabasses, creolefish, coney, hinds, hamlets, anthiines, and soap
fishes). In: Carpenter KE (Ed.) The living marine resources of the
Western Central Atlantic. Vol. 2: bony fishes, part 1 (Acipenseri
dae to Grammatidae). FAO Species Identification Guide for Fish
ery Purposes and American Society of Ichthyologist and Herpe
tologists Special Publication, Rome, Italy, 601–1374. Acknowledgements We thank the local fishermen, A. Campos Pérez and E. Campos Vicente, who collected specimens for us, and
the community of Salinas Roca Partida for its help dur
ing with the fieldwork. We also acknowledge the finan
cial support of SNI-CONACyT. This work was financed
by the project PAPIIT-UNAM IA207820. Heemstra PC, Randall JE (1993) FAO species catalogue. Vol. 16. Groupers of the world (family Serranidae, subfamily Epinepheli
nae). An annotated and illustrated catalogue of the grouper, rock
cod, hind, coral grouper and lyretail species known to date. FAO
Fisheries Synopsis 125 (16): 1–382. Discussion Gonioplectrus hispanus occurs in moderately deep
waters down to depths of 460 m (Oliveira and Barbosa
2010; Baldwin et al. 2018) and co-exists with other ser
ranids, usually at the periphery of tropical reefs (Bullock
and Smith 1991). Specifically, it lives in coral reefs of the
rariphotic zone (139–309 m; Baldwin et al. 2018). This
species has no value as human food due to its small size,
but it is often used as ornamental fish in the aquarium
trade (Oliveira and Barbosa 2010). Its capture is difficult
below 100 m, and barriers, such as thermoclines at these
depths, function as biological boundaries (Heemstra and
Randall 1993; Oliveira and Barbosa 2010). The species has been recorded sporadically along 1302 Check List 17 (5) the Caribbean coasts of Colombia and Venezuela, as
well as in the Caribbean Islands (Cervigón 1971; Gui
tart 1977; Thompson and Munro 1978; Betancur-R. et al. 2001; Baldwin et al. 2018). It has been recently recorded
in Brazilian waters as a result of deep-sea fishing and
mesophotic exploration (Oliveira and Barbosa 2010; Gar
cia-Júnior et al. 2015; Sampaio et al. 2017). Bullock and
Smith (1991) reported this species in the north-western
Atlantic, and in Mexico, it has been detected in the south
ern Gulf of Mexico, between Laguna de Términos and
the Yucatán Peninsula, although the location was not
specified (Lara-Domínguez et al. 1993). Despite recent
ichthyofaunal surveys in reef areas in the south-eastern
Gulf of Mexico and on the Yucatán continental shelf,
G. hispanus has not been detected (Colás-Marrufo et al. 1998; Robertson et al. 2019; González-Gandara 2020). letin of the Florida State Museum, Biological Sciences Series 2
(8): 223–318. letin of the Florida State Museum, Biological Sciences Series 2
(8): 223–318. Briggs JC, Hoese HD, Hadley WF, Jones RS (1964) Twenty-two new
fish records for the north–western Gulf of Mexico. Texas Journal
of Science 16 (1): 113–116. Bullock LH, Smith GB (1991) Seabasses (Pisces: Serranidae). Mem
oirs of the Hourglass Cruises 8 (2): 1–243. Cervigón F (1971) Los peces marinos de Venezuela. Complemento i Cervigón F (1971) Los peces marinos de Venezuela. Complemento
II. Contribuciones Científicas Universidad de Oriente Porlamar
1: 5–36. Chasqui-Velasco LH, González-Corredor, JD (2019) Peces registrados
en ambientes mesofóticos de Bajo Frijol, la porción más somera del
Parque Nacional Natural Corales de Profundidad, usando buceo
técnico CCR. Boletín de Investigaciones Marinas y Costeras 48
(1): 89–101. References Kendall AW Jr., Fahay MP (1979) Larva of the serranid fish Gonio
plectrus hispanus with comments on its relationships. Bulletin of
Marine Science 29 (1): 117–121. Baldwin CC, Tornabene L, Roberston DR (2018) Below the meso
photic. Scientific Report 8: 4920. https://doi.org/10.1038/s41598-
018-23067-1 Lara–Domínguez AL, Arreguín–Sánchez F, Alvarez–Guillen H
(1993) Biodiversidad y el uso de recursos naturales: las comuni
dades de peces en el sur del Golfo de México. Revista de la Socie
dad Mexicana de Historia Natural 44: 345–385. Baqueiro CE, Mendez RL (1994) Artificial reefs: an alternative to en
hance Mexican littoral commercial fisheries. Bulletin of Marine
Science 55 (2–3): 1014–1020. McEachran JD, Fechhelm JD (2005) Fishes of the Gulf of Mexico. Vol. 2: Scorpaeniformes to Tetraodontiformes. University of Texas,
Austin, USA, 1004 pp.i Betancur-R R, Acero PA, Mejía-Ladino LM (2001) El pez serránido
Gonioplectrus hispanus (Epinephelinae: Epinephelini) en Colom
bia: primer registro para las costas continentales caribeñas. Bo
letín Ecotrópica 35: 25–30.i Moore RH (1975) New records of three marine fish from Texas waters
with notes on some additional species. The Texas Journal of Sci Briggs, J.C. 1958. A list of Florida fishes and their distribution. Bul Del Moral-Flores et al. | Gonioplectrus hispanus from the south-western Gulf of Mexico Del Moral-Flores et al. | Gonioplectrus hispanus from the south-western Gulf of Mexico Del Moral-Flores et al. | Gonioplectrus hispanus from the south-western Gulf of Mexico 1303 ence 26 (1–2): 155–163. (2): 12–17. https://doi.org/10.32360/acmar.v50i2.31142 Oliveira VS, Barbosa JM (2010) Ocorrência do “Spanish flag” Gonio
plectrus hispanus (Cuvieri, 1828) na costa de Pernambuco, Brasil. Revista Brasileira de Engenharia de Pesca 5 (1): 93–97. https://doi. org/10.18817/repesca.v5i1.276 i Schaldach WJ Jr, Huidobro Campos L, Espinosa Pérez H (1997) Peces
marinos. In: González Soriano E, Dirzo R, Vogt RC (Eds.) Histo
ria natural de los Tuxtlas. Instituto de Biología, Comisión Nacio
nal para el Conocimiento y Uso de la Biodiversidad, Universidad
Nacional Autónoma de México, Mexico, 571–588. Parenti P, Randall JE (2020) An annotated checklist of the fishes of
the family Serranidae of the world with description of two new re
lated families of fishes. FishTaxa 15 (1): 1–170. Nacional Autónoma de México, Mexico, 571–588. Simon T, Pinheiro HT, Moura RL, Carvalho-Filho A, Rocha L Simon T, Pinheiro HT, Moura RL, Carvalho-Filho A, Rocha LA, Mar
tins AS, Mazzei E, Francini-Filho RB, Amado-Filho GM, Joyeux
J-C (2016) Mesophotic fishes of the Abrolhos Shelf, the largest
reef ecosystem in the South Atlantic. Journal of Fish Biology 89
(1): 990–1001. https://doi.org/10.1111/jfb.12967 Robertson DR, Pérez-España H, Domínguez-Domínguez O, Estapé
CJ, Estapé AM (2019) An update to the inventory of shore–fishes
from the Parque Nacional Sistema Arrecifal Veracruzano, Ve
racruz, México. ZooKeys 882: 127–157. https://doi.org/10.3897/
zookeys.882.38449 Smith GB (1976) Ecology and distribution of eastern Gulf of Mexico
reef fishes. Florida Marine Research Publications 19: 1–178. Sampaio CLS, Lopes PRD, Oliveira-Silvia JT, Malafaia P, Olavo G
(2017) Registros de Gonioplectrus hispanus (Cuvier, 1828) (Acti
nopterygii: Epinephelidae) no estado da Bahia (nordeste do Bra
sil), océano Atlantico occidental. Arquivos de Ciencias do Mar 50 Thompson R, Munro J (1978) Aspects of the biology and ecol
ogy of Caribbean reef fishes: Serranidae (hinds and grou
pers). Journal of Fish Biology 12 (2): 115–146. https://doi. org/10.1111/j.1095-8649.1978.tb04158.x
|
https://openalex.org/W4230288712
|
https://www.researchsquare.com/article/rs-23362/v1.pdf
|
English
| null |
Transcriptomic analysis of a-linolenic acid content and biosynthesis in Paeonia ostii fruits and seeds
|
Research Square (Research Square)
| 2,020
|
cc-by
| 14,743
|
Results Gas chromatograph-mass spectrometry indicated that ALA content was highest in the kernel, moderate in
the testa, and lowest in the pericarp. Therefore, we used RNA-sequencing to compare ALA synthesis
among these three tissues. We identified 227,837 unigenes, with an average length of 755 bp. Of these,
1371 unigenes were associated with lipid metabolism. The fatty acid (FA) biosynthesis and metabolism
pathways were significantly enriched during the early stages of oil accumulation in the kernel. ALA
biosynthesis was significantly enriched in parallel with increasing ALA content in the testa, but these
metabolic pathways were not significantly enriched during pericarp development. By comparing unigene
transcription profiles with patterns of ALA accumulation, specific unigenes encoding crucial enzymes and
transcription factors (TFs) involved in de novo FA biosynthesis and oil accumulation were identified. Specifically, the bell-shaped expression patterns of genes encoding SAD, FAD2, FAD3, PDCT, PDAT, OLE,
CLE, and SLE in the kernel were similar to the patterns of ALA accumulation in this tissue. Genes
encoding BCCP, BC, KAS I– III, and FATA were also upregulated during the early stages of oil
accumulation in the kernel. In the testa, the upregulation of the genes encoding SAD, FAD2, and FAD3 was
followed by a sharp increase in the concentrations of ALA. In contrast, these genes were minimally
expressed (and ALA content was low) throughout pericarp development. Conclusions In this study, we used three tissues with high, moderate, and low ALA concentrations as an exemplar
system in which to compare tissue-specific ALA accumulation mechanism in P. ostii. The genes and TFs
identified herein might be useful targets for future studies of ALA accumulation in the tree peony. This
study also provides a framework for future studies of FA biosynthesis in other oilseed plants. Background Paeonia ostii is a potentially important oilseed crop because its seed yield is high and the seeds are rich
in α-linolenic acid (ALA). However, the molecular mechanisms underlying ALA biosynthesis during seed
kernel, seed testa, and fruit pericarp development in this plant are unclear. We used transcriptome data to
address this knowledge gap. Version of Record: A version of this preprint was published at BMC Genomics on April 23rd, 2021. See the
published version at https://doi.org/10.1186/s12864-021-07594-2. Version of Record: A version of this preprint was published at BMC Genomics on April 23rd, 2021. See the
published version at https://doi.org/10.1186/s12864-021-07594-2. Page 1/34 Background Edible oils for human consumption are mainly derived from plant seeds, which contain several types of
fatty acids (FAs). Among the FAs, linoleic acid (LA; C18:2Δ9,12, a ω-6 FA) and α-linolenic acid (ALA;
C18:3Δ9,12,15) are essential dietary nutrients for humans; these FAs cannot be synthesized independently
by humans [1]. ω-6 FA and ALA not only exert a hypocholesterolemic effect against coronary heart Page 2/34 Page 2/34 disease and hypertension when used as human dietary supplements, but also are critical for fetal
development and breastfeeding support [1, 2]. It has been suggested that a ratio of ω-6 to ω-3 FAs < 5 is
optimal for human beings [3], and several sources have testaulated that ancient human diets had ω-6 to
ω-3 FA ratios of ~ 1 [4]. Indeed, the high ratio of ω-6 to ω-3 FAs (~ 15:1) in the typical modern human diet
is thought to be a major factor contributing to the high rates of cardiovascular disease in modern human
societies [5]. One explanation for this imbalance is that the bulk of the oil seed crops consumed by
modern humans, including soybean, peanut, maize, sunflower, and rape, have relatively low levels of ω-3
FAs, such as ALA [5]. However, tree peony seed oil, which is rich in ALA, has a ω-6 to ω-3 FA ratio of < 1.0
[6]. For this reason, tree peony seed oil has been recognized as a high-quality edible oil, and was identified
as a new food resource by the Chinese Ministry of Health in 2011 [7]. The tree peony (family Paeoniaceae, genus Paeonia, section Moutan DC), which was historically known
as "The King of Flowers", is a perennial shrub that is widely distributed in China [8]. There are nine wild
species in section Moutan DC, which differ with respect to preferred habitat, but all rich in unsaturated
FAs (>90%) and ALA (26.7–50%) [6, 9]. To date, transcriptomic, proteomic, and microRNA sequencing
have been used to investigate the mechanisms underlying ALA synthesis in tree peony seeds [10–14]. Previous studies have focused on enzymes that are key to ALA biosynthesis in various tree peony
species, including FAD3 in P. rockii, P. potaninii and P. lutea seed [15]; FAD2 and FAD8 in P. ostii [10]; and
ACCase, FATA, LPCAT, FADs, and DGAT in the developing endosperm of P. ostii var. lishizhenii [11]. Wang
et al. Background [14] identified 115 genes and 24 proteins associated with the ALA metabolism in P. ostii seeds. However, despite these studies of FA metabolism in the tree peony, additional functional genes require
further exploration. In oil seed plants, stored lipids accumulate primarily as a result of FA synthesis in the plastids and
triacylglycerol (TAG) assembly in the endoplasmic reticulum (ER). Acetyl-coenzyme A (CoA) is the unique
building block used for FA production in plants [16]. In the plastid, de novo FA synthesis begins when
acetyl-CoA is converted into malonyl-CoA by the acetyl-CoA carboxylase (ACC); malonyl-CoA is then either
elongated to C16 and C18 by a series of enzymes or desaturated to C18:1-ACP by delta-9-stearoyl-ACP
desaturase (SAD) [17, 18]. In most oilseed plants, more than 95% of the newly-synthesized FAs (primarily
C18:1) are exported from plastids to the ER as CoA thioesters[19, 20]. The biosynthesis of
polyunsaturated FAs (PUFAs) primarily relies on the further desaturation of C18:1 by omega-6 fatty acid
desaturase (omega-6 FAD) and omega-3 FAD in the plastid or ER [16]. FAs, both those synthesized de
novo or those have been modified through acyl editing, are assembled into glycerol-3-phosphate (G3P) to
form TAG [21]. Generally, in plants and eukaryotic algae, TAG is mainly synthesized along the acyl-CoA-
dependent Kennedy pathway and the acyl-CoA-independent pathway. Once TAGs have been synthesized,
they are surrounded by a layer of phospholipids and amphipathic proteins, and then released to form oil
bodies in the cytoplasm. The FA metabolic pathway is complex, and the details of this pathway differ substantially among plant
species and tissue types. In castor beans and oil palms, transcriptome analyses showed that differences
in FAs among seed tissues were associated with certain genes and transcription factors (TFs) [22, 23]. Page 3/34 Page 3/34 Interestingly, previous studies have showed that FA composition and relative abundance differ among
wild P. ostii trees [6, 9], as well as among seed kernels and testas [6, 24]. This implies that FA synthesis
and TAG assembly in developing tree peony seeds are differentially regulated among species. Zhang et
al. [13, 15] compared FAD transcription (i.e., SAD, FAD2, and FAD3) among three species of tree peonies,
and found that FAD transcription was greater in P. rockii seeds than in P. potaninii and P. lutea seeds. In
addition, various transcriptomes of P. ostii seeds or endosperms are also available [10, 11, 14]. Background However,
to date, no integrated analysis of the developing pericarp, seed coat, and endosperm in P. ostii has been
performed. In this study, we chose P. ostii as an exemplar tree peony species primarily because, compared with other
tree peonies, P. ostii has higher seed yields and ALA content. This species is also suitable for large-scale
cultivation south of the Yangtze River, China. In this study, we aimed to use comparative transcriptomics
to assess the expression levels of certain genes associated with ALA accumulation during the
development of three P. ostii tissues: the seed kernel, the seed testa, and the fruit pericarp. The
identification of candidate genes that may affect levels of ALA accumulation will provide molecular tools
for future genetic modifications, aimed at increasing the diversity and yield of seed oils produced by the
tree peony and other oilseed plants. In this study, we chose P. ostii as an exemplar tree peony species primarily because, compared with other
tree peonies, P. ostii has higher seed yields and ALA content. This species is also suitable for large-scale
cultivation south of the Yangtze River, China. In this study, we aimed to use comparative transcriptomics
to assess the expression levels of certain genes associated with ALA accumulation during the
development of three P. ostii tissues: the seed kernel, the seed testa, and the fruit pericarp. The
identification of candidate genes that may affect levels of ALA accumulation will provide molecular tools
for future genetic modifications, aimed at increasing the diversity and yield of seed oils produced by the
tree peony and other oilseed plants. Morphological changes in developing P. ostii fruits and seeds At the study location (south of the Yangtze River), P. ostii fruit and seed development lasted about 120
days. P. ostii fruits and seeds grew rapidly between 7 and 49 days after fertilization (DAF), and at 49 DAF
were almost as large as the mature forms (Fig. 1a–c). The fruit peel was green during the early stages of
development, but began to turn yellow at about 70 DAF (Fig. 1a). Examination of the seeds in longitudinal
section indicated that embryogenesis and seed-filling occurred from 0 to 70 DAF; cotyledon embryos were
obvious at 70 DAF. Between 70 and 119 DAF, the seed coat became thinner and browner as compared to
the period from 49 to 70 DAF (Fig. 1d). At 119 DAF, the seeds were mature (Fig. 1b, d). Dynamic changes in FA composition in the pericarp, kernel, and testa All five major FAs [ALA, LA, oleic acid (OA, C18:1Δ9), stearic acid (SA, C18:0) and palmitic acid (PA, C16:0)]
were detected in the pericarp, testa, and kernel samples taken between 21 and 119 DAF (Fig. 2). The three
most abundant UFAs during kernel development were OA, LA, and ALA. In the kernel, OA, LA, and ALA
concentrations were 2.4±0.08 mg/g, 6.35±0.34 mg/g, and 2.5±0.24 mg/g, respectively, at 35 DAF,
increasing to 58.79±2.21 mg/g, 92.56±4.98 mg/g, and 134.29±2.23 mg/g, respectively, at 77 DAF
(Fig. 2a). After 77 DAF, OA, LA, and ALA concentrations decreased gradually, to 52±0.73 mg/g,
77.36±1.39 mg/g, and 131.26±1.68 mg/g, respectively, at 84 DAF, and to 41.96±1.21 mg/g,
58.56±1.94 mg/g, and 94.96±3.64 mg/g, respectively, at 119 DAF (Fig. 2a). Although the concentrations
of all five FAs were similar throughout most of testa development (21–63 DAF and 77–119 DAF; Fig. 2b), Page 4/34 Page 4/34 ALA, LA, and OA peaked sharply at 70 DAF, with ALA concentration showing the greatest increase (to
20.27 ± 3.08 mg/g). FA concentrations fluctuated but remained low throughout pericarp development; PA
and LA were the most abundant FAs (Fig. 2c). In all three tissues, SA concentrations remained
consistently low. ALA, LA, and OA peaked sharply at 70 DAF, with ALA concentration showing the greatest increase (to
20.27 ± 3.08 mg/g). FA concentrations fluctuated but remained low throughout pericarp development; PA
and LA were the most abundant FAs (Fig. 2c). In all three tissues, SA concentrations remained
consistently low. Unigene Assembly, Analysis, And Quantification Of Gene Expression The transcriptomes of the kernel, testa, and pericarp samples taken at 35, 49, 63, 77, 91, and 119 DAF
(T1–T6) from two separate trees (specimens CS0009 and CS0016) were sequenced. Across the 36
samples, we obtained approximately 49.69 M reads per sample (Table 1). A total 227,837 unigenes were
detected, with an average length of 755 bp (Additional file 1: Table S1). The heatmap of the Pearson
correlation coefficients of gene expression levels between pairs of genes showed that the gene
expression levels were significantly correlated throughout pericarp development and during rapid oil
accumulation in the kernel (Additional file 2: Figure S1). Across the six developmental stages, more genes
were co-expressed in all three tissues than were expressed only in one tissue (Additional file 3: Figure S2). In addition, more genes were expressed specifically in the pericarp than in either the kernel or the testa. More genes were specifically expressed in the seed kernel at 49 DAF than at any other stage(Additional
file 3: Figure S2). Page 5/34 Table 1 Sequencing statistics for the 36 samples taken during the development of Paeonia ostii seeds. Unigene Assembly, Analysis, And Quantification Of Gene Expression Sample
Total Raw
Reads(Mb)
Total Clean
Reads(Mb)
Total Clean
Bases(Gb)
Clean
Reads
Q20(%)
Clean
Reads
Q30(%)
Clean
Reads
Ratio(%)
CS0009_p_35
65.12
51.42
7.71
97.09
91.51
78.96
CS0009_p_49
53.48
41.89
6.28
97.08
91.53
78.32
CS0009_p_63
79.5
63.54
9.53
97.22
91.84
79.92
CS0009_p_77
85.27
63.95
9.59
96.95
91.22
74.99
CS0009_p_91
75.27
59.6
8.94
97.1
91.58
79.18
CS0009_p_119
79.37
58.11
8.72
97
91.2
73.22
CS0009_t_35
42.71
33.45
5.02
97.35
91.95
78.32
CS0009_t_49
68.7
51.46
7.72
96.62
90.23
74.9
CS0009_t_63
56.31
43.43
6.51
97.09
91.59
77.12
CS0009_t_77
56.61
41.72
6.26
96.93
91.16
73.69
CS0009_t_91
65.53
48.97
7.35
96.94
91.21
74.73
CS0009_t_119
49.76
41.65
6.25
98.13
94.01
83.71
CS0009_k_35
51.22
40.63
6.1
97.12
91.56
79.34
CS0009_k_49
55.96
44.77
6.72
97.22
91.84
80.01
CS0009_k_63
80.62
61.78
9.27
96.94
91.19
76.63
CS0009_k_77
56.87
42.97
6.45
96.99
91.24
75.55
CS0009_k_91
66.07
48.77
7.32
96.86
91.06
73.82
CS0009_k_119
58.84
43.22
6.48
96.76
90.8
73.45
CS0016_p_35
64.68
49.97
7.49
96.95
91.25
77.25
CS0016_p_49
70.79
55.9
8.38
97.05
91.41
78.95
CS0016_p_63
59.62
46.02
6.9
96.92
91.18
77.19
CS0016_p_77
72.86
56.46
8.47
96.99
91.3
77.48
CS0016_p_91
81.99
64.04
9.61
96.95
91.24
78.1
CS0016_p_119
42.01
31.77
4.77
96.89
90.75
75.62
CS0016_t_35
104.07
83.87
12.58
97.81
93.25
80.59
CS0016_t_49
66.34
50.45
7.57
97.06
91.51
76.04 Page 6/34 Page 6/34 Sample
Total Raw
Reads(Mb)
Total Clean
Reads(Mb)
Total Clean
Bases(Gb)
Clean
Reads
Q20(%)
Clean
Reads
Q30(%)
Clean
Reads
Ratio(%)
CS0016_t_63
61.83
48.98
7.35
97.06
91.39
79.22
CS0016_t_77
73.54
57.14
8.57
97.15
91.65
77.71
CS0016_t_91
61.93
47.81
7.17
97.19
91.81
77.2
CS0016_t_119
109.95
81.97
12.3
97.34
92.47
74.56
CS0016_k_35
52.81
41.03
6.15
97
91.31
77.69
CS0016_k_49
61.03
48.59
7.29
97.2
91.78
79.61
CS0016_k_63
52.57
39.83
5.97
96.97
91.28
75.76
CS0016_k_77
57.55
48.62
7.29
98.16
94.06
84.47
CS0016_k_91
50.84
37.42
5.61
96.84
90.95
73.6
CS0016_k_119
47.16
34.85
5.23
96.75
90.75
73.9
Ontology and KEGG pathway enrichment of the differentially expressed genes Ontology and KEGG pathway enrichment of the differentially expressed genes Ontology and KEGG pathway enrichment of the differentially expressed genes The up- and downregulated genes at each of the six time points in the kernel, testa, and pericarp are
shown in Additional file 4: Table S2. We found that the proportion of genes up- and downregulated varied
among tissues and time points. The heatmap of the hierarchical DEG clusters indicated that more genes
were differently expressed during the first three stages of kernel development (Additional file 5: Figure
S3). We then determined which gene ontology (GO) functions were enriched in the DEGs. GO functions
are grouped in three categories: molecular function, cell component and biological process. At 49 DAF,
twice as many genes in the kernel and testa were enriched in metabolic process as compared to the
pericarp (Additional file 6: Figure S4). In the kernel, 49 DAF was a period of rapid oil accumulation
(Fig. 2a). Consistent with this, the genes differentially expressed in the kernel at 49 DAF as compared to
35 DAF were primarily associated with the lipid metabolism (226 DEGs) and the carbohydrate
metabolism (309 genes; Fig. 3a). Other metabolic pathways, such as fatty acid metabolism, fatty acid
biosynthesis, linoleic acid metabolism, glycosphingolipid biosynthesis-ganglioseries, glycosphingolipid
synthesis-globo and isoglobe series, starch and sucrose metabolism were also significantly enriched in
these DEGs (Fig. 3b). Most of the metabolic pathways were overrepresented in the genes downregulated
between 49 DAF and 35 DAF, including the linoleic acid metabolism pathway; linoleic acid is the substrate
of ALA synthesis (Fig. 3c). However, the fatty acid metabolism, fatty acid biosynthesis, pyruvate
metabolism, and plant hormone signal transduction pathways were overrepresented in the genes
upregulated between 49 DAF and 35 DAF (Fig. 3c). With respect to the genes differently expressed
between 63 DAF and 49 DAF, more pathways were significantly enriched in the downregulated genes than
in the upregulated genes (Fig. 3d). Interestingly, the fatty acid metabolism and fatty acid biosynthesis Page 7/34 Page 7/34 pathways, which were significantly enriched at 49 DAF, were not significantly enriched at any other time
point (Additional file 7: Figure S5). In the testa, 187 genes associated with the lipid metabolism were differentially expressed between 49 In the testa, 187 genes associated with the lipid metabolism were differentially expressed between 49
DAF and 35 DAF. Ontology and KEGG pathway enrichment of the differentially expressed genes Pathways related to the lipid metabolism (i.e., glycerolipid metabolism,
glycerophospholipid metabolism, glycosphingolipid biosynthesis-ganglio series, and cutin/suberine and
wax biosynthesis) and to the carbohydrate metabolism (i.e., starch/sucrose metabolism and galactose
metabolism) were significantly enriched in these DEGs (Fig. 4a). The glycerolipid metabolism pathway
was overrepresented in similar numbers of up- and downregulated DEGs at 49 DAF (Fig. 4b). Several
other lipid-associated pathways (i.e., glycerophospholipid metabolism, glycosphingolipid biosynthesis-
ganglio series, and steroid biosynthesis) were more enriched in the upregulated DEGs than in the
downregulated DEGs at 49 DAF (Fig. 4b). Similarly, pathways related to starch accumulation and sugar
synthesis (i.e., fructose/mannose metabolism and galactose metabolism) were also enriched in more
upregulated than downregulated DEGs at 49 DAF (Fig. 4b). This might be due to the intensive membrane
lipid synthesis, seed coat thickening, and dry matter accumulation that occurs during this stage in the
testa. Compared with 35 DAF, 287 genes associated with the lipid metabolism were differently expressed
at 63 DAF in the testa; the metabolic pathways significantly enriched in these DEGs included fatty acid
elongation, glycerolipid metabolism, sphingolipid metabolism, glycosphingolipid biosynthesis-ganglio
series, and cutin/suberine and wax biosynthesis (Fig. 4c). At 63 DAF, most of the DEGs were associated
with the alpha linolenic acid pathway; in this pathway, the number of upregulated genes was similar to
the number of downregulated genes (Fig. 4d). No lipid metabolism pathways were enriched at any other
stage of testa development (Additional file 8: Figure S6). DAF and 35 DAF. Pathways related to the lipid metabolism (i.e., glycerolipid metabolism, In the pericarp, certain lipid metabolism pathways, including glycerolipid metabolism, fatty acid
elongation, sphingolipid metabolism, and cutin/suberine and wax biosynthesis, were similarly enriched in
all DEGs in different stages (Additional file 9: Figure S7 and Additional file 10: Figure S8). Expression Patterns Of Unigenes Associated With Oil Accumulation Based on the functional annotations of the DEGs, we identified 1373 unigenes associated with lipid
metabolism (Additional file 11: Table S3). These unigenes were associated with 10 metabolic pathways:
de novo plastid FA synthesis, elongation, desaturation and export (138 unigenes); triacylglycerol
biosynthesis and eukaryotic phospholipid synthesis & editing (176 unigenes); prokaryotic and eukaryotic
galactolipid, sulfolipid, and phospholipid synthesis (25 unigenes); sphingolipid biosynthesis (58
unigenes); fatty acid elongation, wax biosynthesis, cutin synthesis, and suberin synthesis and transport
(251 unigenes); triacylglycerol and fatty acid degradation (99 unigenes); phospholipid signaling (108
unigenes); lipid trafficking (27 unigenes); oxylipin metabolism (39 unigenes); and mitochondrial fatty acid
synthesis (43 unigenes) (Additional file 12: Table S4). Of these 1373 unigenes, 314 were associated with FA and TAG biosynthesis (Additional file 12: Table S4). Specifically, 13 unigenes were associated with the pyruvate dehydrogenase complex (PDHC), which
catalyzes the oxidative decarboxylation of pyruvate to produce acetyl-CoA. The PDHC contains three Page 8/34 Page 8/34 subunits: pyruvate dehydrogenase (PDH, 10 unigenes), dihydrolipoyl acyltransferase (DHLAT, 2
unigenes), and dihydrolipoamide dehydrogenase (LPD, 1 unigene). These unigenes were highly expressed
throughout the development of the pericarp, testa, and kernel. In addition, 25 unigenes encoded subunits
of ACC, a multi-subunit enzyme that includes biotin carboxylase (BC), the biotin carboxyl carrier protein
(BCCP), and carboxyltransferase (CT). Finally, seven unigenes were homologous to α-CT; one unigene
was homologous to β-CT; 13 unigenes were homologous to BC; and four unigenes were homologous to
BCCP (Additional file 12: Table S4). In particular, CL15202, homologous to α-CT, was highly expressed
across all three tissues, but was most strongly upregulated in the kernel. Similarly, CL9207.Contig2,
homologous to homomeric ACC (HmACC), was also highly expressed during the development of all three
tissues, but was most highly expressed in the testa. CL16462 (homologous to α-CT) and unigene 719
(homologous to β-CT) were strongly upregulated in the pericarp, but expressed only at low levels in the
kernel and the testa. In addition, homologs to BC (CL7345.contig8) and BCCP (CL18970.contig3 and
unigene17464) were significantly upregulated in the kernel at 35 and 49 DAF, as compared to later
developmental stages (63–119 DAF); these genes were expressed only at low levels throughout the
development of the testa and pericarp (Additional file 13: Figure S9). Three unigenes were associated
with the catalysis of the malonyl-ACP elongation cycle: CL8489.Contig2, associated with 3-ketoacyl-ACP
synthase isoform I (KAS I); CL1611.Contig2, associated with KAS II; and unigene32463, associated with
KAS III. Expression Patterns Of Unigenes Associated With Oil Accumulation In the kernel, unigene32372 expression increased from 35–49 DAF, and then decreased
fairly steadily until the end of the experiment (Fig. 5b). Three unigenes encoding
lysophosphatidylcholineacyltransferase (LPCAT) (unigene39796, CL18110.Contig2, and
CL18110.Contig24) and two unigenes encoding lysophosphatidylethanolamineacyltransferase (LPEAT)
(CL10703.Contig3 and CL20149.Contig2) were identified as DEGs. We also identified 19 unigenes
encoding PLD. In the kernel, the expression profile of one of these unigenes (CL1337.Contig6) resembled
a bell-shaped curve, peaking at 63 DAF (Fig. 5b). Other phospholipase D (PLD) genes were also identified
as DEGs, but did not exhibit a smooth peak in expression. cholinephosphotransferase (PDCT) (unigene32372), was significantly differentially expressed among
these tissues. In the kernel, unigene32372 expression increased from 35–49 DAF, and then decreased
fairly steadily until the end of the experiment (Fig. 5b). Three unigenes encoding lysophosphatidylcholineacyltransferase (LPCAT) (unigene39796, CL18110.Contig2, and
CL18110.Contig24) and two unigenes encoding lysophosphatidylethanolamineacyltransferase (LPEAT)
(CL10703.Contig3 and CL20149.Contig2) were identified as DEGs. We also identified 19 unigenes
encoding PLD. In the kernel, the expression profile of one of these unigenes (CL1337.Contig6) resembled
a bell-shaped curve, peaking at 63 DAF (Fig. 5b). Other phospholipase D (PLD) genes were also identified
as DEGs, but did not exhibit a smooth peak in expression. We identified 45 unigenes associated with TAG biosynthesis. Of these, 16 unigenes encoded glycerol-3-
phosphate acyltransferase (GPAT), 10 unigenes encoded lysophosphatidic acid acyltransferase (LPAAT),
10 unigenes encoded phosphatidate phosphatase (PAP), and 4 unigenes encoded diacylglycerol
acyltransferase (DGAT). Specifically, four unigenes were strongly upregulated in the kernel at 49 DAF as
compared to 35 DAF: unigene42999 encoding GPAT; CL1842.Contig1 and CL1842.Contig2 encoding
LPAAT; CL4004.Contig1 and CL18619.Contig1 encoding PAP; and CL5087.Contig1 encoding DGAT
(CL5087.Contig1) (Additional file 13: Figure S9). This pattern was observed in neither the testa nor the
pericarp. Five unigenes encoding phospholipid:diacylglycerol acyltransferase (PDAT) were identified as
DEGs. One of these (CL7876.Contig4) was highly expressed throughout pericarp and testa development,
but was downregulated at 49 DAF as compared to 35 DAF in the kernel. In the kernel, the expression of
another PDAT-encoding unigene (unigene24505) increased dramatically from 35–49 DAF and then again
from 77–91 DAF (Fig. 5b). This gene was highly expressed in neither the testa nor the pericarp
(Additional file 13: Figure S9). We identified 29 unigenes associated with the formation of oil bodies in
the cytoplasm that were also DEGs. Of these unigenes, six encoded oleosin (OLE), 19 encoded caleosin
(CLE), and four encoded steroleosin (SLE) (Additional file 12: Table S4). Expression Patterns Of Unigenes Associated With Oil Accumulation These unigenes were strongly upregulated during pericarp and testa development, but were highly
expressed in the kernel at 49 DAF only (Additional file 13: Figure S9). Eighteen unigenes encoding SAD
were also identified as DEGs. Of these, four (CL2824.Contig13, CL2824.Contig14, CL2824.Contig25, and
CL2824.Contig26) were more highly expressed in the kernel and the testa than in the pericarp (Additional
file 13: Figure S9). In addition, in the kernel, the expression levels of CL2824.Contig25 steadily increased
from 35–63 DAF, remained constant from 63–77 DAF, and decreased steadily from 77–119 DAF
(Fig. 5b). One unigene was shown to encode fatty acyl-ACP thioesterase A (FATA) (unigene17393), and
three were shown to encode FATB (unigene2022, unigene5608, and unigene6721). Unigene6721 was
differently expressed among the different developmental stages of the pericarp, testa, and kernel, but
unigene17393 was only highly expressed in the kernel (with an expression peak at 49 DAF; Additional file
13: Figure S9). We also identified 10 unigenes encoding long-chain acyl-CoA synthetase (LACS)
(Additional file 12: Table S4). Ten unigenes were associated with PUFA biosynthesis: three unigenes encoded FAD2 (CL15521.Contig1,
CL15521.Contig2, and CL15521.Contig3), and two unigenes encoded FAD6 (CL5945.Contig3 and
CL5945.Contig8). In the kernel, the expression level of one of the FAD2-encoding genes
(CL15521.Contig3) increased from 35–77 DAF, and decreased from 77–119 DAF (Fig. 5b). This unigene
was also sharply upregulated at 77 DAF in the testa (Additional file 13: Figure S9). An additional four
unigenes encoded FAD3 and FAD8 (unigene2598, unigene43483, CL1023.Contig1, and CL1023.Contig4),
and one unigenes encoded FAD7 (unigene21286) (Additional file 11: Table S3). Based on our
phylogenetic analysis of ω-3 FADs from Arabidopsis thaliana and P. ostii (Additional file 14: Figure S10),
we identified four microsomal FAD3 genes and one plastid FAD7/8 gene in P. ostii. In the kernel, the
expression level of one of these FAD3-encoding genes (CL1023.Contig4) increased steadily from 39–91 Page 9/34 Page 9/34 DAF, followed by an abrupt decrease (Fig. 5b). Similarly, CL1023.Contig4 was sharply upregulated at 77
DAF in the testa (Additional file 13: Figure S9). In contrast, CL1023.Contig4 was expressed only at low
levels throughout pericarp development. Several unigenes encoding enzymes important for acyl editing and the Lands cycle were also identified. The three unigenes encoding CDP-choline:DAG choline phosphotransferase (CPT) (unigene7809,
unigene16109, and unigene16131) were differentially expressed during the development of the kernel,
pericarp, and testa. One unigene, encoding phosphatidylcholine:diacylglycerol
cholinephosphotransferase (PDCT) (unigene32372), was significantly differentially expressed among
these tissues. Expression Patterns Of Unigenes Associated With Oil Accumulation All of these unigenes were stably
expressed at very low levels during pericarp development, but were highly expressed throughout the
development of the testa and very highly expressed throughout the development of the kernel (Additional
file 13: Figure S9). In the kernel, bell-shaped expression profiles were observed for one CLE-encoding
unigene (CL18131.Contig3), two OLE-encoding unigenes (unigene5123 and unigene17798), and one SLE-
encoding unigene (CL19272.Contig1) (Fig. 5b). TFs associated with FA and TAG biosynthesis TFs associated with FA and TAG biosynthesis Page 10/34 We predicted the DEGs that might encode TFs, and classified these into TF families (Fig. 6a). Of these TF
DEGs, 301 belonged to the MYB family, 164 to the AP2-EREBP family, 161 to the FAR1 family, 132 to the
NAC family, 115 to the MADS family, 114 to the bHLH family, 70 to the WRKY family, and 14 to the bZIP
family. Within the TF DEGs, we also identified the unigenes related to the FA metabolism: nine in the WRI
family, 20 in the bZIP family, one in the LEAFY family, six in the FUSCA (FUS) family, five in the ABSCISIC
ACID-INSENSITIVE family, and 82 in the MYB family (Additional file 15: Table S5). WRI1 (CL22311) was more strongly upregulated in the kernel than in the testa and the pericarp, especially
during the early stages of FA accumulation (Fig. 6b). FUSCA (unigene25640) was also more strongly
upregulated in the kernel than in the pericarp or the testa (Fig. 6b). The two unigenes encoding bZIP
(unigeneCL3725 and unigeneCL15124) were constitutively expressed in all three tissues (Additional file
16: Fig. 11). The expression patters of the many TF DEGs in the MYB family differed among tissues. One
unigenes (CL14921.Contig3) was highly expressed in the pericarp, but expressed only at low levels in the
kernel and testa. Similarly, unigene25046 was also differentially expressed among tissues. In the kernel,
unigene25046 expression increased from 35–49 DAF, and then decreased sharply until the end of the
experiment (Additional file 16: Fig. 11). In the testa, unigene25046 was upregulated at 49 DAF, this high
level of expression was maintained until the end of the experiment. In the pericarp, unigene25046 was
expressed at low levels throughout the experiment. We also considered the unigenes encoding zinc finger
CCCH domain-containing protein 54 (C3H54) and ethylene-responsive transcription factor (RA212). Unigene32844, encoding C3H54, and unigene43518, encodingRA212, were both upregulated during FA
accumulation in the kernel, but were expressed only at low levels in the pericarp and testa (Fig. 6b). Quantitative real-time PCR (qRT-PCR) validation of key DEGs In general, the expression patterns of the 13 key genes chosen for validation were similar between the
qRT-PCR and RNA-seq analyses (Fig. 5b and 7; Additional file Figure S12-15). For example, seven
unigenes (encoding SAD, FAD2, FAD3, PDAT, OLE, CLE, and SLE), which exhibited bell-shaped patterns of
expression during kernel development in the RNA-seq data, were observed to exhibit similar expression
patterns in the qRT-PCR data. Importantly, both RNA-seq and qRT-PCR indicated that these seven
unigenes were only expressed at low levels throughout the development of the testa and pericarp. However, the qRT-PCR data indicated that FAD3 expression in the testa increased sharply at 63 DAF
(Fig. 7b). RNA-seq and qRT-PCR analyses showed that the remaining six unigenes were upregulated in the
kernel, but expressed at low levels throughout the development of the pericarp and testa. Discussion The patterns of FA accumulation differed noticeably among the pericarp, testa, and kernel. Throughout
development, FA content in the pericarp was generally low, as was FA content in the testa. In contrast,
concentrations of UFAs, particularly ALA, increased rapidly in the testa beginning at 63 DAF, peaking at 70
DAF. At 70 DAF, ALA content in the testa was 10-fold that of ALA content at 56 DAF; this coincided with
the maximum thickening of the testa (Fig. 1d). This is interesting because the concentrations of UFAs, Page 11/34 Page 11/34 especially ALA, exhibited an inverted V-shaped pattern of change during testa development. To our
knowledge, this is the first demonstration of changes in ALA content during testa development. The
kernel began to accumulate oil rapidly once the external shape and size of the seed had stabilized. The
concentrations of UFAs, especially ALA, followed a bell-like curve as the kernel matured. ALA
concentration peaked at 77 DAF; at this point, the cotyledon embryo had formed completely, the seed had
reached maximum size, and the pericarp was yellow. In contrast, Li et al. [10] found that ALA content was
greatest at the seed-browning stage. Our results showed ALA concentrations in the P. ostii were high in
the endosperm, moderate in the testa, and low in the pericarp. As these three tissues have different ALA
concentrations, they represent good research systems for comparative studies of the mechanisms of ALA
synthesis and accumulation in P. ostii. Transcriptomes have previously been used to study the mechanisms underlying ALA synthesis in tree
peony seeds [10–14]. Here, we identified genes associated with the lipid metabolism that were
differentially expressed during the development of the seed kernel, testa, and pericarp. Although the FA
metabolism is the primary metabolic process in plant cells, the pathways associated with FA biosynthesis
and metabolism were not significantly enriched during pericarp and testa development. The metabolic
pathways that were most significantly enriched in the pericarp and testa DEGs (e.g., fatty acid elongation,
glycerolipid metabolism, cutin/suberine and wax biosynthesis, and sphingolipid metabolism) were
primarily associated with the wax and cutin biosynthesis that is required for the development of the
pericarp and the testa. However, at stage 63 DAF in the testa (and not at any other stages), ALA content
increased sharply, and the genes encoding SAD, FAD2, and FAD3 were strongly upregulated (Fig. 7). Discussion In
addition, the ALA biosynthesis and fatty acid biosynthesis pathways were significantly enriched at this
stage in the testa. The inverted V-shape of the change in ALA content in the testa at 56–84 DAF was
significantly correlated with the increase in ALA and FA biosynthesis. More upregulated genes than
downregulated genes were identified in the kernel at 49 DAF, possibly because oil rapidly accumulates in
the kernel during this period. Consistent with this, some of the genes upregulated in the kernel at 49 DAF
were significantly enriched in lipid metabolism pathways, especially FA biosynthesis and metabolism. The genes significantly upregulated at 49 DAF in the kernel included those encoding α-PDH, BCCP, BC,
FATA, KAS I–III, PLD, PDCT, and LPAAT. Most of these genes are known to play major roles in de novo FA
biosynthesis. Genes associated with the pyruvate metabolism and with plant hormone signal
transduction also tended to be upregulated, rather than downregulated, at 49 DAF in the kernel. These two
pathways are also related to the FA metabolism: pyruvate metabolism is the precursor of acetyl CoA
production, while plant hormone signaling pathways may activate a variety of TFs to regulate the FA
metabolism [16]. Thus, the rapid synthesis and accumulation of FAs at 49 DAF in the kernel might be
primarily due to the activation of the FA synthesis pathway and the upregulation of key genes. Two types of ACCs, heteromeric ACC (HeACC) and HmACC, were expressed during the development of the
P. ostii tissues. HeACC forms a multisubunit enzyme with BC, BCCP, alpha-CT, and beta-CT [25]. The genes
encoding these subunits were differentially expressed among the three tissues throughout development. The genes encoding HmACC were highly expressed during testa development, but expressed at low levels
in the other two tissues. Specifically, ACC in plant plastids is heterogeneous, while ACC in plant cytoplasm Page 12/34 Page 12/34 is homogenous; ACC in the cytoplasm catalyzes malonyl monoacyl CoA to produce long-chain FAs,
flavonoids, anthocyanins, and other secondary metabolites [26, 27]. Thus, we speculated that the step of
de novo FA biosynthesis catalyzed by ACCs is specifically catalyzed by HmACC in the testa and by
HeACC in the kernel. However, in the Gramineae, the ACC in the plastids is homogenous [28]. Discussion The genes
encoding KAS I, KAS II, and KAS III, which are important catalysts of FA synthesis, were significantly
upregulated at 49 DAF in the kernel (Additional file 13: Figure S9), while the genes encoding SAD
(CL2824.Contig14 and CL2824.Contig25) exhibited a bell-shaped expression pattern. These genes were
not similarly upregulated in the testa and the pericarp. The upregulation of the SAD and KAS genes
during kernel development might be related to the rapid production of 18:1-ACP in this tissue. Consistent
with this, the upregulation of the gene encoding FATA in the kernel, particularly at 49 DAF, might ensure
that 18:1-ACP is produced rapidly via SAD catalysis and transported to the ER for downstream reactions. Thus, the rapid upregulation of KAS I, KAS III, KAS III, SAD, and FATA, as well as BCCP and BC, at 49 DAF
might be required to ensure that sufficient C18:1-dominated fatty acyl flux are transported to the ER for
acyl editing or TAG assembly. ω-3 FADs are located in the plastid or the ER, and are encoded by a multigene family [20]. ω-3 FADs act
on C18:2 to produce C18:3 (or on C16:2 to produce C16:3). There are two types of ω-3 FADs: ω-3 FADs are located in the plastid or the ER, and are encoded by a multigene family [20]. ω-3 FADs act
on C18:2 to produce C18:3 (or on C16:2 to produce C16:3). There are two types of ω-3 FADs:
microsomatic FAD3, which is located in the ER and acts on PC or other phospholipids with cytochrome b
as the electronic donor, and plastid FAD7/8, which is located in the plastid and acts on
phosphatidylglycerol and galactose lipids with ferritin as the electronic donor [16, 20]. In A. thaliana,
FAD7/8 were shown to be plastid isozymes of FAD3; FAD3, FAD7, and FAD8 were all present as single
copies in A. thaliana [29–31]. Here, based on gene annotation, we identified five unigenes (unigene21286,
unigene2598, unigene43483, CL1023.Contig1, and CL1023.Contig4) as encoding the temperature-
sensitive ω-3 FAD7/8 (Additional file 11: Table S3). However, our phylogenetic analysis suggested that
four microsomal FAD3 genes and one plastid FAD7/8 gene were expressed in P. ostii. One of the genes
encoding a microsomal FAD3 (CL1023.Contig4) was most highly expressed at 77 DAF in the kernel
(Fig. 7). One gene encoding a plastid FAD7/8 (Unigene21286) was expressed during kernel development
at a low level. Discussion ALA content in the kernel also peaked at 77 DAF, suggesting that FAD3 might play a vital
role in the formation of ALA. In the testa, CL1023.Contig4 was also highly expressed at 63 DAF, followed
by in a sharp increase in ALA content at 70 DAF. In contrast, FAD3 was not highly expressed in the
pericarp, and ALA content in the pericarp was very low. Although a previous study concluded that the high
concentrations of ALA in P. ostii seeds were due to FAD8 activity and abundance [10], our results
suggested that high concentrations of ALA in the kernel might be associated with the activity and
abundance of FAD3, not FAD8. Other plants with high concentrations of ALA, such as flax, perilla, and
sea-buckthorn, invariably express both FAD3 and FAD7/8 [32–35]. For example, the upregulation of FAD3
in flax was associated with high ALA content during seed development [32, 33]. In Perilla frutescens,
FAD3 was more highly expressed than FAD7/8 during seed development, but FAD7/8 was more highly
expressed than FAD3 in the leaves [34]. In the fruits of the sea-buckthorn (Hippophae rhamnoides) levels
of FAD3 and FAD7/8 expression were similarly upregulated, leading to high levels of ALA [35]. However, Page 13/34 Page 13/34 even among species with high ALA content, the enzymes that play key roles in ALA synthesis differ,
suggesting that ALA synthesis pathways also differ among plants, or even among tissues. De novo FA synthesis, and the subsequent acyl flux through the Kennedy pathway, produces oils
containing saturated and monounsaturated FAs [16]. However, the production of more diverse TAG acyl
compositions in plants requires the flux of acyl groups through the membrane lipids PC or PE [16, 21]. Therefore, the accumulation of TAG assemblies containing various PC/PE-modified FAs requires the
coordinated flux of acyl groups into PC/PE for desaturation, and the subsequent flux of acyl groups out
of the PC/PE and into TAG assemblies (Fig. 5a). In conjunction, acyl editing and the Lands cycle regulate
acyl flux, to ensure that acyl groups are modified to increase the diversity of TAG acyl compositions [21]. In the three P. ostii tissues, we detected 28 unigenes associated with acyl editing and the Lands cycle,
including three CPT-encoding unigenes, one PDCT-encoding unigene, three LPCAT-encoding unigenes, two
LPEAT-encoding unigenes, and 19 PLD-encoding unigenes. Discussion In particular, one PDCT-encoding unigene
(unigene32372) and one PLD-encoding unigene (CL1337.Contig6) showed bell-shaped expression
patterns during kernel development, but were expressed minimally in the pericarp and testa (Additional
file 13: Figure S9). This indicated that, compared with the testa and pericarp, the high expression of these
genes in kernel was very important for overall carbon flux into TAG in P. ostii in the form of PUFAs. To our
knowledge, unigene32372 and CL1337.Contig6 have not previously been identified as key genes in tree
peony seed development. In A. thaliana, the reduced oleate desaturase 1 gene (rod1) was shown to encode a novel enzyme (PDCT);
when rod1 expression was inhibited, the accumulation of PC-modified fatty acids in the TAG assembly
decreased ~ 40%, although overall levels of TAG remained constant [36]. As PC/PE is the site for extra-
plastidial FA modification, the flux of the de novo DAG moiety through PC can provide a PC-derived DAG
pool with a different acyl composition than that of de novo DAG (Fig. 5a). However, the relative proportion
of TAG synthesized from the Kennedy pathway using de novo DAG to TAG synthesized from PC-derived
DAG is unclear in most plants. Indeed, the PC-derived DAG pool, rather than the de novo DAG pool, might
be the primary source of DAG for TAG synthesis in various plants [36, 37]. The DGAT enzyme has also
been implicated in the formation of TAG from de novo DAG and acyl-CoA (the final step of the Kennedy
pathway; Fig. 5a, blue line). For example, DGAT1, DGAT2, and DGAT3 drive TAG production in Arabidopsis
[38], the peanuts [39], and tung tree [40]. Here, although four DGAT-encoding unigenes were identified in P. ostii, only one unigene (CL5087.Contig1) was strongly upregulated at 49 DAF in the testa, weakly
upregulated at same stage in the kernel, and expressed minimally throughout development in the pericarp
(Additional file 19: Figure S14). TAG synthesis may also be catalyzed by PDAT, using FAs from the PC
pool and PC-derived DAG as the substrate (Fig. 5a, green line). Here, the PDAT-encoding unigene
(unigene24505) showed a bell-shaped expression pattern in the developing kernel, but was minimally
expressed in the testa and the pericarp (Additional file 19: Figure S14). This indicated that in the P. ostii
kernel, PDAT reflected the amount of PFUA-containing TAG more accurately than DGAT. Thus, our results
indicated that the PDAT pathway (Fig. Discussion 5a, green line), rather than the Kennedy pathway (Fig. 5a, blue line),
might be the primary mechanism of TAG generation in the P. ostii kernel. Zhang et al. [13] reported that
PDAT levels were significantly associated with TAG accumulation in P. rokii. However, a previous study Page 14/34 Page 14/34 concluded that the PDAT pathway was probably not an important pathway for TAG generation in tree
peony seeds, due to the low abundance of PDAT-encoding gene transcripts [10]. In mature seeds, TAGs can be stored as oil bodies, surrounded by a phospholipid monolayer and
abundant amphipathic proteins (e.g., OLE, CLE, and SLE) [41–43]. Throughout development, the
expression levels of the gene homologs encoding OLE, CLE, and SLE either increased or were consistently
high in the kernel and the testa, ensuring efficient ALA accumulation, but remained low in the pericarp
(Additional file 20: Figure S15). In all three tissues, the CLE-encoding genes were more strongly
upregulated than the OLE- or SLE-encoding genes. However, during the development of Brassica napus
seeds and Prunus sibirica kernels, OLE was much more highly expressed than CLE and SLE [41, 44]. This
suggested that mechanisms of oil formation differ among plants. In mature seeds, TAGs can be stored as oil bodies, surrounded by a phospholipid monolayer and
abundant amphipathic proteins (e.g., OLE, CLE, and SLE) [41–43]. Throughout development, the
expression levels of the gene homologs encoding OLE, CLE, and SLE either increased or were consistently
high in the kernel and the testa, ensuring efficient ALA accumulation, but remained low in the pericarp
(Additional file 20: Figure S15). In all three tissues, the CLE-encoding genes were more strongly
upregulated than the OLE- or SLE-encoding genes. However, during the development of Brassica napus
seeds and Prunus sibirica kernels, OLE was much more highly expressed than CLE and SLE [41, 44]. This
suggested that mechanisms of oil formation differ among plants. TFs, such as WRI1, FUS3, ABI3, bZIP, LEC1, LEC2, MYB, and GL2, have previously been shown to play key
roles in seed oil synthesis and deposition [45]. Here, transcriptional profiling revealed that WRI1 and
FUSCA participated in the positive regulation of genes associated with oil synthesis during kernel
development (Fig. 7b). Notably, the AP2 TF WRI1 is upregulated by LEC1, LEC2, ABI3, and FUS3 in
Arabidopsis [45]. During kernel development, both FUS3 and WRI1 were upregulated (Fig. Discussion 6b), suggesting
that the gene encoding WRI1 might be a direct target of FUS3 in the developing kernel. Additionally, the
co-upregulation of plastidial ACC (BCCP and BC), KAS I–III, SAD, and FATA with WRI1 during kernel
development (Additional file 13: Figure S9) suggested that WRI1 might be involved in the transcriptional
regulation of these target genes, as was shown in Arabidopsis seeds. Thus, WRI1 (CL22311) might play a
critical role in the regulatory network controlling ALA accumulation during kernel development. An
important regulator of gene transcription in the soybean is zinc finger CCCH domain containing protein
(GmZF351). Transgenic experiments using Arabidopsis showed that GmZF351 directly regulated WRI1,
BCCP, KAS III, OLE and other genes; in addition, GmZF351 overexpression in soybeans increased oil
content [46]. Here, we found that the expression pattern of unigene32844, which encoded the TF zinc
finger CCCH domain-containing protein 54 (C3H54), in the seed kernel were consistent with the changes
in ALA content (Figs. 2 and 6b). Therefore, this TF might be a useful research target for future studies of
the regulation of ALA biosynthesis. Plant materials Fruits and seeds of P. ostii were collected in 2016 and 2017 at Shanghai Chenshan Botanical Garden
(31°4′52″N, 121°10′14″E), Shanghai, China. These P. ostii were introduced to the garden more than 10
years ago, and have been grown under the same environmental and cultivation conditions since that
time. We observed the development of the fruits and seeds produced by these trees over two growing
seasons (April–August, 2016 and 2017). Each year, the budded flowers of both selected plants (CS0016
and CS0009) were hand-pollinated with the pollen collected from a third P. ostii tree (CS0010). The
pollination date was recorded as 0 DAF. Between 0 and 119 DAF, seeds were collected every seven days
from both trees, for a total of 17 samples. After seed collection, the pericarp, testa, and kernel were
separated manually, frozen in liquid nitrogen, and stored at -80°C. Separated samples were used for FA
detection, transcriptome sequencing, and qRT-PCR. Conclusions Our results indicated that the optimal time for tree peony oil collection was 77 DAF, when the fruit peel
was yellow and oil quantity and quality were greatest. That is, FA concentration increased dramatically
during cotyledon embryogenesis, and continued to increase steadily to peak at 77 DAF. This rapid
increase in seed oil content was associated with the differential expression of many genes associated
with FA and TAG synthesis during embryogenesis. Many FA biosynthesis and metabolism pathways were
significantly enriched in these DEGs in the kernel at the early stages of rapid oil accumulation. ALA
content also increased sharply in the testa during development; many of the genes differentially
expressed in the testa at this stage were significantly enriched in ALA and FA biosynthesis. In total, we
identified 1373 unigenes related to lipid metabolism in P. ostii. Of these, unigenes with bell-shaped Page 15/34 Page 15/34 expression patterns (e.g., those encoding SAD, FAD2, FAD3, PDCT, PDAT, OLE, CLE, and SLE) and
unigenes that were upregulated at 49 DAF (e.g., those encoding BCCP, BC, KAS I–III, and FATA) may be
useful targets for future investigations of lipid metabolism in the tree peony. Similarly, many regulatory
enzymes (e.g., PDH, ACC, KAS I–III, SAD, FATA, FAD2, FAD3 LPCAT/LPEAT, CPT, PDCT, DGAT, PDAT, OLE,
CLE, and SLE) and TFs (WRI1 and FUS3), which are crucial for the biosynthesis of acetyl-CoA, FA, TAG,
and the oil body, and which were significantly enriched during kernel development, deserve further
investigation. In this study, we used three tissues with high, moderate, and low ALA concentrations as an
exemplar system in which to compare tissue-specific ALA accumulation mechanism in P. ostii. We found
that key genes related to ALA synthesis were differentially expressed among the three tissues. Thus, our
results help to provide a framework for future studies of the tree peony, particularly those aimed at
improving tree peony seed oil production through breeding, genetic diversification, and gene excavation. Measurement of FA accumulation To explore how FA composition changed in the pericarp, testa, and kernel throughout development, we
measured the FA concentrations in the pericarp, testa, and kernel samples taken from both trees between
21 and 119 DAF (a total of 42 samples per tree per year). The samples used for FA analysis were dried at
60°C to a constant weight. Total lipids were extracted from the dried biomass as previously described
[47], and FAs were methylated as previously described in spinach [48], with minor modifications. First, the
dried, powdered sample was added to 3 mL of a 1:2 chloroform-methanol (v/v) mixture. Next, this
solution was incubated in a water bath at 35°C for 1 h at 120 rpm for lipid extraction. After the full
extraction, 1.0 mL supplementary chloroform was added to the solution, and the mixture was vortexed. Then, 1.8 mL ddH2O was added, to generate a solution with a final chloroform:methanol:ddH2O ratio of
1:1:0.9 (v/v). The solution was then centrifuged at 4000 g for 15 min. The chloroform layer was
withdrawn, and then dried with sample concentrators under a nitrogen evaporator. The concentrated
lipids were then re-dissolved in 2 mL of an H2SO4-methanol solution (2% H2SO4). After charging with Page 16/34 Page 16/34 nitrogen gas, the solution was vortexed for 1 min, and then incubated in a 90°C water bath for 1 h. After
incubation, 1 mL ddH2O and 1 mL hexane were added to the solution. The solution was vortexed, and
then centrifuged at 4000 g for 15 min. The supernatant was transferred to a new tube, concentrated using
bubbling nitrogen, and stored at 4°C for gas chromatograph-mass spectrometry (GC-MS) analysis. We
used 50 mg/mL nonadecanoic acid in hexane as the internal standard. The FA methyl esters were
measured using a GC-MS (GC7890/MS5975, Agilent) on a HP-88 capillary column (60 m long x 0.25 mm
internal diameter; 0.2 mm; Agilent). FA methylation was tested and analyzed following Yu et al.6 Unigene assembly and annotation After sequencing, the raw reads were preprocessed to remove clipped adapter sequences, low-quality
reads (Q value £ 20 or containing ambiguous nucleotides), and contaminated sequences. The clean
reads were de novo assembled using Trinity [49]. Redundant sequences were removed, and remaining
sequences were clustered based on homology. Based on overlap, the fragments were merged or extended
into longer transcripts to form a set of non-redundant unigenes. To annotate the obtained unigenes, we
used BLASTX (e-value < 0.00001) to search several public databases in the following order: NCBI non-
redundant (Nr), Swiss-Prot, KEGG, and COG/KOG. The Gene Ontology (GO) [50] annotation for unigenes
was obtained using Blast2GO [51]. GO classification and enrichment analyses were performed using
WEGO [52]. RNA extraction and cDNA library construction As preliminary results indicated that patterns of FA accumulation differed substantially among the three
tissues, we sequenced the transcriptomes of the kernel, testa, and pericarp throughout development to
explore the molecular mechanisms underlying FA accumulation in the three tissues. RNA was extracted
from kernel, testa, and pericarp samples taken at 35, 49, 63, 77, 91, and 119 DAF from CS0016 in 2016
and 2017, and CS0009 in 2017. Total RNA was extracted from these 54 samples using RNA Exaction Kits
(E.Z.N.A. HP Plant RNA Kit, Omega Bio-Tek), and then purified using the RNeasy Plant Mini Kit (Qiagen),
following the manufacturer’s protocols. The concentration and quality of each RNA sample was
determined using an Agilent 2100 Bioanalyzer (Agilent Technologies). All samples had an OD260/OD280
ratio of 2.0–2.1, and an RNA integrity number >7.0. The extracted total RNA was treated with DNase I, and
Oligo(dT) primers were used to isolate mRNA. Then, cDNA was synthesized using these mRNA fragments
as templates. Short fragments were purified and resolved with EB buffer for end repair. Single A (adenine)
nucleotides were then added. Next, the short fragments were connected with adapters, and suitable
fragments were selected for PCR amplification. The Agilent 2100 Bioanalyzer and the ABI StepOnePlus
Real-Time PCR System were used for the quality control of the sample library. The generated cDNA
libraries were sequenced at BGI-Shenzhen (XX), using an Illumina HiSeq 4000 system. Identification of DEGs We mapped the clean reads to unigenes using Bowtie2 [53], and then calculated gene expression level
with RSEM [54]. Unigene expression levels were calculated and normalized using the Fragments Per Page 17/34 Kilobase per Million mapped fragments (FPKM) method. We then used several methods to cluster the
gene expression data; clustering genes based on expression patterns helps to identify genes with similar
functions, which may be associated with the same biological functions. We performed principal
components analysis (PCA) of all samples using the princomp function in R. We used Cluster [55] and the
Euclidean matrix formula to cluster the expressed genes and sample schemes simultaneously. The
resulting clusters were visualized using Java Treeview. We also used the heatmap function in R to draw a
complete clustering graph. We used Mfuzz, which employs a loose clustering algorithm, to analyze the
time series data. Kilobase per Million mapped fragments (FPKM) method. We then used several methods to cluster the
gene expression data; clustering genes based on expression patterns helps to identify genes with similar
functions, which may be associated with the same biological functions. We performed principal
components analysis (PCA) of all samples using the princomp function in R. We used Cluster [55] and the
Euclidean matrix formula to cluster the expressed genes and sample schemes simultaneously. The
resulting clusters were visualized using Java Treeview. We also used the heatmap function in R to draw a
complete clustering graph. We used Mfuzz, which employs a loose clustering algorithm, to analyze the
time series data. We identified DEGs using DEseq2, EBseq, NOIseq, and PossionDis as required. DEseq2, which is based on
the negative binomial distribution, was used as described by Michael et al. [56]. EBseq, which is based on
the empirical Bayesian model, was used as described by Leng et al. [57]. NOIseq, which is based on the
noisy distribution model, was used as described by Tarazona et al. [58]. PossionDis, which is based on
the Poisson distribution, was used as described by Audic et al. [59]. We next classified the identified DEGs
based on GO and KEGG annotations. We calculated GO functional enrichment and KEGG pathway
enrichment using phyper in R. Analysis of unigene expression patterns and associated TFs All protein sequences were downloaded from Swiss-Prot. Based on the functional annotations of the
DEGs, we selected all DEGs associated with the lipid metabolism. To better understand the molecular
factors underlying ALA accumulation during kernel development, we focused on the DEGs associated
with FA and TAG biosynthesis. The sequence of three ω-3 FADs from Arabidopsis thaliana (AtFAD3_AT2G29980, AtFAD7_AT3G11170,
and AtFAD8_AT5G05580) were downloaded from the NCBI. We aligned the A. thaliana ω-3 FADs with
those from P. ostii (unigene21286, unigene2598, unigene43483, CL1023.Contig1, and CL1023.Contig4)
using clustalX [60]. We then constructed a phylogenetic tree based on this multiple sequence alignment
using FastTree2 [61]. To investigate how TFs regulated tree peony oil biosynthesis, we analyzed all of the DEGs that were also
TFs. First, we used Getorf to detect the ORF of each DEG. These ORFs were then mapped to the
transcription factor protein domain database (from PlntfDB) using Hmmsearch [62]. We predicted the TF
coding ability of each DEG based on the characteristics of each TF family. Based on the Swiss-Prot
annotations of the DEGs, we identified the TF DEGs related to FA metabolism. qRT-PCR validation of the expression patterns of key DEGs involved in oil accumulation Each 20 µL reaction mixture contained 10 μl of TB Green
Premix Ex Taq II (Tli RNaseH Plus) (Takara), 0.8 μl of each primer (10 μM), 0.3 μl of cDNA template (1 μg),
and 8.1 μl of RNase-free water. PCRs for each gene were performed in triplicate, with the following
thermal cycling conditions: 95°C for 30 s; 40 cycles of 95°C for 5 s and 64°C for 30s; and 95°C for 15 s. Primer specificity was confirmed by melting curve analysis. The relative expression levels of the tested
genes were calculated using the 2-ΔΔCt method [63], with the actin genes as internal controls. Abbreviations ACC: acetyl-CoA carboxylase; ACP: acyl carrier protein; ALA: alpha linolenic acid; BC: biotin carboxylase;
BCCP: biotin carboxyl carrier protein; CT: carboxyltransferase; CLE: caleosins; CPT: CDP-choline:DAG
choline phosphotransferase; DAG: diacylglycerol; DAF: days after fertilization; DEG: differentially
expressed gene; DGAT: diacylglycerol acyltransferase; EPT: CDP-ethanolamine:DAG
ethanolaminephosphotransferase; ER: endoplasmic reticulum; FA: fatty acid; FAS: fatty acid synthase;
FATA/B: acyl-ACP thioesterase A/B; FAD2: oleate desaturase; FAD3: linoleate desaturase; FPKM:
fragments per kilobase per million mapped fragments; G3P: glycerol-3-phosphate; GPAT: glycerol-3-
phosphate acyltransferase; KAS I, II, III: ketoacyl-ACP synthase I, II, III; KEGG: kyoto encyclopedia of genes
and genomes; LA: linoleic acid; LACS: long-chain acyl-CoA synthetase; LEC1: leafy cotyledon1; LPA:
lysophosphatidic acid; LPAAT: lysophosphatidyl acyltransferase; LPC: lysophosphatidylcholine; LPE:
lysophosphatidylethanolamine; LPCAT: lysophosphatidylcholineacyltransferase; LPEAT:
lysophosphatidylethanolamineacyltransferase; MCMT: malonyl-CoA ACP transacylase; OA: oleic acid;
OLE: oleosin; PA: phosphatidic acid; PAP: phosphatidic acid phosphohydrolase; PC: phosphatidylcholine;
PE: phosphatidylethanolamine; PDAT: phospholipid: diacylglycerol acyltransferase; PDCT:
phosphatidylcholine: diacylglycerol cholinephosphotransferase; PDH: pyruvate dehydrogenase; PLC:
phospholipase C; PLD: phospholipase D; PUFA: polyunsaturated fatty acids; SAD: stearoyl-ACP
desaturase; SLE: steroleosin ; TAG: triacylglycerol; TF: transcription factor; UFA: unsaturated fatty acid. qRT-PCR validation of the expression patterns of key DEGs involved in oil accumulation To experimentally validate our RNA-seq data, we selected 13 unigenes associated with FA biosynthesis
and TAG assembly, in particular those related to ALA, for qRT-PCR validation. The unigenes selected were
CL2824.Contig25, encoding SAD; CL15521.Contig3, encoding FAD2; CL1023.Contig4, encoding FAD3;
CL5945.Contig8, encoding FAD6; Unigene21286, encoding FAD7/8; CL5087.Contig1, encoding DGAT; Page 18/34 Page 18/34 Unigene24505, encoding PDAT; Unigene5123, encoding OLE; CL18131.Contig3, encoding CLE; and
Unigene19272, encoding SLE. The expression levels of these unigenes were quantified using qRT-PCR in
the kernel, testa and pericarp at 35, 49, 63, 77, 91, and 119 DAF (18 samples in total). Total RNA was
isolated from each tissue at each time point using RNA Exaction Kits (E.Z.N.A. HP Plant RNA Kit, Omega
Bio-Tek). First-strand cDNA was prepared from 1 μg of total RNA per sample using a FastKing RT Kit with
gDNase (Tiangen). Specific primers were designed for each of the 13 unigenes (Additional file 21: Table
S6). PCRs were performed on an ABI StepOnePlus Real-Time PCR System (Applied Biosystems),
following the manufacturer’s instructions. Each 20 µL reaction mixture contained 10 μl of TB Green
Premix Ex Taq II (Tli RNaseH Plus) (Takara), 0.8 μl of each primer (10 μM), 0.3 μl of cDNA template (1 μg),
and 8.1 μl of RNase-free water. PCRs for each gene were performed in triplicate, with the following
thermal cycling conditions: 95°C for 30 s; 40 cycles of 95°C for 5 s and 64°C for 30s; and 95°C for 15 s. Primer specificity was confirmed by melting curve analysis. The relative expression levels of the tested
genes were calculated using the 2-ΔΔCt method [63], with the actin genes as internal controls. Unigene24505, encoding PDAT; Unigene5123, encoding OLE; CL18131.Contig3, encoding CLE; and
Unigene19272, encoding SLE. The expression levels of these unigenes were quantified using qRT-PCR in
the kernel, testa and pericarp at 35, 49, 63, 77, 91, and 119 DAF (18 samples in total). Total RNA was
isolated from each tissue at each time point using RNA Exaction Kits (E.Z.N.A. HP Plant RNA Kit, Omega
Bio-Tek). First-strand cDNA was prepared from 1 μg of total RNA per sample using a FastKing RT Kit with
gDNase (Tiangen). Specific primers were designed for each of the 13 unigenes (Additional file 21: Table
S6). PCRs were performed on an ABI StepOnePlus Real-Time PCR System (Applied Biosystems),
following the manufacturer’s instructions. Ethics approval and consent to participate Not applicable. Consent for publication Declarations Ethics approval and consent to participate Competing interests The authors declare that they have no competing interests. Authors¢ contributions Y-HH and J-HY designed all the experiments. S-YY collected all samples. S-YY and XZ analyzed all
phenotypic and molecular data. XZ quantified FA composition, RNA extraction, performed qRT-PCR
experiment. YZ cultivated tree peony plants. S-YY and L-BH carried out transcriptome data analysis. Y-PL
and Z-JY assisted with the RNA extraction and qRT-PCR experiment. X-XZ assisted with transcriptome
data analysis. S-YY and XZ drafted the manuscript. JY and YH managed the experiments, organized the
manuscript and revised manuscript. All authors read and approved the final manuscript. S-YY and XZ contributed equally to this work. Consent for publication Page 19/34 Page 19/34 Not applicable. Funding This research was funded by the Chenshan Key Scientific Research Projects of Shanghai Municipal
Administration of Landscaping and City Appearance (G172401, G182406, G182407, G192411 and
G192419), Science and Technology Service Network Plan of Chinese Academy of Sciences (KFJ-STS-
QYZD-035). Funders provided financial support only, they hand no role in the design of the study, analysis
and interpretation of data, decision to publish, or the preparation of the manuscript. Acknowledgements The authors thank Plant Metabolic Platform of Shanghai Key Laboratory of Plant Functional Genomics
and Resources and Dr. Yu Kong for helps with detecting fatty acids by GC-MS. Also, we extend our thanks
to Mr. Binjie Ge for his assistance in taking photograph of fruits and seeds. Availability of data and materials Illumina read data used for expression profiling of the Paeonia ostii reference genes have been submitted
to the NCBI Sequence Read Archive (SRA) under the accession number PRJNA595001. All other data
supporting our findings can be found in Additional files 1-21. References 1. Ramaprasad TR, Srinivasan K, Baskaran V, Sambaiah K, Lokesh BR. Spraydried milk supplemented
with alpha-linolenic acid or eicosapentaenoic acid and docosahexaenoic acid decreases HMG CoA
reductase activity and increases biliary secretion of lipids in rats. Steroids. 2006;71(5):409–15. 2. Simopoulos AP. Omega-3 fatty acids in wild plants, nuts and seeds. Asia Pac J Clin Nutr. 2002;11:163–73. Page 20/34 Page 20/34 3. FAO & WHO. Fats and oils in human nutrition: Report of a joint expert consultation. FAO Food and
Nutrition. Rome, 1994;No. 57. 4. Simopoulos AP. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomed
Pharmacother. 2002;56:365–79. 4. Simopoulos AP. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomed
Pharmacother. 2002;56:365–79. 5. Simopoulos AP. Human requirement for n-3 polyunsaturated fatty acids. Poultry Sci. 2000;79(7):961–70. 6. Yu SY, Du SB, Yuan JH, Hu YH. Fatty acid profile in the seeds and seed tissues of Paeonia L. species
as new oil plant resources. Sci Rep. 2016;6:26944. 6. Yu SY, Du SB, Yuan JH, Hu YH. Fatty acid profile in the seeds and seed tissues of Paeonia L. species
as new oil plant resources. Sci Rep. 2016;6:26944. 7. NHFPCC (National Health and Family Planning Commission of China). Notice on the Approval of
Acer Truncatum Seed Oil and Peony Seed Oil as New Resource Food. Available online:
tp://www.nhfpc.gov.cn/sps/s7891/201103/cd9def6007444ea271189c18063b54.shtml (accessed
on 22 March 2011). 7. NHFPCC (National Health and Family Planning Commission of China). Notice on the Approval of
Acer Truncatum Seed Oil and Peony Seed Oil as New Resource Food. Available online:
tp://www.nhfpc.gov.cn/sps/s7891/201103/cd9def6007444ea271189c18063b54.shtml (accessed
on 22 March 2011). tp://www.nhfpc.gov.cn/sps/s7891/201103/cd9def6007444ea271189c18063b54.shtml (accessed
on 22 March 2011). 8. Hong DY, Pan KY. Taxonomical history and revision of Paconia sect. Moutan (Paeoniaceae). Acta
Phytotaxon Sin. 1999;37:351–68. 8. Hong DY, Pan KY. Taxonomical history and revision of Paconia sect. Moutan (Paeoniaceae). Acta
Phytotaxon Sin. 1999;37:351–68. 9. Zhang XX, Zhang YL, Niu LX, Sun JY, Li LH, Zhang J, et al. Chemometric classification of different
tree peony species native to China based on the assessment of major fatty acids of seed oil and
phenotypic characteristics of the seeds. Chem Biodiversity. 2017;14:e1600111. 10. Li SS, Wang LS, Shu QY, Wu J, Chen LG, Shao S, et al. Fatty acid composition of developing tree
peony (Paeonia section Moutan DC.) seeds and transcriptome analysis during seed development. BMC Genom. 2015;16:208. 11. Xiu Y, Wu GD, Tang W, Peng ZF, Bu XP, Chao LJ, et al. References Oil biosynthesis and transcriptome profiles
indeveloping endosperm and oil characteristic analyses in Paeonia ostii var. lishizhenii. J Plant
Physiol. 2018;228:121–33. 12. Yin DD, Li SS, Shu QY, Gu ZY, Wu Q, Feng CY, et al. Identification of microRNAs and long non-coding
RNAs involved in fatty acid biosynthesis in tree peony seeds. Gene. 2018;666:72–82. 12. Yin DD, Li SS, Shu QY, Gu ZY, Wu Q, Feng CY, et al. Identification of microRNAs and long non-coding
RNAs involved in fatty acid biosynthesis in tree peony seeds. Gene. 2018;666:72–82. 13. Zhang QY, Yu R, Sun DY, Rahman MM, Xie LH, Hu JY, et al. Comparative transcriptome analysis
reveals an efficient mechanism for α-linolenic acid synthesis in tree peony seeds. Int J Mol Sci. 2019;20:65. 13. Zhang QY, Yu R, Sun DY, Rahman MM, Xie LH, Hu JY, et al. Comparative transcriptome analysis
reveals an efficient mechanism for α-linolenic acid synthesis in tree peony seeds. Int J Mol Sci. 2019;20:65. 14. Wang XJ, Liang HY, Guo DL, Guo LL, Duan XG, Jia QS, et al. Integrated analysis of transcriptomic
and proteomic data from tree peony (P. ostii) seeds reveals key developmental stages and candidate
genes related to oil biosynthesis and fatty acid metabolism. Hoticul Res. 2019;6:111. 15. Zhang QY, Yu R, Xie LH, Rahman MM, Kilaru A, Niu LX, et al. Fatty acid and associated gene
expression analyses of three tree peony species reveal key genes for α-linolenic acid synthesis in
seeds. Front Plant Sci. 2018;9:106. 16. Li-Beisson Y, Shorrosh B, Beisson F, Andersson MX, Arondel V, Bates PD, et al. Acyl-lipid metabolism. The Arabidopsis book / American Society of Plant Biologists 2010;8:e0133. 16. Li-Beisson Y, Shorrosh B, Beisson F, Andersson MX, Arondel V, Bates PD, et al. Acyl-lipid metabolism. The Arabidopsis book / American Society of Plant Biologists 2010;8:e0133. Page 21/34
17. Konishi T, Shinohara K, Yamada K, Sasaki Y. Acetyl- CoA carboxylase in higher plants: Most plants
other than gramineae have both the prokaryotic and the eukaryotic forms of this enzyme. Plant Cell Page 21/34
17. Konishi T, Shinohara K, Yamada K, Sasaki Y. Acetyl- CoA carboxylase in higher plants: Most plants
other than gramineae have both the prokaryotic and the eukaryotic forms of this enzyme. Plant Cell Physiol. 1996;37:117–22. 18. Yasuno R, von Wettstein-Knowles P, Wada H. Identification and molecular characterization of the β-
ketoacyl-[acyl carrier protein] synthase component of the Arabidopsis mitochondrial fatty acid
synthase. J Biol Chem. 2004;279:8242–51. 19. References Roughan PG, Slack CR. Cellular-organization of glycerolipid metabolism. Annu Rev Plant Phys. 1982;33:97–132. 20. Ohlrogge J, Browse J. Lipid biosynthesis. Plant Cell. 1995;7(7):957–70. 20. Ohlrogge J, Browse J. Lipid biosynthesis. Plant Cell. 1995;7(7):957–70. 21. Bates PD. Understanding the control of acyl flux through the lipid metabolic network of plant oil
biosynthesis. Bioch et Biophy Acta. 2016;1861:1214–25. 21. Bates PD. Understanding the control of acyl flux through the lipid metabolic network of plant oil
biosynthesis. Bioch et Biophy Acta. 2016;1861:1214–25. 22. Dussert S, Guerin C, Andersson M, Joet T, Tranbarger TJ, Pizot M, et al. Comparative transcriptome
analysis of three oil palm fruit and seed tissues that differ in oil content and fatty acid composition. Plant Physiol. 2013;162:1337–58. 23. Brown AP, Kroon JTM, Swarbreck D, Febrer M, Larson TR, Graham IA, et al. Tissue-specific whole
transcriptome sequencing in Castor, directed at understanding triacylglycerol lipid biosynthetic
pathways. PLoS ONE. 2012;7(2):e30100. 23. Brown AP, Kroon JTM, Swarbreck D, Febrer M, Larson TR, Graham IA, et al. Tissue-specific whole
transcriptome sequencing in Castor, directed at understanding triacylglycerol lipid biosynthetic
pathways. PLoS ONE. 2012;7(2):e30100. 24. Peng LP, Men SQ, Liu ZA, Tong NN, Imran M, Shu QY. Fatty acid composition, phytochemistry,
antioxidant activity on seed coat and kernel of Paeonia ostii from main geographic production areas. Foods. 2020;9:30. 24. Peng LP, Men SQ, Liu ZA, Tong NN, Imran M, Shu QY. Fatty acid composition, phytochemistry,
antioxidant activity on seed coat and kernel of Paeonia ostii from main geographic production areas. Foods. 2020;9:30. 25. Harwood JL. Recent advances in the biosynthesis of plant fatty acids. Biochim Biophys Acta Lipids
Lipid Metab. 1996;1301:7–56. 26. Gronwald JW. Lipid biosynthesis inhibitors. Weed Sci. 1991;39:435–49. 26. Gronwald JW. Lipid biosynthesis inhibitors. Weed Sci. 1991;39:435–49. 27. Nikolau BJ, Ohlrogge JB. Wur tele ES. Plant biotin-containing carboxylases. Arch Bioch Biophy. 2003;414(2):211–22. 28. Turner JA, Pernich DJ. Origin of enantiomeric selectivity in the aryloxyphenoxypropionic acid class of
herbicidal acetyl coenzyme A carboxylase (ACCase) inhibitors. J Agric Food Chem. 2002;50(16):4554–66. 29. Arondel V, Lemieux B, Hwang I, Gibson S, Goodman HM, Somerville CR. Map-based cloning of a gene
controlling omega-3 fatty acid desaturation in Arabidopsis. Science. 1992;258:1353–5. 30. Iba K, Gibson S, Nishiuchi T, Fuse T, Nishimura M, Arondel V, et al. A gene encoding a chloroplast
omega-3 fatty acid desaturase complements alterations in fatty acid desaturation and chloroplast
copy number of the fad7 mutant of Arabidopsis thaliana. J Biol Chem. 1993;268:24099–105. 31. References Saha S, Enugutti B, Rajakumari S, Rajasekharan R. Cytosolic triacylglycerol biosynthetic pathway in
oilseeds. Molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase. Plant
Physiol. 2006;141:1533–43. 40. Shockey JM, Gidda SK, Chapital DC, Kuan J-C, Dhanoa PK, Bland JM, et al. Tung tree DGAT1 and
DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different
subdomains of the endoplasmic reticulum. Plant Cell. 2006;18:2294–313. 41. Troncoso-Ponce MA, Kilaru A, Cao X, Durrett TP, Fan J, Jensen JK, et al. Comparative deep
transcriptional profiling of four developing oilseeds. Plant J. 2011;68:1014–27. 41. Troncoso-Ponce MA, Kilaru A, Cao X, Durrett TP, Fan J, Jensen JK, et al. Comparative deep
transcriptional profiling of four developing oilseeds. Plant J. 2011;68:1014–27. 42. Bourgis F, Kilaru A, Cao X, Ngando-Ebongue GF, Drira N, Ohlrogge JB, et al. Comparative
transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically
in carbon partitioning. Proc Natl Acad Sci U S A. 2011;108:12527–32. 42. Bourgis F, Kilaru A, Cao X, Ngando-Ebongue GF, Drira N, Ohlrogge JB, et al. Comparative
transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically
in carbon partitioning. Proc Natl Acad Sci U S A. 2011;108:12527–32. 43. Jolivet P, Roux E, d’Andrea S, Davanture M, Negroni L, Zivy M, et al. Protein composition of oil bodies
in Arabidopsis thaliana ecotype WS. Plant Physiol Bioch. 2004;42:501–9. 43. Jolivet P, Roux E, d’Andrea S, Davanture M, Negroni L, Zivy M, et al. Protein composition of oil bodies
in Arabidopsis thaliana ecotype WS. Plant Physiol Bioch. 2004;42:501–9. 44. Niu J, An JY, Wang LB, Fang CL, Ha DL, Fu CY, et al. Transcriptomic analysis revealed the mechanism
of oil dynamic accumulation during developing Siberian apricot (Prunus sibirica L.) seed kernels for
the development of woody biodiesel. Biotech for Biofu. 2015;8:29. 44. Niu J, An JY, Wang LB, Fang CL, Ha DL, Fu CY, et al. Transcriptomic analysis revealed the mechanism
of oil dynamic accumulation during developing Siberian apricot (Prunus sibirica L.) seed kernels for
the development of woody biodiesel. Biotech for Biofu. 2015;8:29. 45. Kong Q, Ma W. WRINKLED1 transcription factor: How much do we know about its regulatory
mechanism? Plant Sci. 2018;272:153–6. 45. Kong Q, Ma W. WRINKLED1 transcription factor: How much do we know about its regulatory
mechanism? Plant Sci. 2018;272:153–6. 46. Li QT, Lu X, Song QX, Chen HW, Wei W, Tao JJ, et al. References Gibson S, Arondel V, Iba K, Somerville C. Cloning of a temperature-regulated gene encoding a
chloroplast omega-3 desaturase from Arabidopsis thaliana. Plant Physiol. 1994;106:1615–21. 31. Gibson S, Arondel V, Iba K, Somerville C. Cloning of a temperature-regulated gene encoding a
chloroplast omega-3 desaturase from Arabidopsis thaliana. Plant Physiol. 1994;106:1615–21. 32. Prakash V, Xiang DQ, Qiu SQ, Stone SL, Tibiche C, Cram D, et al. Gene expression analysis of flax
seed development. BMC Plant Biol. 2011;11:74–87. 32. Prakash V, Xiang DQ, Qiu SQ, Stone SL, Tibiche C, Cram D, et al. Gene expression analysis of flax
seed development. BMC Plant Biol. 2011;11:74–87. 33. Banik M, Duguid S, Cloutier S. Transcript profiling and gene characterization of three fatty acid
desaturase genes in high, moderated, and low linolenic acid genotypes of flax (Linum usitatissimum 33. Banik M, Duguid S, Cloutier S. Transcript profiling and gene characterization of three fatty acid
desaturase genes in high, moderated, and low linolenic acid genotypes of flax (Linum usitatissimum 33. Banik M, Duguid S, Cloutier S. Transcript profiling and gene characterization of three fatty acid
desaturase genes in high, moderated, and low linolenic acid genotypes of flax (Linum usitatissimum Page 22/34 L.) and their role in linolenic acid accumulation. Genome. 2011;54:471–83. L.) and their role in linolenic acid accumulation. Genome. 2011;54:471–83. 34. Kim HU, Lee KR, Shim D, Lee JH, Chen GQ, Hwang S. Transcriptome analysis and identification of
genes associated with omega3 fatty acid biosynthesis in Perilla frutescens (L.) var. frutescens. BMC
Genom. 2016;17:474. 35. Fatima T, Snyder CL, Schroeder WR, Cram D, Datla R, Wishart D, et al. Fatty acid composition of
developing sea buckthorn (Hippophae rhamnoides L.) berry and the transcriptome of the mature
seed. PLoS ONE. 2012;7(4):e34099. 36. Lu CF, Xin ZG, Ren ZH, Miquel M, Browse J. An enzyme regulating triacylglycerol composition is
encoded by the ROD1 gene of Arabidopsis. Proc Natl Acad Sci. 2009;106:18837–42. 37. Bates PD, Durrett TP, Ohlrogge JB, Pollard M. Analysis of acyl fluxes through multiple pathways of
triacylglycerol synthesis in developing soybean embryos. Plant Physiol. 2009;150:55–72. 38. Zhang M, Fan J, Taylor DC, Ohlrogge JB. DGAT1 and PDAT1 acyltransferases have overlapping
functions in Arabidopsis triacylglycerol biosynthesis and are essential for normal pollen and seed
development. Plant Cell. 2009;21:3885–901. 39. Saha S, Enugutti B, Rajakumari S, Rajasekharan R. Cytosolic triacylglycerol biosynthetic pathway in
oilseeds. Molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase. Plant
Physiol. 2006;141:1533–43. 39. References FastTree 2-approximately maximum-likelihood trees for large
alignments. PloS one. 2010;5(3):e9490. 61. Price MN, Dehal PS, Arkin AP. FastTree 2-approximately maximum-likelihood trees for large
alignments. PloS one. 2010;5(3):e9490. 62. Eddy SR. Profile hidden Markov models. Bioinformatics. 1998,14:755 – 63. 62. Eddy SR. Profile hidden Markov models. Bioinformatics. 1998,14:755 – 63. 63. Livak KJ, Schmittgen T. Analysis of relative gene expression data using realtime quantitative PCR
and the 2–∆∆Ct method. Methods. 2001;25:402–8. References Selection for zinc-finger protein contributes to
seed oil increase during soybean domestication. Plant Physiol. 2017;173:2208–24. 46. Li QT, Lu X, Song QX, Chen HW, Wei W, Tao JJ, et al. Selection for zinc-finger protein contributes to
seed oil increase during soybean domestication. Plant Physiol. 2017;173:2208–24. 47. Folch J, Lees M, Sloane GMA. Simple method for the isolation and purification of total lipids from
animal tissues. J Biol Chem. 1957;26:497–509. 47. Folch J, Lees M, Sloane GMA. Simple method for the isolation and purification of total lipids from
animal tissues. J Biol Chem. 1957;26:497–509. Page 23/34 48. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Bioch Physiol. 1959;37:911–7. 49. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome
assembly from RNA-seq data without a reference genome. Nat Biotech. 2011;29(7):644–52. 50. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene Ontology: tool for the
unification of biology. Nat Genet. 2000;25:25–9. 51. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M. Blast2GO: a universal tool for
annotation, visualization and analysis in functional genomics research. Bioinformatics. 2005;21(18):3674–6. 52. Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, et al. WEGO: a web tool for plotting GO
annotations. Nucleic Acids Res. 2006;34:293–7. 53. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9. 54. Dewey CN, Li B. RSEM: accurate transcript quantification from RNA-Seq data with or without a
reference genome. BMC Bioinformatics. 2011;4:12–323. 55. Hoon MJL, Imoto S, Nolan J, Miyano S. Open Source Clustering Software Bioinformatics. 2004 20(9) 1453 4 55. Hoon MJL, Imoto S, Nolan J, Miyano S. Open Source Clustering Software Bioinformatics. 2004;20(9):1453–4. 56. Love MI, Wolfgang H, Simon A. Moderated estimation of fold change and dispersion for RNA-seq
data with DESeq2. Genome Biol. 2014;15:550–5. 57. Leng N, EBSeq. An R package for gene and isoform differential expression analysis of RNA-seq data. Bioinformatics. 2015;29(8):1035–43. 58. Tarazona S, Garcia-Alcalde F, Dopazo J. Differential expression in RNA-seq: a matter of depth. Genome research. 2011;21(12):4436–41. 59. Audic S, Claverie JM. The significance of digital gene expression profiles. Genome Res. 1997;7(10):986–95. 60. Higgins J, Wilm A, Gibson L, Chenna R, Mcwilliam H, Brown B, et al. Clustal W and Clustal X version
2.0. Bioinformatics. 2007;23:2947–8. 61. Price MN, Dehal PS, Arkin AP. Supplementary Materials Additional file 1 Table S1. Statistics for the unigene sets assembled from Paeonia ostii. Additional file 1 Table S1. Statistics for the unigene sets assembled from Paeonia ostii. Additional file 1 Table S1. Statistics for the unigene sets assembled from P Additional file 2 Figure S1. Heatmap showing correlations among samples. Samples are plotted along the
x- and y-axes. Higher correlations are indicated by darker blues. Additional file 2 Figure S1. Heatmap showing correlations among samples. Samples are plotted along the
x- and y-axes. Higher correlations are indicated by darker blues. Page 24/34 Page 24/34 Page 24/34 Additional file 3 Figure S2 Venn diagrams showing the numbers of expressed genes unique and shared
among the seed kernel, seed testa, and fruit pericarp of Paeonia ostii at six different developmental
stages. Additional file 3 Figure S2 Venn diagrams showing the numbers of expressed genes unique and shared
among the seed kernel, seed testa, and fruit pericarp of Paeonia ostii at six different developmental
stages. Additional file 4 Table S2. Total number of the differentially expressed genes (DEGs) among the three
Paeonia ostii tissues at various time points. Additional file 4 Table S2. Total number of the differentially expressed genes (DEGs) among the three
Paeonia ostii tissues at various time points. Additional file 5 Figure S3. Heatmap showing the hierarchical clustering of the differentially expressed
genes. The samples compared are plotted along the x-axis; the DEGs are plotted along the y-axis. The
color of each intercept represents the log2-transformed fold change value (high: red, low: blue). Additional file 6 Figure S4. GO classification of differentially expressed genes. The number of DEGs is
plotted along the x-axis; the GO terms is plotted along the y-axis. a T1 vs. T2 in the kernel. b T1 vs. T2 in
the testa. c T1 vs. T2 in the pericarp. Additional file 7 Figure S5. KEGG pathways enriched in the genes differentially expressed in the
developing kernel at various time points. The enrichment factor is plotted on the x-axis; pathway names
are shown on the y-axis. The color of each dot reflects the Qvalue, while the size of the dot represents the
number of DEGs. a T1 vs. T3. b T1 vs. T4. c T1 vs. T5. d T1 vs. T6. Additional file 8 Figure S6. KEGG pathways enriched in the genes differentially expressed in the
developing testa at various time points. Supplementary Materials a Kernel. b
Testa. c Pericarp. Additional file 20 Figure S15. Quantitative real-time PCR validation of oil-associated genes. a Kernel. b
Testa. c Pericarp. Additional file 21 Table S6. Primer sequences used for qRT-PCR. Additional file 21 Table S6. Primer sequences used for qRT-PCR. Supplementary Materials Heat map showing the expression patterns of the unigenes associated with
lipid metabolism during the development of the seed kernel (k), seed testa (t), and fruit pericarp (p) of
Paeonia ostii. Labels on the y-axis indicate which plant was used (CS0009 or CS0016), the tissue (k, t, or
p), and the developmental period (35–119 days after fertilization, DAF). Additional file 13 Figure S9. Heat map showing the expression patterns of the unigenes associated with
lipid metabolism during the development of the seed kernel (k), seed testa (t), and fruit pericarp (p) of
Paeonia ostii. Labels on the y-axis indicate which plant was used (CS0009 or CS0016), the tissue (k, t, or
p), and the developmental period (35–119 days after fertilization, DAF). Additional file 14 Figure S10. Phylogenetic relationships among ω-3 FADs from Paeonia ostii and
Arabidopsis thaliana. Additional file 14 Figure S10. Phylogenetic relationships among ω-3 FADs from Paeonia ostii and
Arabidopsis thaliana. Additional file 15 Table S5. Annotations and FPKM values for the 125 transcription factors associated
with FA biosynthesis during the development of the three Paeonia ostii tissues. Additional file 15 Table S5. Annotations and FPKM values for the 125 transcription factors associated
with FA biosynthesis during the development of the three Paeonia ostii tissues. Additional file 15 Table S5. Annotations and FPKM values for the 125 transcription factors associated
with FA biosynthesis during the development of the three Paeonia ostii tissues. Additional file 16 Figure S11. The relative expression levels of genes associated with key TFs. a Kernel. b
Testa. c Pericarp. Additional file 16 Figure S11. The relative expression levels of genes associated with key TFs. a Kernel. b
Testa. c Pericarp. Additional file 17 Figure S12. Quantitative real-time PCR validation of ACCase genes. a Kernel. b Testa. c
Pericarp. Additional file 17 Figure S12. Quantitative real-time PCR validation of ACCase genes. a Kernel. b Testa. c
Pericarp. Additional file 18 Figure S13. Quantitative real-time PCR validation of FAD genes. a Kernel. b Testa. c
Pericarp. Additional file 18 Figure S13. Quantitative real-time PCR validation of FAD genes. a Kernel. b Testa. c
Pericarp. Additional file 19 Figure S14. Quantitative real-time PCR validation of PDAT and DGAT genes. a Kernel. b
Testa. c Pericarp. Additional file 19 Figure S14. Quantitative real-time PCR validation of PDAT and DGAT genes. a Kernel. b
Testa. c Pericarp. Additional file 20 Figure S15. Quantitative real-time PCR validation of oil-associated genes. Supplementary Materials The enrichment factor is plotted on the x-axis; pathway names
are shown on the y-axis. The color of each dot reflects the Qvalue, while the size of the dot represents the
number of DEGs. a T1 vs. T4. b T1 vs. T5. c T1 vs. T6. Additional file 9 Figure S7. KEGG pathways enriched in the genes differentially expressed in the
developing pericarp at various time points. a The number of genes associated with various KEGG
pathways differentially expressed between T1 and T2. b The KEGG pathways overrepresented in the
genes differentially expressed between T1 and T2. The color of each dot reflects the Qvalue, while the
size of the dot represents the number of DEGs. c The up- and downregulated genes in T2 as compared to
T1 that were associated with each KEGG pathway. Additional file 10 Figure S8. KEGG pathways enriched in the genes differentially expressed in the
developing pericarp at various time points. The enrichment factor is plotted on the x-axis; pathway names
are shown on the y-axis. The color of each dot reflects the Qvalue, while the size of the dot represents the
number of DEGs. a T1 vs. T3. b T1 vs. T4. c T1 vs. T5. d T1 vs. T6. Additional file 10 Figure S8. KEGG pathways enriched in the genes differentially expressed in the
developing pericarp at various time points. The enrichment factor is plotted on the x-axis; pathway names
are shown on the y-axis. The color of each dot reflects the Qvalue, while the size of the dot represents the
number of DEGs. a T1 vs. T3. b T1 vs. T4. c T1 vs. T5. d T1 vs. T6. Additional file 11 Table S3. The 1373 unigenes related to lipid metabolism identified in this study,
annotated using Nr, Nt, Swiss-Prot, KEGG, and GO. Additional file 11 Table S3. The 1373 unigenes related to lipid metabolism identified in this study,
annotated using Nr, Nt, Swiss-Prot, KEGG, and GO. Additional file 12 Table S4. The relative expression (in FPKM) of Paeonia ostii genes associated with 10
lipid metabolism pathways. Additional file 12 Table S4. The relative expression (in FPKM) of Paeonia ostii genes associated with 10
lipid metabolism pathways. Page 25/34 Page 25/34 Page 25/34 Additional file 13 Figure S9. Figures Page 26/34 Page 26/34 Figure 1
Development of fruits and seeds in Paeonia ostii. a Fruit development. b Seed and pod development. c
Mature fruit. d Longitudinal sections showing seed development and embryogenesis. DAF, days after
fertilization. Figure 1
Development of fruits and seeds in Paeonia ostii. a Fruit development. b Seed and pod development. c
Mature fruit. d Longitudinal sections showing seed development and embryogenesis. DAF, days after
fertilization. Development of fruits and seeds in Paeonia ostii. a Fruit development. b Seed and pod development. c
Mature fruit. d Longitudinal sections showing seed development and embryogenesis. DAF, days after
fertilization. Page 27/34 Figure 2
Fatty acid (FA) composition of Paeonia ostii fruits and seeds during d
testa. c Fruit pericarp. ALA, α-linolenic acid; LA, linoleic acid; OA, oleic a
acid. Figure 2
Fatty acid (FA) composition of Paeonia ostii fruits and seeds during development. a Seed kernel. b Seed
testa. c Fruit pericarp. ALA, α-linolenic acid; LA, linoleic acid; OA, oleic acid; PA, palmic acid; SA, stearic
acid. Figure 2 Fatty acid (FA) composition of Paeonia ostii fruits and seeds during development. a Seed kernel. b Seed
testa. c Fruit pericarp. ALA, α-linolenic acid; LA, linoleic acid; OA, oleic acid; PA, palmic acid; SA, stearic
acid. Page 28/34 Page 28/34 Figure 3 Figure 3 Figure 3 Figure 3 KEGG pathways enriched in the genes differentially expressed during the development of the Paeonia
ostii seed kernel. a The number of genes differentially expressed between 35 and 49 DAF associated with
various KEGG pathways. b The KEGG pathways overrepresented in the genes differentially expressed
between 35 and 49 DAF. The color of each dot reflects the Qvalue, while the size of the dot represents the
number of DEGs. c The up- and downregulated genes at 49 DAF, as compared to 35 DAF, that were
associated with each KEGG pathway. d The up- and downregulated genes at 63 DAF, as compared to 35
DAF, that were associated with each KEGG pathway. Page 29/34 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 KEGG pathways enriched in the genes differentially expressed during the development of the Paeonia
ostii seed testa. a The KEGG pathways overrepresented in the genes differentially expressed between 35
and 49 DAF. The color of each dot reflects the Qvalue, while the size of the dot represents the number of
DEGs. b The up- and downregulated genes at 49 DAF, as compared to 35 DAF, associated with each
KEGG pathway. c The KEGG pathways overrepresented in the genes differentially expressed between 35
DAF and 63 DAF. d The up- and downregulated genes at 63 DAF, as compared to 35 DAF, associated with
each KEGG pathway. KEGG pathways enriched in the genes differentially expressed during the development of the Paeonia
ostii seed testa. a The KEGG pathways overrepresented in the genes differentially expressed between 35
and 49 DAF. The color of each dot reflects the Qvalue, while the size of the dot represents the number of
DEGs. b The up- and downregulated genes at 49 DAF, as compared to 35 DAF, associated with each
KEGG pathway. c The KEGG pathways overrepresented in the genes differentially expressed between 35
DAF and 63 DAF. d The up- and downregulated genes at 63 DAF, as compared to 35 DAF, associated with
each KEGG pathway. Page 30/34 Figure 5 Figure 5
Genes putatively associated with the lipid metabolism in Paeonia ostii. a Lipid metabolism in P. ostii,
showing putative unigenes associated with FA biosynthesis, TAG biosynthesis, and oil formation. b
Expression patterns of key genes associated with ALA biosynthesis in the P. ostii seed kernel. Figure 5
Genes putatively associated with the lipid metabolism in Paeonia ostii. a Lipid metabolism in P. ostii,
showing putative unigenes associated with FA biosynthesis, TAG biosynthesis, and oil formation. b
Expression patterns of key genes associated with ALA biosynthesis in the P. ostii seed kernel. Figure 5 Figure 5 Genes putatively associated with the lipid metabolism in Paeonia ostii. a Lipid metabolism in P. ostii,
showing putative unigenes associated with FA biosynthesis, TAG biosynthesis, and oil formation. b
Expression patterns of key genes associated with ALA biosynthesis in the P. ostii seed kernel. Page 31/34 Page 31/34 Figure 6
Expression profiles of Paeonia ostii unigenes that are also transcription factors (TFs). a The
of unigenes among TF families. Figure 4 b, c, and d The relative expression levels of genes associate
TFs in the developing kernel (b), testa (c), and pericarp (d). Figure 7 Quantitative real-time PCR validation of fatty acid desaturase genes. a Kernel. b Testa. c Pericarp. Actin
was used as reference genes. Expression values were normalized such that the expression level at 21
DAF was set to 1. Values shown are means of three biological replicates; error bars indicate standard
deviation. One-way ANOVAs were used to compare means among groups, and p value <0.005 was
calculated in the bar chart are shown in Figure 7. Quantitative real-time PCR validation of fatty acid desaturase genes. a Kernel. b Testa. c Pericarp. Actin
was used as reference genes. Expression values were normalized such that the expression level at 21
DAF was set to 1. Values shown are means of three biological replicates; error bars indicate standard
deviation. One-way ANOVAs were used to compare means among groups, and p value <0.005 was
calculated in the bar chart are shown in Figure 7. Figure 6 Expression profiles of Paeonia ostii unigenes that are also transcription factors (TFs). a The distribution
of unigenes among TF families. b, c, and d The relative expression levels of genes associated with key
TFs in the developing kernel (b), testa (c), and pericarp (d). Page 32/34 Page 32/34 Figure 7
Quantitative real-time PCR validation of fatty acid desaturase genes. a Kernel. b Testa. c Pericarp. Actin
was used as reference genes. Expression values were normalized such that the expression level at 21
DAF was set to 1. Values shown are means of three biological replicates; error bars indicate standard
deviation. One-way ANOVAs were used to compare means among groups, and p value <0.005 was
calculated in the bar chart are shown in Figure 7. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Additionalfile2FigureS1.pdf Additionalfile14FigureS10.pdf Additionalfile12TableS4.xlsx Additionalfile15TableS5.xls Page 33/34 Page 33/34 Page 33/34 Additionalfile10FigureS8.pdf
Additionalfile16FigureS11.pdf
Additionalfile6FigureS4.pdf
Additionalfile9FigureS7.pdf
Additionalfile5FigureS3.pdf
Additionalfile19FigureS14.pdf
Additionalfile21TableS6.pdf
Additionalfile18FigureS13.pdf
Additionalfile20FigureS15.pdf
Additionalfile17FigureS12.pdf
Additionalfile13FigureS9.pdf
Additionalfile8FigureS6.pdf
Additionalfile3FigureS2.pdf Page 34/34
|
https://openalex.org/W4387452988
|
https://www.nature.com/articles/s41467-023-42168-8.pdf
|
English
| null |
Author Correction: IFNγ-Stat1 axis drives aging-associated loss of intestinal tissue homeostasis and regeneration
|
Nature communications
| 2,023
|
cc-by
| 254
|
Author Correction: IFNγ-Stat1 axis drives aging-a
intestinal tissue homeostasis and regeneration Omid Omrani
, Anna Krepelova, Seyed Mohammad Mahdi Rasa,
Dovydas Sirvinskas, Jing Lu, Francesco Annunziata, George Garside, Seerat Bajwa,
Susanne Reinhardt, Lisa Adam, Sandra Käppel, Nadia Ducano, Daniela Donna,
Alessandro Ori, Salvatore Oliviero
, Karl Lenhard Rudolph & Francesco Neri Correction to: Nature Communications
https://doi.org/10.1038/s41467-023-41683-y,
published online 30 September 2023
https://doi.org/10.1038/s41467-023-42168-8
Check for updates In this article the author name Dovydas Sirvinskas was incorrectly written as Dovydas Syr-
vinskas. The original article has been corrected. 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 license, and indicate if changes were made. The images or other
third party material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the article’s
Creative Commons license and your intended use is not permitted by statutory regulation or
exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2023 Corrections&amendments Corrections&amendments Author Correction: IFNγ-Stat1 axis drives aging-associated loss of
intestinal tissue homeostasis and regeneration Author Correction: IFNγ-Stat1 axis drives aging-associated loss of
intestinal tissue homeostasis and regeneration nature communications (2023) 14:6302 | 1 1
|
https://openalex.org/W2998168244
|
https://roj.igb.ru/jour/article/download/164/165
|
Kirghiz, Kyrgyz
| null |
A new strategy of keratoplasty: laminating and splitting the donor cornea.
|
Rossijskij oftalʹmologičeskij žurnal
| 2,018
|
cc-by
| 6,488
|
Íîâàÿ ñòðàòåãèÿ êåðàòîïëàñòèêè:
ðàññëîåíèå è ðàçäåëåíèå
ðîãîâèöû äîíîðà Î.Ã. Îãàíåñÿí — ä-ð ìåä. íàóê, âåäóùèé íàó÷íûé ñîòðóäíèê îòäåëà òðàâìàòîëîãèè
è ðåêîíñòðóêòèâíîé õèðóðãèè
Ï.Â. Ìàêàðîâ — ä-ð ìåä. íàóê, âåäóùèé íàó÷íûé ñîòðóäíèê îòäåëà òðàâìàòîëîãèè
è ðåêîíñòðóêòèâíîé õèðóðãèè
À.À. Ãðäèêàíÿí — àñïèðàíò îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè
Â.Ð. Ãåòàäàðÿí — àñïèðàíò îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè
ФГБУ «Московский НИИ глазных болезней им. Гельмгольца» Минздрава России,
105062, Москва, ул. Садовая-Черногрязская, д. 14/19 Î.Ã. Îãàíåñÿí — ä-ð ìåä. íàóê, âåäóùèé íàó÷íûé ñîòðóäíèê îòäåëà òðàâìàòîëîãèè
è ðåêîíñòðóêòèâíîé õèðóðãèè
Ï.Â. Ìàêàðîâ — ä-ð ìåä. íàóê, âåäóùèé íàó÷íûé ñîòðóäíèê îòäåëà òðàâìàòîëîãèè
è ðåêîíñòðóêòèâíîé õèðóðãèè
À.À. Ãðäèêàíÿí — àñïèðàíò îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè
Â.Ð. Ãåòàäàðÿí — àñïèðàíò îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè
ФГБУ «Московский НИИ глазных болезней им. Гельмгольца» Минздрава России,
105062, Москва, ул. Садовая-Черногрязская, д. 14/19 Цель работы — описать первый случай применения одной донорской роговицы для 5 последовательных транс-
плантаций в течение одного дня у пациентов с эндотелиальной дистрофией и кератоконусом. Материал и методы. Полностью отслоенная от стромы донора десцеметова мембрана (ДМ) с эндотелием иссекалась циркулярным и
далее прямым высекателем роговицы донора с эндотелиальной стороны, с формированием 4 частичных десцеме-
тотрансплантатов треугольной формы (1/4 десцеметотрансплантата). Строма роговицы донора иссекалась
циркулярным высекателем заданного диаметра. Одному пациенту с кератоконусом была проведена глубокая перед-
няя послойная кератопластика (DALK) по методике Big Bubble. Затем каждый из ¼ десцеметотрансплантатов
был последовательно пересажен по методике трансплантации десцеметовой мембраны с эндотелием (DMEK) 4
пациентам с дистрофией Фукса и артифакией. Результаты. Во время и после хирургических операций не наблюда-
лось каких-либо осложнений, требующих дополнительного вмешательства. Через 6 мес после DALK максимально
корригируемая острота зрения (МКОЗ) составила 0,66, а после 4 операций ¼ DMEK МКОЗ составила 0,95 ± 0,10
(от 0,8 до 1,0). Плотность эндотелиальных клеток (ПЭК) через 6 мес составила 2839 и 1680 ± 254 клеток/мм2
(от 1492 до 2039 клеток/мм2) соответственно. Заключение. Одна донорская роговица может быть использована
для трансплантации минимум 5 пациентам. Несмотря на то, что выполнение DALK и нескольких операций по
методике ¼ DMEK в течение одного дня технически сложно, такой подход может способствовать кардиналь-
ному сокращению дефицита донорской ткани. Подобный подход требует строгого подбора пациентов и большого
хирургического опыта. ючевые слова: роговица донора, кератопластика, трансплантация роговицы, транспл
цеметовой мембраны, DMEK, Q-DMEK, ¼ DMEK. Для цитирования: Оганесян О.Г., Макаров П.В., Грдиканян А.А., Гетадарян В.Р. Новая стратегия керато-
пластики: расслоение и разделение роговицы донора. Российский офтальмологический журнал. 2018; 11
(3):11-8. Клинические исследования Клинические исследования 11
© О.Г. Оганесян, П.В. Макаров, А.А. Грдиканян, В.Р. Гетадарян
ФГБУ «Московский НИИ глазных болезней им. Гельмгольца» Минздрава России Для цитирования: Оганесян О.Г., Макаров П.В., Грдиканян А.А., Гетадарян В.Р. Новая стратегия керато-
пластики: расслоение и разделение роговицы донора. Российский офтальмологический журнал. 2018; 11
(3):11-8. doi: 10.21516/2072-0076-2018-11-3-11-18 Î.Ã. Îãàíåñÿí — ä-ð ìåä. íàóê, âåäóùèé íàó÷íûé ñîòðóäíèê îòäåëà òðàâìàòîëîãèè
è ðåêîíñòðóêòèâíîé õèðóðãèè
Ï.Â. Ìàêàðîâ — ä-ð ìåä. íàóê, âåäóùèé íàó÷íûé ñîòðóäíèê îòäåëà òðàâìàòîëîãèè
è ðåêîíñòðóêòèâíîé õèðóðãèè
À.À. Ãðäèêàíÿí — àñïèðàíò îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè
Â.Ð. Ãåòàäàðÿí — àñïèðàíò îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè
ФГБУ «Московский НИИ глазных болезней им. Гельмгольца» Минздрава России,
105062, Москва, ул. Садовая-Черногрязская, д. 14/19 Íîâàÿ ñòðàòåãèÿ êåðàòîïëàñòèêè:
ðàññëîåíèå è ðàçäåëåíèå
ðîãîâèöû äîíîðà doi: 10.21516/2072-0076-2018-11-3-11-18 В последние годы различные методы передней
и задней послойной кератопластики стали первой
операцией выбора при лечении патологии рого-
вицы. Благодаря быстрой реабилитации, высоким функциональным результатам и закрытому харак-
теру вмешательства, отмечается ежегодный рост
числа выполняемых эндотелиальных кератопластик
с десцеметорексисом (DSEK) и трансплантаций десцеметовой мембраны с эндотелием (DMEK). Все
чаще эти операции выполняются на более ранних
стадиях развития заболеваний. По мере увеличения
продолжительности жизни населения растет также и
число пациентов, нуждающихся в пересадке рогови-
цы. Параллельно увеличивается возраст доноров, а
количество интактных донорских роговиц при этом
уменьшается по причине увеличивающегося количе-
ства хирургических вмешательств (факоэмульсифика-
ция, имплантация интраокулярных линз, кросслин-
кинг, имплантация интрастромальных сегментов,
колец и инлаев, рефракционная хирургия и др.). Все эти факторы, очевидно, будут усугублять имею-
щийся дефицит донорской ткани, особенно интакт-
ной. По некоторым данным, в настоящее время при-
мерно 2 % роговиц, поступающих в банк тканей, ранее
перенесли те или иные рефракционные операции [1]. скопию глаза на щелевой лампе (Opton 30 SL-M,
Германия), оценку состояния заднего сегмента глаза,
кератоанализирование (Galilei 6, Ziemer Ophthalmic
Systems AG, Швейцария). Аналогичные обследова-
ния, а также измерение ПЭК (Confoscan 4, Nidek Co. Ltd., Япония) проводились во всех 5 случаях через
3 и 6 мес после операции. Минимальный срок на-
блюдения всех пациентов составил 6 мес. скопию глаза на щелевой лампе (Opton 30 SL-M,
Германия), оценку состояния заднего сегмента глаза,
кератоанализирование (Galilei 6, Ziemer Ophthalmic
Systems AG, Швейцария). Аналогичные обследова-
ния, а также измерение ПЭК (Confoscan 4, Nidek Co. Ltd., Япония) проводились во всех 5 случаях через
3 и 6 мес после операции. Минимальный срок на-
блюдения всех пациентов составил 6 мес. Операция DALK проводилась по очередности
первой и под общей анестезией. Затем поочеред-
но выполнялись операции ¼ DMЕK под местной
инстилляционной анестезией. Формирование всех
трансплантатов и все хирургические вмешательства
выполнены одним хирургом. Все пациенты давали добровольное информиро-
ванное согласие. Исследования проводились в соот-
ветствии с положениями Хельсинкской декларации. Подготовка трансплантатов. Для выполнения
5 операций использована одна роговица донора
48 лет, мужского пола, с ПЭК = 2774 клеток/мм2. На сегодняшний день во многих странах мира
наблюдается нехватка донорского материала. В раз-
вивающихся странах ежегодная потребность в до-
норской ткани достигает 300 тыс. роговиц, при этом
доступно только 15 тыс. роговиц [2, 3]. Отслаивание десцеметовой мембраны (ДМ) с
эндотелием выполняли по ранее описанной методи-
ке [5]. Вкратце техника операции была следующей. Новая стратегия кератопластики:
расслоение и разделение роговицы донора
12 Íîâàÿ ñòðàòåãèÿ êåðàòîïëàñòèêè:
ðàññëîåíèå è ðàçäåëåíèå
ðîãîâèöû äîíîðà После аспирационной фиксации корнеосклераль-
ного диска донора эндотелием вверх ДМ с пери-
ферической тканью, включающей трабекулярную
сеть, полностью отслаивалась от задней поверх-
ности стромы (рис. 1, А). После окрашивания ДМ
0,06 % раствором трипанового синего (Vision Blue,
DORC International, Нидерланды) последняя поме-
щалась на мягкую контактную линзу, в свою очередь
расположенную в высекателе донорской роговицы
(Barron vacuum donor Punch; Katena Products Inc,
США) (рис. 1, Б). По данным Федеральной службы государствен-
ной статистики, в 2015 г. в России было проведено
3453 кератопластики, в том числе 155 операций у
детей (сведения с официального сайта Федеральной
службы государственной статистики РФ: http://www. gks.ru/). Очевидно, что для увеличения количества и
доступности донорской ткани требуется системный
подход, поддержка государства и общества, внуши-
тельные инвестиции, время и т. д. На наш взгляд,
наиболее быстрым и очевидным способом увеличе-
ния количества трансплантаций является рациональ-
ное и экономное использование донорской ткани,
внедрение современных методов кератопластики, а
также индивидуальный подход к хирургии роговицы
в каждом клиническом случае [4]. Циркулярным стандартным высекателем Barron
диаметром 9,5 мм вырезали ДМ с эндотелиальной
стороны вместе с контактной линзой. Не перемещая
ДМ с подлежащей контактной линзой с помощью
разработанного и изготовленного на заказ высека-
теля (заявка на полезную модель, регистрационный
№ 2017137958) (рис. 1, В, 2) разрезали ДМ с эндо-
телиальной стороны вместе с подлежащей контакт-
ной линзой на 4 равные четверти (рис. 1, Г—Е). Все
4 части ДМ помещали во флакон, где они самопро-
извольно сворачивали в рулон эндотелием наружу и
свободно плавали в консерванте. ЦЕЛЬ работы — описать первый случай при-
менения одной донорской роговицы для 5 по-
следовательных трансплантаций в течение одного
операционного дня у пациентов с эндотелиальной
дистрофией и кератоконусом. Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 МАТЕРИАЛ И МЕТОДЫ А
Г
Б
Д
В
Е А
Г
Б
Д А
Г
Б
Д В Е Д Е Рис. 1. Этапы формирования частичных (1/4) десцеметотрансплантатов. А — отслоение десцеметовой мембраны с каймой трабеку-
лярной сети от корнеосклерального диска донора. Б — отслоенная десцеметова мембрана с каймой трабекулярной сети расположена
на контактной линзе в высекателе Barron. В — десцеметова мембрана с подлежащей контактной линзой разделяется на 2 половины
разработанным высекателем (вид сверху) с полукруглым лезвием (черные стрелки). Г — после 90-градусного поворота режущего
блока каждая половина десцеметовой мембраны (1/2) затем снова разделена на 2 половины тем же высекателем. После двойного
взаимно перпендикулярного разреза получены 4 равные части контактной линзы (Д) и 4 десцеметотрансплантата для ¼ DMEK (Е). Fig. 1. Images of surgical steps of Q-grafts preparation. А — DM stripping with the adjacent trabecular meshwork from the corneoscleral
button. Б — isolated DM with adjacent trabecular meshwork was placed onto contact lens which was located on the cutting block of Barron
punch. В — the DM with underlying contact lens was first divided into 2 equal halves with a custom made single used punch for linear cutting of
DM (view from above) with semicircular blade (black arrows). Г — after a 90-degree turn of the cutting block, each half of DM was then again
divided into 2 equal halves with the same custom-made punch. After a double cut 4 pieces of contact lens (Д) and 4 pieces of Q-grafts of DM
for Q-DMEK were obtained (Е). g. 1. Images of surgical steps of Q grafts preparation. А
DM stripping with the adjacent trabecular meshwork from the corneoscleral
utton. Б — isolated DM with adjacent trabecular meshwork was placed onto contact lens which was located on the cutting block of Barron
unch. В — the DM with underlying contact lens was first divided into 2 equal halves with a custom made single used punch for linear cutting of
M (view from above) with semicircular blade (black arrows). Г — after a 90-degree turn of the cutting block, each half of DM was then again
vided into 2 equal halves with the same custom-made punch. After a double cut 4 pieces of contact lens (Д) and 4 pieces of Q-grafts of DM
or Q-DMEK were obtained (Е) Рис. 2. Разработанный высекатель для линейного прорезывания десцеметовой мем-
браны. A — продольный вид высекателя с полукруглым лезвием (черные стрелки). Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 МАТЕРИАЛ И МЕТОДЫ Б —
поперечный вид высекателя с полукруглым лезвием (черные стрелки). Fig. 2. Custom-made punch for linear cutting of DM. А — longitudinal view of punch with
semicircular blade (black arrows). Б — transverse view of punch with semicircular blade
(black arrows). А
Б 30G формировали канал длиной
2–3 мм, в который вводили каню-
лю (Sarnicola, ASICO, Inc, США)
для DALK диаметром 27G, при-
соединенную к шприцу объемом
5 мл. Канюлю проводили к центру
роговицы максимально близко
к ДМ и, надавливая на поршень
шприца, производили пневмати-
зацию роговицы до формирования
«большого пузыря» диаметром
меньше диаметра кератэктомии. Не извлекая канюли, выполняли
парацентез и частично опорож-
няли переднюю камеру глаза. После этого продолжали введение
воздуха в роговицу до достижения
диаметра «большого пузыря»,
диаметра кератэктомии (рис. 3, А). Лезвием осуществляли вскрытие
«большого пузыря» и вводили в его
вный вискоэластик (Healon GV, AMO,
чная часть стромы удалялась тупоко-
охирургическими ножницами после
ием крест-накрест.Поверхность ДМ
мывалась от вискоэластика (рис. 3, Б). 30G формировали канал длиной
2–3 мм, в который вводили каню-
лю (Sarnicola, ASICO, Inc, США)
для DALK диаметром 27G, при-
соединенную к шприцу объемом
5 мл. Канюлю проводили к центру
роговицы максимально близко
к ДМ и, надавливая на поршень
шприца, производили пневмати-
зацию роговицы до формирования
«большого пузыря» диаметром
меньше диаметра кератэктомии. Не извлекая канюли, выполняли
парацентез и частично опорож-
няли переднюю камеру глаза. После этого продолжали введение
воздуха в роговицу до достижения
диаметра «большого пузыря»,
диаметра кератэктомии (рис. 3, А). Лезвием осуществляли вскрытие
«большого пузыря» и вводили в его
полость когезивный вискоэластик (Healon GV, AMO,
США). Остаточная часть стромы удалялась тупоко-
нечными микрохирургическими ножницами после
ее разреза лезвием крест-накрест.Поверхность ДМ
тщательно промывалась от вискоэластика (рис. 3, Б). десцеметовой мем-
ерные стрелки). Б —
ки). al view of punch with
h semicircular blade
Б А Б Рис. 2. Разработанный высекатель для линейного прорезывания десцеметовой мем-
браны. A — продольный вид высекателя с полукруглым лезвием (черные стрелки). Б —
поперечный вид высекателя с полукруглым лезвием (черные стрелки). Fig. 2. Custom-made punch for linear cutting of DM. А — longitudinal view of punch with
semicircular blade (black arrows). Б — transverse view of punch with semicircular blade
(black arrows). роговицы реципиента вакуумным трепаном Hes-
sburg — Barron (Katena Products Inc, США) диаметром
9 мм делали насечку роговицы глубиной до 80 %
от толщины стромы роговицы. Новая стратегия кератопластики:
расслоение и разделение роговицы донора МАТЕРИАЛ И МЕТОДЫ В течение одного операционного дня мы по-
следовательно выполнили глубокую переднюю
послойную кератопластику (DALK) пациенту с ке-
ратоконусом (КК) и 4 операции методикой ¼ DMEK
4 пациентам с дистрофией Фукса и артифакией. В
послеоперационном периоде фиксировалось коли-
чество и характер осложнений, показатели макси-
мально корригированной остроты зрения (МКОЗ)
и плотности эндотелиальных клеток (ПЭК) спустя
6 мес после операции. Оставшийся корнеосклеральный диск без ДМ
после маркировки центра и 4 основных меридианов
переносили в пустой контейнер для донорской рого-
вицы и помещали на хранение в холодильную камеру. На случай выбраковки донорской ткани в про-
цессе формирования трансплантатов либо интраопе-
рационных осложнений резервная роговичная ткань
имелась в наличии. Глубокая передняя послойная кератопластика. Операцию DALK выполняли методикой «большого
пузыря», описанной M. Anwar и K. Teichmann [6]. После маркировки центра и 4 основных меридианов Предоперационное обследование пациентов
включало определение МКОЗ на проекторе опто-
типов (SZP 350, Zeiss, Германия) и внутриглазного
давления (iCare, Finland Oy, Финляндия), биомикро- Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Рис. 1. Этапы формирования частичных (1/4) десцеметотрансплантатов. А — отслоение десцеметовой мембраны с каймой трабеку-
лярной сети от корнеосклерального диска донора. Б — отслоенная десцеметова мембрана с каймой трабекулярной сети расположена
на контактной линзе в высекателе Barron. В — десцеметова мембрана с подлежащей контактной линзой разделяется на 2 половины
разработанным высекателем (вид сверху) с полукруглым лезвием (черные стрелки). Г — после 90-градусного поворота режущего
блока каждая половина десцеметовой мембраны (1/2) затем снова разделена на 2 половины тем же высекателем. После двойного
взаимно перпендикулярного разреза получены 4 равные части контактной линзы (Д) и 4 десцеметотрансплантата для ¼ DMEK (Е). Fig. 1. Images of surgical steps of Q-grafts preparation. А — DM stripping with the adjacent trabecular meshwork from the corneoscleral
button. Б — isolated DM with adjacent trabecular meshwork was placed onto contact lens which was located on the cutting block of Barron
punch. В — the DM with underlying contact lens was first divided into 2 equal halves with a custom made single used punch for linear cutting of
DM (view from above) with semicircular blade (black arrows). Г — after a 90-degree turn of the cutting block, each half of DM was then again
divided into 2 equal halves with the same custom-made punch. After a double cut 4 pieces of contact lens (Д) and 4 pieces of Q-grafts of DM
for Q-DMEK were obtained (Е). Новая стратегия кератопластики:
расслоение и разделение роговицы донора
14 РЕЗУЛЬТАТЫ Возраст пациента, перенесшего операцию
DALK, составлял 52 года. МКОЗ до операции со-
ставляла 0,16, а через 6 мес после операции она
улучшилась до 0,66. На момент последнего осмотра
у пациента не наблюдалось никаких осложнений,
трансплантат сохранял прозрачность. Конфокальная
микроскопия показала ПЭК = 2839 клеток/мм2. Частичная трансплантация десцеметовой мембра-
ны с эндотелием (1/4 DMEK). Техника ¼ DMEK во всех
4 случаях была идентичной и в целом аналогичной тех-
нике стандартной DMEK, описанной нами ранее [7]. После выполнения единственного парацен-
теза в меридиане 10 ч выполняли десцеметорексис
(7–8 мм) под воздухом при помощи обратного крюч-
ка Sinskey (DORC International, Нидерланды). Лим-
бальный тоннельный разрез шириной 1,6 мм выпол-
няли в меридиане 12 ч с помощью кератома (MANI
inc., Япония). Частичные десцеметотрансплантаты
окрашивали 0,06 % раствором трипанового синего и
аспирировали в браунюлю диаметром 18G (Apexmed
International B.V., Нидерланды), прикрепленную к
шприцу объемом 2 мл и заполненную раствором BSS
(«Способ трансплантации десцеметовой мембра-
ны — микроинвазивная десцеметопластика», патент
РФ № 2394532 от 20.07.2010). Путем ирригации
трансплантат вводили в переднюю камеру глаза
реципиента через сформированный разрез в мери-
диане 12 ч. Манипулируя ab externo, трансплантат
центрировали, раскрывали рулон эндотелием вниз,
и переднюю камеру полностью заполняли возду-
хом, который оставляли не менее чем на 2 ч. Через
2 ч после операции часть воздуха выпускалась через
парацентез под биомикроскопом. Средний возраст пациентов, перенесших опе-
рации ¼DMEK, составлял 69,0 ± 1,1 года (от 52 до
70 лет). Средняя МКОЗ перед операцией составляла
0,35 ± 0,10, а через 6 мес после ¼ DMEK повыси-
лась до 0,95 ± 0,10. На момент последнего осмотра
все роговицы сохраняли прозрачность. Через 6 мес
после операции средняя ПЭК с дооперационных
2774 клеток/мм2 уменьшилась до 1680 ± 254 клеток/мм2
(от 1492 до 2039 клеток/мм2). Частичное неприлегание десцеметотран-
сплантата имело место в 2 из 4 случаев ¼ DMEK. Тем не менее ни в одном случае не потребовалось
повторного введения воздуха в переднюю камеру. Обследование под щелевым биомикроскопом через
6 мес после операции показало полное прилегание
всех частичных десцеметотрансплантатов. Других
осложнений, таких как реакция отторжения, несо-
стоятельность эндотелия трансплантата или синдром
Urrets-Zavalia, за весь период наблюдения не зафик-
сировано (рис. 4, 5). Рис. 3. Этапы глубокой передней послойной кератопластики. A — формирование big bubble, равного диаметру трепанационного от-
верстия. Б — обнаженная десцеметова мембрана реципиента после полной стромэктомии. В — строма донора фиксирована в ложе
8 узловыми швами. Fig. 3. Images of surgical steps of DALK. МАТЕРИАЛ И МЕТОДЫ После послойной ке-
ратэктомии с помощью одноразовой иглы диаметром роговицы реципиента вакуумным трепаном Hes-
sburg — Barron (Katena Products Inc, США) диаметром
9 мм делали насечку роговицы глубиной до 80 %
от толщины стромы роговицы. После послойной ке-
ратэктомии с помощью одноразовой иглы диаметром 13 Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Корнеосклеральный диск донора без ДМ распо-
лагали в высекателе Barron и вырезали трансплантат
диаметром 9 мм, который фиксировали в ложе ро-
говицы реципиента 8 узловыми (рис. 3, В) и одним
непрерывным нейлоновым швом 10/0. Во время и после хирургических операций не
отмечалось развития каких-либо осложнений, тре-
бовавших вмешательств. После операции во всех слу-
чаях назначались глазные капли 0,1 % дексаметазона
каждые 4 ч, глазные капли 0,5 % моксифлоксацина
гидрохлорида 3 раза в сутки и заменители слезы без
консервантов каждые 4 ч. Антибиотикотерапию
прекращали через 2 нед после операции, а кратность
инстилляций остальных медикаментов постепенно
сокращалась в течение 6 мес. В послеоперационном периоде медикаментоз-
ная терапия включала глазные капли 0,1 % дексаме-
тазона каждые 4 ч, глазные капли 0,5 % моксифлокса-
цина гидрохлорида 3 раза в сутки и заменители слезы
без консервантов каждые 4 ч. Антибиотикотерапию
прекращали через 3 нед после операции, кратность
инстилляций остальных препаратов постепенно
снижалась в течение 6 мес. Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Рис. 3. Этапы глубокой передней послойной кератопластики. A — формирование big bubble, равного диаметру трепанационного от-
верстия. Б — обнаженная десцеметова мембрана реципиента после полной стромэктомии. В — строма донора фиксирована в ложе
8 узловыми швами.
Fig. 3. Images of surgical steps of DALK. A — formation of a “big bubble” equal to the trephination diameter. Б — the stroma was removed and
DM of the host is denuded В — the donor button was sutured in place using 8 interrupted 10-0 monofilament nylon sutures РЕЗУЛЬТАТЫ A — formation of a “big bubble” equal to the trephination diameter. Б — the stroma was removed and
DM of the host is denuded. В — the donor button was sutured in place using 8 interrupted 10-0 monofilament nylon sutures. А
Б
В А
Б В А Б Б В А Рис. 3. Этапы глубокой передней послойной кератопластики. A — формирование big bubble, равного диаметру трепанационного от-
верстия. Б — обнаженная десцеметова мембрана реципиента после полной стромэктомии. В — строма донора фиксирована в ложе
8 узловыми швами. Fig 3 Images of surgical steps of DALK A
formation of a “big bubble” equal to the trephination diameter Б
the stroma was removed and 8 узловыми швами. Fig. 3. Images of surgical steps of DALK. A — formation of a “big bubble” equal to the trephination diameter. Б — the stroma was removed and
DM of the host is denuded. В — the donor button was sutured in place using 8 interrupted 10-0 monofilament nylon sutures. узловыми швами. ig. 3. Images of surgical steps of DALK. A — formation of a “big bubble” equal to the trephination diameter. Б —
M of the host is denuded. В — the donor button was sutured in place using 8 interrupted 10-0 monofilament n Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Рис. 4. Клинический случай ¼ DMEK № 1. Биомикроскопическая картина и карта пахи-
метрии до операции (A, Г), через 3 дня (Б, Д) и через 6 мес (В, Е) после ¼ DMEK. Карты
пахиметрии, а также изображения щелевого биомикроскопа демонстрируют постепенное
истончение роговицы. Fig. 4. Q-DMEK case № 1. Slit-lamp images and pachymetry maps preoperatively (A, Г), at
3 days (Б, Д), and at 6 months (В, Е) after Q-DMEK. Pachymetry maps, as well as slit-lamp
images show gradual cornea thinning. А
Г
Б
Д
В
Е Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 ОБСУЖДЕНИЕ А ОБСУЖДЕНИЕ
Первая успешная пересад-
ка роговицы была проведена
Eduard Zirm в 1905 г. Он так-
же первым применил принцип
рационального использования
донорской ткани, выполнив
2 частичные сквозные кератопла-
стики из одной роговицы доно-
ра [8]. Есть несколько публика-
ций, посвященных использова-
нию одной донорской рогови-
цы для выполнения нескольких
трансплантаций. R. Vajpayee и
соавт. [4] описывают примене-
ние одного корнеосклерального
диска донора для лечения 3 па-
циентов, которым выполнили
DALK, DSEK и пересадку лим-
бальных стволовых клеток. J. Lie
и соавт. [9] разработали методику
одновременного выполнения
2 операций из одной роговицы
донора. Существуют и другие
публикации, касающиеся рацио-
нального использования донор-
ской ткани, указывающие на то,
что значительный дефицит до-
норских роговиц во многих частях
мира является по-настоящему
большой проблемой [10–15]. Рас-
слоение и разделение донор-
ской ткани может являться од-
ним из путей сокращения дефицита донорс-
кой ткани. Рис. 4. Клинический
метрии до операции
пахиметрии, а также и
истончение роговицы
Fig. 4. Q-DMEK case
3 days (Б, Д), and at 6
images show gradual Первая успешная пересад-
ка роговицы была проведена
Eduard Zirm в 1905 г. Он так-
же первым применил принцип
рационального использования
донорской ткани, выполнив
2 частичные сквозные кератопла-
стики из одной роговицы доно-
ра [8]. Есть несколько публика-
ций, посвященных использова-
нию одной донорской рогови-
цы для выполнения нескольких
трансплантаций. R. Vajpayee и
соавт. [4] описывают примене-
ние одного корнеосклерального
диска донора для лечения 3 па-
циентов, которым выполнили
DALK, DSEK и пересадку лим-
бальных стволовых клеток. J. Lie
и соавт. [9] разработали методику
одновременного выполнения
2 операций из одной роговицы
донора. Существуют и другие
публикации, касающиеся рацио-
нального использования донор-
ской ткани, указывающие на то,
что значительный дефицит до-
норских роговиц во многих частях
мира является по-настоящему
большой проблемой [10–15]. Рас-
слоение и разделение донор-
ской ткани может являться од-
ним из путей сокращения деф
кой ткани. А Б В Г
Д
Е Д
Е Г Е Д Рис. 4. Клинический случай ¼ DMEK № 1. Биомикроскопическая картина и карта пахи-
метрии до операции (A, Г), через 3 дня (Б, Д) и через 6 мес (В, Е) после ¼ DMEK. Карты
пахиметрии, а также изображения щелевого биомикроскопа демонстрируют постепенное
истончение роговицы. Fig. 4. Q-DMEK case № 1. Slit-lamp images and pachymetry maps preoperatively (A, Г), at
3 days (Б, Д), and at 6 months (В, Е) after Q-DMEK. Pachymetry maps, as well as slit-lamp
images show gradual cornea thinning. Рис. 4. Новая стратегия кератопластики:
расслоение и разделение роговицы донора Новая стратегия кератопластики:
расслоение и разделение роговицы донора
16 ОБСУЖДЕНИЕ Клинический случай ¼ DMEK № 1. Биомикроскопическая картина и карта пахи-
метрии до операции (A, Г), через 3 дня (Б, Д) и через 6 мес (В, Е) после ¼ DMEK. Карты
пахиметрии, а также изображения щелевого биомикроскопа демонстрируют постепенное
истончение роговицы. Fig. 4. Q-DMEK case № 1. Slit-lamp images and pachymetry maps preoperatively (A, Г), at
3 days (Б, Д), and at 6 months (В, Е) after Q-DMEK. Pachymetry maps, as well as slit-lamp
images show gradual cornea thinning. МКОЗ и ПЭК, как и стандартная операция DMEK. Стратегия использования одной донорской рогови-
цы для проведения нескольких пересадок, впервые
реализованная E. Zirm, открывает возможности
значительного увеличения количества выполняемых
трансплантаций имеющимися в распоряжении объ-
емами донорских роговиц. Теоретически можно еще
увеличить количество (до 6) проводимых трансплан-
таций при условии использования донорской лим-
бальной ткани для аллолимбальной трансплантации. Разделение на части донорской ткани активно
применяется в других областях клинической транс-
плантологии. Например, разделение печени умень-
шает потребность в ней примерно на 40 % [16, 17]. Ежегодная потребность развивающихся стран в до-
норской роговице составляет примерно 300 тыс.,
тогда как доступно только 15 тыс. трансплантатов, и
почти половина из них не подходит для пересадки [4]. Мы считаем, что следует пересмотреть критерии
оценки пригодности донорской ткани для пересад-
ки, поскольку на сегодняшний день можно успешно
пересаживать как донорскую роговицу целиком, так
и отдельные слои, которые ранее выбраковывались
[1, 18–25]. Применение ламеллярной кератопла-
стики в ургентной хирургии может являться про-
явлением рационального использования донорских
тканей [26–30]. В рамках настоящего исследования мы опи-
сываем первый случай получения 5 трансплантатов
из одной донорской роговицы. Метод ¼ DMEK со-
вмещает в себе преимущества стандартной DMEK и
имплантации ДМ с эндотелием (DMET). Основным
преимуществом DMEK по сравнению с другими
методами является быстрое восстановление зрения. Основным преимуществом ¼ DMEK, по сравнению с
обычным DMEK и ½ DMEK, является максимально
эффективное использование донорской роговицы. Кроме того, ¼ DMEK позволяет сохранить больше
клеток реципиента и в то же время создает благопри-
ятные условия для эндотелизации, поскольку дес-
цеметорексис при ¼ DMEK всегда больше размера
частичного десцеметотрансплантата. Выполнение Представленные клинические случаи демон-
стрируют возможность последовательного проведе-
ния 5 операций: DALK и 4 ¼ DMEK в течение одного
дня — с использованием одной роговицы донора. Обследование через 6 мес показало, что частичный
DMEK позволил достичь таких же показателей 15 Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Новая стратегия кератопластики:
расслоение и разделение роговицы донора А
Г
Б
Д
В
Е
Рис. 5. ОБСУЖДЕНИЕ Клинический случай ¼ DMEK № 2 (A, Б), № 3 (В, Г) и № 4 (Д, Е). Биомикроскопи-
ческая картина до операции (A, В, Д) и спустя 6 мес (Б, Г, Е) после ¼ DMEK. Биомикро-
скопическая картина свидетельствует об отсутствии участков неприлегания частичного
десцеметотрансплантата и об уменьшении толщины роговицы после операции ¼ DMEK. Fig. 5. Q-DMEK cases № 2 (A, Б), № 3 (В, Г) and № 4 (Д, Е). Slit-lamp images preoperatively
(A, В, Д) and at 6 months (Б, Г, Е) after Q-DMEK. Slit-lamp images show absence of any
detachment of Q-graft and decreasing the thickness of cornea after surgery. А
Б
В А
Б А
Б
В В донорского материала, професси-
онализма хирургической бригады. Тем не менее, несмотря на слож-
ности такой стратегии, ее преиму-
щества очевидны. Рациональное
использование донорской ткани
является одним из важнейших
очевидных и доступных способов
быстрого увеличения количества
пересадок и, следовательно, выле-
ченных пациентов. Такой подход
с применением расслоения и раз-
деления донорской роговицы спо-
собен значимо снизить дефицит
донорских тканей. Мы считаем,
что предварительная подготовка
и загрузка частичных десцеме-
тотрансплантатов сотрудниками
глазных банков может существен-
но облегчить методику ¼ DMEK. донорского материала, професси-
онализма хирургической бригады. Тем не менее, несмотря на слож-
ности такой стратегии, ее преиму-
щества очевидны. Рациональное
использование донорской ткани
является одним из важнейших
очевидных и доступных способов
быстрого увеличения количества
пересадок и, следовательно, выле-
ченных пациентов. Такой подход
с применением расслоения и раз-
деления донорской роговицы спо-
собен значимо снизить дефицит
донорских тканей. Мы считаем,
что предварительная подготовка
и загрузка частичных десцеме-
тотрансплантатов сотрудниками
глазных банков может существен-
но облегчить методику ¼ DMEK. В А Б Г
Д
Е Г
Д
Е
Рис. 5. Клинический случай ¼ DMEK № 2 (A, Б), № 3 (В, Г) и № 4 (Д, Е). Биомикроскопи-
ческая картина до операции (A, В, Д) и спустя 6 мес (Б, Г, Е) после ¼ DMEK. Биомикро-
скопическая картина свидетельствует об отсутствии участков неприлегания частичного
десцеметотрансплантата и об уменьшении толщины роговицы после операции ¼ DMEK. Fig. 5. Q-DMEK cases № 2 (A, Б), № 3 (В, Г) and № 4 (Д, Е). Slit-lamp images preoperatively
(A, В, Д) and at 6 months (Б, Г, Е) after Q-DMEK. Slit-lamp images show absence of any
detachment of Q-graft and decreasing the thickness of cornea after surgery. Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Е Г Д Д Рис. 5. Клинический случай ¼ DMEK № 2 (A, Б), № 3 (В, Г) и № 4 (Д, Е). Биомикроскопи-
ческая картина до операции (A, В, Д) и спустя 6 мес (Б, Г, Е) после ¼ DMEK. Биомикро-
скопическая картина свидетельствует об отсутствии участков неприлегания частичного
десцеметотрансплантата и об уменьшении толщины роговицы после операции ¼ DMEK. Fig. 5. Q-DMEK cases № 2 (A, Б), № 3 (В, Г) and № 4 (Д, Е). Slit-lamp images preoperatively
(A, В, Д) and at 6 months (Б, Г, Е) after Q-DMEK. Slit-lamp images show absence of any
detachment of Q-graft and decreasing the thickness of cornea after surgery. Конфликт интересов: отсут-
ствует. ¼ DMEK является вполне оправданным, так как
патологические изменения при дистрофии Фукса
в основном затрагивают ограниченную область,
расположенную, как правило, в центре роговицы. Для проведения ¼ DMEK необходим строгий подбор
пациентов исходя из потенциала остроты зрения,
глубины передней камеры, площади и степени эн-
дотелиальной дистрофии. Среднее значение МКОЗ
всех пациентов с дистрофией Фукса и артифакией
перед ¼ DMEK составляло 0,35 ± 0,10 (от 0,2 до 0,4),
что указывает на наличие начальных и незначитель-
ных изменений в структуре роговицы. Прозрачность финансовой деятельности: никто из
авторов не имеет финансовой заинтересованности в
представленных материалах или методах. ЗАКЛЮЧЕНИЕ Представленные клиниче-
ские случаи показывают, что
одна роговица донора может быть
успешно пересажена как мини-
мум 5 пациентам. Несмотря на то,
что последовательное выполне-
ние DALK и 4 ¼ DMEK в течение
одного операционного дня пред-
ставляет технические и логисти-
ческие сложности, тем не менее
подобная стратегия способствует
кардинальному сокращению де-
фицита роговичной ткани. При
этом необходим строгий выбор
пациентов для ¼ DMEK. Прозрачность финансовой деятельности: никто из
авторов не имеет финансовой заинтересованности в
представленных материалах или методах. Eine erfolgreiche totale keratoplastik. Arch. Ophthalmol. 1906; 64: 580–93 24. Оганесян О.Г., Яковлева С.С., Харлампиди М.П. и др. Рацио-
нальное использование донорского материала: собственный
десятилетний опыт, возможные пути развития и данные
литературы. Медицинский журнал Российской Федерации. 2016; 22: 193–7
Oganesyan O.G., Yakovleva S.S., Kharlampidi M.P., et al. The rationale application of donor material: original 251 ten
years’ experience, possible ways of development and literature
data. Meditsinskij zhurnal Rossijskoj Federatsii. 2016; 22: 193–7
(in Russian) 9. Lie J.T., Groeneveld-van Beek E.A., Ham L., et al. More efficient
use of donor corneal tissue with Descemet membrane endothelial
keratoplasty (DMEK): two lamellar keratoplasty procedures with
one donor cornea. Br. J. Ophthalmol. 2010; 94: 1265–6. doi:
10.1136/bjo.2009.171629 10. Heindl L.M., Riss S., Bachmann B.O., et al. Split cornea
transplantation for 2 recipients: a new strategy to reduce corneal
tissue cost and shortage. Ophthalmology. 2011; 118: 294–301. doi:
10.1016/j.ophtha.2010.05.025 11. Sharma N., Agarwal P., Titiyal J., et al. Optimal use of donor corneal
tissue: one cornea for two recipients. Cornea. 2011; 30: 1140–4. doi: 10.1097/ICO.0b013e318209d23c 25. Нероев В.В., Оганесян О.Г., Гундорова Р.А. и др. Использова-
ние глаз с кератотомиями в качестве трансплантационного
материала. Российский медицинский журнал. 2011; 5: 32–4. Neroev V.V., Oganesyan O.G., Gundorova R.A., et al. Use of
keratotomy eyes as a transplant. Rossijskij meditsinskij zhurnal. 2011; 5: 32–4 (in Russian) 12. Müller T.M., Lavy I., Baydoun L., et al. Case report of Quarter-
Descemet membrane endothelial keratoplasty for Fuchs
endothelial dystrophy. Cornea. 2017; 36: 104–7. doi: 10.1097/
ICO.0000000000001008 13. Yoeruek E., Schmidt K.U. Current approaches to combat the shor-
tage of corneal tissues: split-DMEK and double-split keratoplasty. Cornea. 2015; 34: 6-9. doi: 10.1097/ICO.0000000000000341 26. Fang W., Xiuming J., Yesheng X., et al. Treatment of corneal
perforation with lenticules from small incision lenticule extraction
surgery: a preliminary study of 6 patients. Cornea. 2015; 34: 658–63. doi: 10.1097/ICO.0000000000000397 14. Rasouli M., Caraiscos V.B., Slomovic A.R. Efficacy of routine
notification and request on reducing corneal transplantation wait
times in Canada. Can. J. Ophthalmol. 2009; 44: 31–5. doi: 10.3129/
i08–187 27. Graue-Hernandez E.O., Zuniga-Gonzalez I., Hernandez-Camare-
na J.C., et al. Tectonic DSAEK for the management of impend-
ing corneal perforation. Case Reports in Ophthalmol. Med. 2012;
Article ID 916528, 4 pages. http://dx.doi.org/10.1155/2012/916528 15. De By T.M. Shortage in the face of plenty: improving the allocation
of corneas for transplantation. Dev. Ophthalmol. 2003; 36: 56–61. 28. Nahum Y., Bahar I., Busin M. Прозрачность финансовой деятельности: никто из
авторов не имеет финансовой заинтересованности в
представленных материалах или методах. Литература/References 1. Phillips P., Terry M., Shamie N., et al. Descemet’s stripping
automated endothelial keratoplasty (DSAEK) using corneal donor
tissue not acceptable for use in penetrating keratoplasty as a result
of anterior stromal scars, pterygia, and previous corneal refractive
surgical procedures. Cornea. 2009; 28: 871–6. doi: 10.1097/
ICO.0b013e318199f8d7 2. Tandon R., Verma K., Vanathi M., et al. Factors affecting eye
donation from postmortem cases in a tertiary care hospital. Cornea. 2004; 23: 597–601 Проведение в течение одного дня операции
DALK и последующих операций ¼ DMEK требует
сложной логистики — одновременного присутствия
всех пациентов в клинике, быстрой и адекватной
дооперационной подготовки пациентов, своевре-
менного послеоперационного осмотра, резервного ;
3. Krishnaiah S., Kovai V., Nutheti R., et al. Awareness of eye donation
in the rural population of India. Indian J. Ophthalmol. 2004; 52:
73–8. 4. Vajpayee R.B., Sharma N., Jhanji V., et al. One donor cornea for 3 re-
cipients: a new concept for corneal transplantation surgery. Arch. Ophthalmol. 2007; 125: 552–4. doi: 10.1001/archopht.125.4.552 Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 previous laser in situ keratomileusis or photorefractive keratectomy:
a case series and donor cap histopathology. Cornea. 2012; 31:
533–7. doi: 10.1097/ICO.0b013e31820142be 5. Groeneveld-van Beek E.A., Lie J.T., von der Wees J., et al. Standardized ‘no-touch’ donor tissue preparation for DALK and
DMEK: harvesting undamaged anterior and posterior transplants
from the same donor cornea. Acta Ophthalmol. 2013: 91: 145–50. doi: 10.1111/j.1755-3768.2012.02462.x /
20. Maharana P., Jhanji V., Vajpayee R.B. Inadvertent use of cornea
with previous LASIK in deep lamellar keratoplasty. Optom. Vis. Sci. 2014; 91: 59–62. doi: 10.1097/OPX.0000000000000162 6. Anwar M., Teichmann K.D. Deep lamellar keratoplasty: surgical
techniques for anterior lamellar keratoplasty with and without
baring of Descemet’s membrane. Cornea. 2002; 21: 374–83 21. Huang T., Wang Y., Hu A., et al. Use of pediatric donor tissue in
Descemet’s stripping endothelial keratoplasty. Br. J. Ophthalmol. 2009; 93: 1625–8. doi: 10.1136/bjo.2009.158477 7. Оганесян О.Г., Нероев В.В., Гундорова Р.А., Воробьева М.А. Первый опыт трансплантации десцеметовой мембраны. Офтальмология. 2008; 5 (4): 18–22
Oganesyan O.G., Neroev V.V., Gundorova R.A., Vorobjeva M.A. The first experience of descemet's membrane transplantation. Oftal’mologija. 2008. 5: 18–22 (in Russian) 22. Kim P., Yeung S.N., Lichtinger A., et al. Descemet stripping
automated endothelial keratoplasty using infant donor tissue. Cornea. 2012; 31: 52–4. doi: 10.1097/ICO.0b013e31821eeac0 23. Sun Y.X., Hao Y.S., Hong J. Descemet membrane stripping
endothelial keratoplasty with neonate donors in two cases. Br. J. Ophthalmol. 2009; 93: 1692–9. http://dx.doi.org/10.1136/
bjo.2009.158188 8. Zirm E. Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Новая стратегия кератопластики:
расслоение и разделение роговицы донора Прозрачность финансовой деятельности: никто из
авторов не имеет финансовой заинтересованности в
представленных материалах или методах. Tectonic Descemet stripping
automated endothelial keratoplasty for the management of sterile
corneal perforations in decompensated corneas. Cornea. 2016; 35:
1516–9. doi: 10.1097/ICO.0000000000001037 16. Otte J.B., de Ville de Goyet J., Alberti D., et al. The concept and
technique of the split liver in clinical transplantation. Surgery. 1990;
107: 605–12 17. Cintorino D., Spada M., Gruttadauria S., et al. In situ split liver
transplantation for adult and pediatric recipients: an answer to
organ shortage. Transplant Proc. 2006; 38: 1096–8. doi:10.1016/j. transproceed.2006.02.146 29. Tu E.Y. Descemet membrane endothelial keratoplasty patch
for persistent corneal hydrops. Cornea. 2017; 36: 1559–61. doi:
10.1097/ICO.0000000000001351 30. Kolomeyer A.M., Chu D.S. Descemet stripping endothelial
keratoplasty in a patient with keratoglobus and chronic hydrops
secondary to a spontaneous Descemet membrane tear. Case
Reports in Ophthalmol. Med. 2013; ID 697403. doi: 10.1155/2013/
697403 18. Armour R., Ousley P., Wall J., et al. Endothelial keratoplasty using
donor tissue not suitable for full-thickness penetrating keratoplasty. Cornea. 2007; 26: 515–9. doi:10.1097/ICO.0b013e3180335511 19. Moshirfar M., Khalifa Y., Davis D., et al. Descemet stripping
automated endothelial keratoplasty using donor corneas with Поступила: 19.12.2017 Новая стратегия кератопластики:
расслоение и разделение роговицы донора A new strategy of keratoplasty: laminating and splitting the donor cornea A new strategy of keratoplasty: laminating and splitting the donor cornea
O.G. Oganesyan — Dr. Med. Sci., leading researcher, department of eye traumatology and
reconstructive surgery
P.V. Makarov — Dr. Med. Sci., leading researcher, department of eye traumatology and
reconstructive surgery
d k
h
d
d
f
l
d O.G. Oganesyan — Dr. Med. Sci., leading researcher, department of eye traumatology and
reconstructive surgery
P.V. Makarov — Dr. Med. Sci., leading researcher, department of eye traumatology and
reconstructive surgery
À.À. Grdikanyan — PhD student, department of eye traumatology and reconstructive surgery
V.R. Getadaryan — PhD student, department of eye traumatology and reconstructive surgery À.À. Grdikanyan — PhD student, department of eye traumatology and reconstructive surgery
V.R. Getadaryan — PhD student, department of eye traumatology and reconstructive surgery Moscow Helmholtz Research Institute of Eye Diseases, 14/19, Sadovaya-Chernogryazskaya St., 105062,
Moscow, Russia
oftalmolog@mail.ru Moscow Helmholtz Research Institute of Eye Diseases, 14/19, Sadovaya-Chernogryazskaya St., 105062,
Moscow, Russia
oftalmolog@mail.ru Purpose: to report the first case of the same day use of a single donor cornea tissue for 5 consecutive transplantations
in patients with endothelial dystrophy and keratoconus. Material and methods. Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Для контактов: Оганесян Оганес Георгиевич
E-mail: oftalmolog@mail.ru Прозрачность финансовой деятельности: никто из
авторов не имеет финансовой заинтересованности в
представленных материалах или методах. The Descemet membrane (DM) and the
endothelium were fully detached from the stroma of the donor on the endothelial side, using first a circular and then a direct
corneal cutter, forming four partial triangle-shaped Descemet grafts (each constituted a 1/4 Descemet graft). The stroma
of the donor's cornea was separated by a circular cutter of a preset diameter. One patient with keratoconus underwent deep
anterior lamellar keratoplasty (DALK) using the big-bubble technique. Then, each of the four Descemet grafts was sequen-
tially transplanted by the Descemet membrane endothelial keratoplasty (DMEK) method to 4 patients with Fuchs dystrophy
and pseudophakia. Results. During and after the surgery, there were no complications requiring additional intervention. Six
months after DALK, the best corrected visual acuity (BCVA) was 0.66. Six months after four operations each using ¼ DMEK
the average BCVA was 0.95 ± 0.1 (from 0.8 to 1.0) and the endothelial cell density was, respectively, 2839 after DALK and
1680 ± 254 cells/mm2 (from 1492 to 2039 cells/mm2) after ¼ DMEK. Conclusion. One donor cornea can be successfully
transplanted to at least 5 patients. Despite the fact that the implementation of DALK and several operations using the ¼
DMEK technique within one day is technically difficult, this approach can contribute to a drastic reduction in the deficit of
donor tissue. This approach requires a thorough selection of patients and an extensive surgical experience. Keywords: donor cornea, keratoplasty, corneal transplantation, Descemet membrane transplantation, DMEK,
Q DMEK ¼ DMEK Keywords: donor cornea, keratoplasty, corneal transplantation, Descemet membrane transplantation, DMEK,
Q-DMEK, ¼ DMEK The full English version is available online at http://roj.igb.ru/eng/issues The full English version is available online at http://roj.igb.ru/eng/issues The full English version is available online at http://roj.igb.ru/eng/issues For citation: Oganesyan O.G., Makarov P.V., Grdikanyan А.А., Getadaryan V.R. A new strategy of keratoplasty:
laminating and splitting the donor cornea. Russian ophthalmological journal. 2018; 11 (3): 11–8 (In Russian). doi: 10.21516/2072-0076-2018-11-3-11-18 Conflict of interests: there is no conflict of interests. Conflict of interests: there is no conflict of interests. Financial disclosure: No author has a financial or property interest in any material or method mentioned. Financial disclosure: No author has a financial or property interest in any material or method mentioned. Для контактов: Оганесян Оганес Георгиевич
E-mail: oftalmolog@mail.ru Новая стратегия кератопластики:
расслоение и разделение роговицы донора
18 Новая стратегия кератопластики:
расслоение и разделение роговицы донора
18
|
https://openalex.org/W3095979265
|
https://discovery.ucl.ac.uk/id/eprint/10142201/1/Lovering_gkaa921.pdf
|
English
| null |
RNAcentral 2021: secondary structure integration, improved sequence search and new member databases
|
Nucleic acids research
| 2,020
|
cc-by
| 6,321
|
RNAcentral 2021: secondary structure integration,
improved sequence search and new member nsortium1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,* RNAcentral Consortium1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26 1European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton,
Cambridge CB10 1SD, UK, 2Center for non-coding RNA in Technology and Health, Department of Veterinary and
Animal Sciences, University of Copenhagen, Copenhagen, DK-1871, Denmark, 3Department of Integrative Biology,
University of Texas at Austin, Austin, Texas 78712, USA, 4Department of Physiology, Development and Neuroscience,
University of Cambridge, Downing Street, Cambridge CB2 3DY, UK, 5Department of Molecular Genetics, Weizmann
Institute of Science, Rehovot 7610001, Israel, 6Department of Biomolecular Engineering, University of California
Santa Cruz, Santa Cruz, CA, 95064, USA, 7Department of Haematology, University of Cambridge School of Clinical
Medicine, Cambridge, CB2 0AW, UK, 8Universit`a della Calabria, Dipartimento di Biologia, Ecologia e Scienze della
Terra, Via Pietro Bucci Cubo 6/C, Rende, CS, 87036, Italy, 9DIANA-LAB, Department of Computer Science and
Biomedical Informatics, University of Thessaly, Greece & Hellenic Pasteur Institute, 351 31, Greece, 10China National
Center for Bioinformation & National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of
Sciences, 100101, China, 11Cancer Research Institute Ghent (CRIG), 9000 Ghent, Belgium, 12Department of
Biomolecular Medicine, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium, 13Faculty
of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester, M13 9PT, UK, 14Science for
Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm,
S-10691, Sweden, 15Department of Biological Sciences, Dartmouth College, Hanover, NH, 03755, USA, 16National
Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894
USA, 17Center for the Origins of Life, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta,
GA 30032, USA, 18Department of Genetics, Stanford University, Palo Alto, CA 94303, USA, 19Department of
Biochemistry and Functional Genomics, Universit´e de Sherbrooke, Sherbrooke, QCJ1H 5N4, Canada,
20Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA,
94720, USA, 21Department of Software Engineering, Faculty of Mathematics and Physics, Charles Univesity, Prague,
11800 Praha 1, Czech Republic, 22Functional Gene Annotation, Preclinical and Fundamental Science, UCL Institute
of Cardiovascular Science, University College London, London, WC1E 6BT, UK, 23The Institute of Neuroscience,
University of Oregon, Eugene, OR 97403-1254, USA, 24Bioinformatics Group, Department of Computer Science and
Interdisciplinary Centre for Bioinformatics, Leipzig University, H¨artelstraße 16–18, 04107, Leipzig, Germany,
25Department of Computational Biology, Adam Mickiewicz University in Poznan, Poznan, 61-614, Poland and 26The
Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA method that uses consistent, reproducible and rec-
ognizable layouts for related RNAs. RNAcentral 2021: secondary structure integration,
improved sequence search and new member The sequence
similarity search has been updated with a faster in-
terface featuring facets for filtering search results by
RNA type, organism, source database or any key-
word. This sequence search tool is available as a
reusable web component, and has been integrated
into several RNAcentral member databases, includ-
ing Rfam, miRBase and snoDB. To allow for a more
fine-grained assignment of RNA types and subtypes, C⃝The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
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. D212–D220
Nucleic Acids Research, 2021, Vol. 49, Database issue
doi: 10.1093/nar/gkaa921 D212–D220
Nucleic Acids Research, 2021, Vol. 49, Database issue
doi: 10.1093/nar/gkaa921 Published online 27 October 2020 Received September 15, 2020; Editorial Decision October 01, 2020; Accepted October 05, 2020 *To whom correspondence should be addressed. Tel: +44 1223 492550; Fax: +44 1223 494468; Email: apetrov@ebi.ac.uk RNA 2D STRUCTURE INTEGRATION Since 2017 RNAcentral has included 2D structure informa-
tion starting with a tRNA dataset submitted by Genomic
tRNA Database (GtRNAdb) (7). However, for the vast ma-
jority of RNAcentral sequences no secondary structure is
available in the source database (e.g., ENA or RefSeq). In
addition, there are accepted layouts and orientations for
the display of secondary structures of well-known families
(such as rRNA and tRNA) (8,9), but existing automated 2D
visualization tools do not account for these layouts, mak-
ing it difficult to analyze and compare structured RNAs. As these large families of well-known RNAs constitute the
majority of sequences in RNAcentral, we set out to develop
a new method for producing 2D structure diagrams in stan-
dard orientations called R2DT (RNA 2D Templates) (10). The R2DT software automatically selects the best match-
ing template from a library of 3632 2D templates that repre-
sent a wide range of RNA types, such as rRNA (both small
and large subunit), tRNA, as well as 2675 RNA families
from Rfam. A template encapsulates a reference sequence
along with cartesian coordinates for each nucleotide and a Since 2017 RNAcentral has included 2D structure informa-
tion starting with a tRNA dataset submitted by Genomic
tRNA Database (GtRNAdb) (7). However, for the vast ma-
jority of RNAcentral sequences no secondary structure is
available in the source database (e.g., ENA or RefSeq). In
addition, there are accepted layouts and orientations for
the display of secondary structures of well-known families
(such as rRNA and tRNA) (8,9), but existing automated 2D
visualization tools do not account for these layouts, mak-
ing it difficult to analyze and compare structured RNAs. As these large families of well-known RNAs constitute the
majority of sequences in RNAcentral, we set out to develop
a new method for producing 2D structure diagrams in stan-
dard orientations called R2DT (RNA 2D Templates) (10). The new search features an updated interface that enables
exploring the results using facets, such as species, RNA
type and source database (Figure 3). The results can also
be filtered by any keyword, similar to the RNAcentral text
search, and sorted by E-value, sequence identity, query and
target coverage and other parameters. The query sequence is also automatically searched
against the Rfam families (3024 as of Rfam 14.2) using
Infernal (15). The Rfam results are post-processed to se-
lect the top scoring families from the same Rfam clan (16). UPDATED SEQUENCE SIMILARITY SEARCH Since 2015, RNAcentral has been hosting a sequence simi-
larity search tool powered by the nhmmer software (14), to
enable users to compare any query sequence against a com-
prehensive collection of ncRNAs (https://rnacentral.org/
sequence-search). As RNAcentral grew in size, the search
time increased and users experienced wait times of up to an
hour to get the results. In 2019, an updated version of the
search was launched using a scalable cloud infrastructure
hosted at the Embassy Cloud platform provided by EMBL-
EBI. The searches are executed in parallel and complete
more quickly. For example, we repeated all searches submit-
ted in 2019 using the new infrastructure and saw a decrease
in the average search time from 4.5 min to 13 s, an approxi-
mately 20-fold increase in speed. Since the new launch was
launched, the number of searches increased from around
600 to 3000 searches per month. 1. Newly integrated 2D structure information 2. Improved sequence similarity search 3. Transition to Sequence Ontology to annotate RNA
types INTRODUCTION RNAcentral is the non-coding RNA (ncRNA) sequence
database that currently integrates 44 specialist ncRNA
databases, known as Expert Databases, to provide uni-
fied access to >18 million ncRNA sequences spanning a
broad range of functions and species (1). In addition to se-
quences, RNAcentral provides a wide range of annotation
types, such as genome coordinates, microRNA–target in-
teractions (2,3), Gene Ontology (GO) terms (4), orthologs
and paralogs (5), RNA family classification from Rfam
(6) and more. Data can be accessed via text search, se-
quence similarity search, integrated genome browser and
bulk data downloads from the FTP archive. The primary
goal of RNAcentral is to provide open access to a compre-
hensive set of ncRNA sequences for a wide range of species,
enabling the users to find what is known about individ-
ual sequences or download ncRNA sequences and their ge-
nomic locations that can be used for a broad range of stud-
ies, such as interpreting the results of RNA-seq experiments
or training bioinformatic algorithms. RNAcentral also pro-
vides stable accessions for distinct RNA sequences, facilitat-
ing the work of other RNA resources. R2DT is now routinely applied to all sequences in RNA-
central. In the most recent release (version 16), we generated
>13 million 2D structure diagrams, representing the world’s
largest collection of RNA 2D structures. The 2D structures
are displayed in the sequence report pages and in the text
search results (Figure 2). In addition, R2DT is available as
a web server (https://rnacentral.org/r2dt) that enables users
to submit sequences and generate 2D diagrams. As new templates are added to the R2DT library (e.g.,
with future Rfam releases), the number and quality of the
2D diagrams will be improved in RNAcentral. We welcome
feedback about individual 2D structures to help prioritize
improvements in R2DT. RNAcentral continues to grow (Figure 1) with the incor-
poration of 16 new Expert Databases since the last publica-
tion (1). In this paper, we discuss the new data and focus on
the following major new features: ABSTRACT RNAcentral is a comprehensive database of non-
coding RNA (ncRNA) sequences that provides a sin-
gle access point to 44 RNA resources and >18 mil-
lion ncRNA sequences from a wide range of organ-
isms and RNA types. RNAcentral now also includes
secondary (2D) structure information for >13 million
sequences, making RNAcentral the world’s largest
RNA 2D structure database. The 2D diagrams are dis-
played using R2DT, a new 2D structure visualization Nucleic Acids Research, 2021, Vol. 49, Database issue D213 2D structure. The best-matching templates are selected us-
ing the Ribovore (https://github.com/nawrockie/ribovore)
and tRNAscan-SE 2.0 (11) software, and are visualized
using Traveler (12). The templates ensure that similar se-
quences are visualized in consistent, reproducible orienta-
tions and can be easily compared across related RNAs. all RNAcentral sequences have been annotated with
Sequence Ontology terms. The RNAcentral database
continues to grow and provide a central data re-
source for the RNA community. RNAcentral is freely
available at https://rnacentral.org. A key strength of the method is the ability to visual-
ize some of the largest structured RNAs, such as the hu-
man large subunit ribosomal rRNAs (LSU) with >5000 nu-
cleotides (Figure 2). The LSU templates are displayed using
a set of new 3D structure based templates from RiboVision
(13). In addition, RiboVision provided a set of 3D structure
based small subunit (SSU) rRNA templates that improves
the representation of species-specific expansion segments in
rRNA. RNA 2D STRUCTURE INTEGRATION For example, a rRNA sequence may match both eukary-
otic and bacterial Rfam families, but the clan competi-
tion procedure keeps only the top scoring family. In ad-
dition, the sequence search is integrated with the R2DT The R2DT software automatically selects the best match-
ing template from a library of 3632 2D templates that repre-
sent a wide range of RNA types, such as rRNA (both small
and large subunit), tRNA, as well as 2675 RNA families
from Rfam. A template encapsulates a reference sequence
along with cartesian coordinates for each nucleotide and a D214 Nucleic Acids Research, 2021, Vol. 49, Database issue Figure 1. Growth in the number of RNAcentral Expert Databases since its launch in 2014 (for an up-to-date list see https://rnacentral.org/expert-databases). The previous NAR publication is marked with a vertical dashed line. Downloaded from https://academic.oup.com/nar/article/49/D1/D212/5940500 by gues Figure 1. Growth in the number of RNAcentral Expert Databases since its launch in 2014 (for an up-to-date list see https://rnacentral.org/expert-databases). The previous NAR publication is marked with a vertical dashed line. In addition, in response to the COVID-19 pandemic,
the cloud-centric approach enabled us to rapidly repur-
pose the RNAcentral infrastructure to search Betacoron-
avirus genomes instead of ncRNA sequences. The Beta-
coronavirus search provides virus-specific facets that en-
able filtering the results by virus, such as SARS-CoV or
SARS-CoV-2, as well as the country of sample origin. The Betacoronavirus sequence search is available at https:
//covid19sequencesearch.github.io. software described above so that a 2D structure (if avail-
able) is visualized alongside similar sequences (Figure 3C). The updated search includes some of the most frequently
requested features that were not available previously. For
example, a batch search mode enables users to submit a
FASTA file with up to 50 sequences in order to launch
multiple searches simultaneously. Users can also down-
load the results in several formats, including plain text
and JSON. software described above so that a 2D structure (if avail-
able) is visualized alongside similar sequences (Figure 3C). The updated search includes some of the most frequently
requested features that were not available previously. For
example, a batch search mode enables users to submit a
FASTA file with up to 50 sequences in order to launch
multiple searches simultaneously. Users can also down-
load the results in several formats, including plain text
and JSON. RNA 2D STRUCTURE INTEGRATION The new interface is implemented as a reusable web
component, enabling other RNAcentral Expert Databases
or anyone else to include it in their websites to pro-
vide sequence similarity search to their users. The em-
beddable component is available at https://github.com/
rnacentral/rnacentral-sequence-search-embed. It can be in-
tegrated into any website with a few lines of code. The
component is highly customizable, for example, it is pos-
sible to select a subset of RNAcentral sequences to be
searched or adjust the widget appearance to match the host
website. Figure 2. (A) The RNAcentral text search results include simplified thumbnails representing the 2D structures. (B) A 2D structure of the human LSU
rRNA displayed on the sequence report page for URS000075EC78 9606. REFINED
RNA
TYPE
ASSIGNMENT
USING
SE-
QUENCE ONTOLOGY The result- classification system that combines the information about
the INSDC RNA types submitted by member databases,
Rfam annotations and other information to expand the
SO term coverage to the entire set of sequences found in
RNAcentral. For example, for rRNA sequences, the R2DT
rRNA template matches are used to transfer the corre-
sponding SO term to the sequence, enabling the classifi-
cation of rRNA subclasses. Consequently, an Arabidopsis
thaliana sequence URS0000AF5D55 3702 previously an-
notated as misc RNA in ENA is now assigned the SO
term for 25S LSU rRNA due to matches to the eukary-
otic large subunit (LSU) rRNA Rfam model (RF02543)
and an eukaryotic LSU R2DT template. For the ‘other’ and
‘misc RNA’ INSDC sequence classes, we use Rfam family
annotations to assign the corresponding SO term to the se-
quences. For all remaining sequences, we map the INSDC
RNA types to the SO terms using the mapping developed
by the SO and the RefSeq groups (https://github.com/The-
Sequence-Ontology/SO-Ontologies/issues/378). The result- REFINED
RNA
TYPE
ASSIGNMENT
USING
SE-
QUENCE ONTOLOGY Downloaded from https://academic.oup.com/nar/article/49/D1/D212/5940500 by guest on 19 January 2022 Downloaded from https://academic.oup.com/nar/article/49/D1/D212/5940500 by gue Figure 3. New RNAcentral sequence similarity search interface. (A) Query sequence, (B) Identical sequence found in RNAcentral, (C) Secondary structur
visualized with R2DT, (D) Rfam classification, including Rfam family annotations and an alignment between the query and the Rfam model, (E) Simila
sequences found in RNAcentral that can be filtered using facets, such as RNA types and Organisms, sorted or downloaded. Figure 3. New RNAcentral sequence similarity search interface. (A) Query sequence, (B) Identical sequence found in RNAcentral, (C) Secondary structure
visualized with R2DT, (D) Rfam classification, including Rfam family annotations and an alignment between the query and the Rfam model, (E) Similar
sequences found in RNAcentral that can be filtered using facets, such as RNA types and Organisms, sorted or downloaded. Figure 3. New RNAcentral sequence similarity search interface. (A) Query sequence, (B) Identical sequence found in RNAcentral, (C) Secondary structure
visualized with R2DT, (D) Rfam classification, including Rfam family annotations and an alignment between the query and the Rfam model, (E) Similar
sequences found in RNAcentral that can be filtered using facets, such as RNA types and Organisms, sorted or downloaded. ing distribution of RNAcentral sequences by SO terms is
shown in Figure 4. ing distribution of RNAcentral sequences by SO terms is
shown in Figure 4. classification system that combines the information about
the INSDC RNA types submitted by member databases,
Rfam annotations and other information to expand the
SO term coverage to the entire set of sequences found in
RNAcentral. For example, for rRNA sequences, the R2DT
rRNA template matches are used to transfer the corre-
sponding SO term to the sequence, enabling the classifi-
cation of rRNA subclasses. Consequently, an Arabidopsis
thaliana sequence URS0000AF5D55 3702 previously an-
notated as misc RNA in ENA is now assigned the SO
term for 25S LSU rRNA due to matches to the eukary-
otic large subunit (LSU) rRNA Rfam model (RF02543)
and an eukaryotic LSU R2DT template. For the ‘other’ and
‘misc RNA’ INSDC sequence classes, we use Rfam family
annotations to assign the corresponding SO term to the se-
quences. For all remaining sequences, we map the INSDC
RNA types to the SO terms using the mapping developed
by the SO and the RefSeq groups (https://github.com/The-
Sequence-Ontology/SO-Ontologies/issues/378). REFINED
RNA
TYPE
ASSIGNMENT
USING
SE-
QUENCE ONTOLOGY Since its inception, RNAcentral has used the INSDC
feature table (http://www.insdc.org/files/feature table.html)
and ncRNA vocabulary (http://www.insdc.org/rna vocab. html) to annotate sequences with different RNA types. However, the INSDC classification lacks precision and does
not distinguish between different rRNA types, such as SSU,
LSU or 5S rRNAs, simply grouping them in a single cate-
gory. Similarly, there were no specific terms for precursor
microRNAs to separate them from other RNA precursors
and maintain their connection with mature microRNAs. However, the Sequence Ontology (SO) (19) provides more
granular terms for rRNAs, microRNAs and other RNA
types. The search has been integrated into Rfam (6), miRBase
(17) and snoDB (18). For example, when a user enters a
query sequence in Rfam, it is not only annotated with Rfam
families but also searched against a comprehensive set of se-
quences from RNAcentral. If a query comes from an RNA
sequence not represented in Rfam, the results will include
hits from RNAcentral, and if a query matches Rfam, the
users will get additional information about matching se-
quences and can explore them using the facets. yp
Several member databases, such as FlyBase or miRBase,
already provide SO terms for their sequences. However,
<10% of sequences have been annotated with SO terms at
the time of submission to RNAcentral. We implemented a Nucleic Acids Research, 2021, Vol. 49, Database issue D215 D215 Downloaded from https://academic.oup.com/nar/article/49/D1/D212/5940500 by gue Downloaded from https://academic.oup.com/nar/article/49/D1/D212/5940500 by guest on 19 January 2022 e RNAcentral text search results include simplified thumbnails representing the 2D structures (B) A 2D structur Figure 2. (A) The RNAcentral text search results include simplified thumbnails representing the 2D structures. (B) A 2D structure of the human LSU
rRNA displayed on the sequence report page for URS000075EC78 9606. Figure 2. (A) The RNAcentral text search results include simplified thumbnails representing the 2D structures. (B) A 2D structure of the human LSU
rRNA displayed on the sequence report page for URS000075EC78 9606. D216 Nucleic Acids Research, 2021, Vol. 49, Database issue Figure 3. New RNAcentral sequence similarity search interface. (A) Query sequence, (B) Identical sequence found in RNAcentral, (C) Secondary structure
visualized with R2DT, (D) Rfam classification, including Rfam family annotations and an alignment between the query and the Rfam model, (E) Similar
sequences found in RNAcentral that can be filtered using facets, such as RNA types and Organisms, sorted or downloaded. NEW DATA AND ANNOTATIONS Since the last publication, the number of imported
databases increased from 28 to 44 databases, integrating 16
additional resources listed in Table 1. To provide detailed human ncRNA annotations, we im-
ported data from LncBook (20) and snoDB (18) that host a
variety of annotations for lncRNAs and snoRNAs, respec-
tively. GeneCards (21) and MalaCards (22) have also been
included into RNAcentral. GeneCards is a human gene
knowledgebase, which aims to consolidate information
about all human genes, coding and non-coding. MalaCards
is an integrated database of human diseases and their an-
notations. MalaCards uses text mining and manual cura-
tion to associate human ncRNAs with information about
diseases and lists the supporting literature. Notably, snoDB Nucleic Acids Research, 2021, Vol. 49, Database issue D217 Figure 4. The distribution of RNAcentral sequences by the SO terms. Downloaded from https://academic.oup.com/nar/article/49/D1/D212/5940500 by gue Figure 4. The distribution of RNAcentral sequences by the SO terms. described. All GO Consortium ncRNA annotations are
available in RNAcentral, as well as via the GO browsers
QuickGO and AmiGO and in other major resources includ-
ing Ensembl, NCBI Gene, miRBase and the web service
PSICQUIC. and GeneCards are also using RNAcentral as a data source. GeneCards also used RNAcentral to produce a compre-
hensive and non-redundant gene-centric view of ncRNAs,
which is available at the ‘GeneCards ncRNAs’ track hub at
the UCSC genome browser (23). Several RNA type specific databases have been included,
such as 5SRNAdb with 5S rRNAs (30), snoRNA Database
with archaeal snoRNAs (31,32), MirGeneDB with mature
and precursor microRNAs (33), as well as CRW with 5S,
SSU and LSU rRNAs (8). A broad range of prokaryotic
ncRNAs has been incorporated from the ZWD database
(34), which includes high-quality sequence alignments for
structured RNAs discovered in a diverse range of habitats
and organisms. We completed the integration of all model organism
databases forming the Alliance of Genome Resources (24)
by importing ZFIN (25), a model organism database that
hosts a wide array of expertly curated, organized and cross-
referenced research data for zebrafish (Danio rerio). In or-
der to provide genomic annotations for a broad range of
organisms, we also imported ncRNAs from Ensembl Fungi,
Metazoa and Protists (26). (
)
We have added several new sources of functional annota-
tions. NEW DATA AND ANNOTATIONS We have integrated IntAct (27) bringing in 1152 inter-
molecular interactions for 382 RNAs, with the majority of
data points coming from human and yeast (168 and 114 an-
notated RNAs, respectively). As curators continue to anno-
tate additional interactions in IntAct, the new data will au-
tomatically flow into RNAcentral. We have also integrated
microRNA–lncRNA interactions from LncBase v2 (3). We have also imported the Conserved RNA Structure
(CRS) resource that computationally screened the hu-
man centered 100-way vertebrate sequence alignment from
UCSC Genome Browser for conserved RNA secondary
structures with CMfinder (35). We have integrated CRSs
with a false discovery rate lower or equal to 10% in 29 ver-
tebrate species and excluded matches to known structured
RNAs from Rfam. In addition to the automatic GO annotations created
by RNAcentral, over 3400 ncRNAs currently are asso-
ciated with GO terms, following the manual curation of
research articles by the GO Consortium (1). Over 80%
of these, 17 000 annotations capture the cellular role of
human and mouse ncRNAs; microRNAs are the most
commonly curated ncRNA. The majority of these anno-
tations describe ‘gene silencing by miRNA’ and ‘mRNA
binding’ and include the target of the ncRNA in the an-
notation extension field. However, downstream processes
such as ‘regulation of epithelial-to-mesenchymal transition’
and ‘regulation of inflammatory response’ (28,29) are also Significant data updates A number of previously integrated resources have provided
significant updates in the last 2 years. Recent changes in
SILVA (36) allowed us to integrate the SILVA-based in-
ferred bacterial taxonomy into RNAcentral, which is dis-
played on the sequence report pages. FlyBase (37) ncRNA annotations have been continuously
updated within RNAcentral. Notably, FlyBase now reflects
gene model annotations for Drosophila melanogaster only, D218 Nucleic Acids Research, 2021, Vol. 49, Database issue Table 1. Sixteen new member databases incorporated into RNAcentral in releases 11–16
Database
Description
Number of annotated sequences
5SrRNAdb
5SrRNAdb is an information resource for 5S ribosomal RNAs. 11 415
CRS
Conserved RNA structures (CRS) are structured elements of RNA
molecules that are conserved across vertebrate species. 250 867
CRW
CRW Site provides comparative sequence and structure information
for ribosomal, intron and other RNAs. 948
Ensembl Fungi,
Metazoa, Protists
The Ensembl Genomes divisions complement the Ensembl database. 16 331; 64 237; 5652
GeneCards
GeneCards is a searchable, integrative database that provides
comprehensive, user-friendly information on all annotated and
predicted human genes. 250 702
IntAct
IntAct provides a freely available, open source database system and
analysis tools for molecular interaction data. All interactions are
derived from literature curation or direct user submissions. 382
LncBase
LncBase is a database of experimentally verified and
computationally predicted microRNA targets on lncRNAs. 1337
LncBook
LncBook is a curated knowledgebase of human lncRNAs. 268 848
MalaCards
MalaCards integrates manually curated and text-mining sources to
associate genes, including ncRNAs, with diseases, and lists the
supporting evidence. 34 087
MirGeneDB
MirGeneDB is a curated microRNA gene database covering 45
metazoan organisms. 29 681
snoDB
snoDB is an interactive database of human snoRNA sequences,
abundance and interactions. 1984
snoRNA Database
The snoRNA Database is a curated collection of archaeal snoRNAs
maintained by the Lowe Lab at UC Santa Cruz. 727
ZFIN
The Zebrafish Information Network (ZFIN) is the database of
genetic and genomic data for the zebrafish as a model organism. 1060
ZWD
ZWD is a collection of non-coding RNA alignments maintained by
Zasha Weinberg. 47 998
Total: 986 256 Table 1. Sixteen new member databases incorporated into RNAcentral in releases 11–16 Number of annotated sequences provements have been made to uniquely link LNCipedia en-
tries with those of other databases such as Ensembl (45) and
HGNC (38). meaning that ncRNA data for non-melanogaster genomes
are no longer submitted by FlyBase to RNAcentral. OTHER IMPROVEMENTS /
q
The HUGO Gene Nomenclature Committee (HGNC)
(38) is the only organization with the authority to approve
human gene symbols, including for ncRNA genes. Since
January 2019, the HGNC has primarily worked on expand-
ing its lncRNA dataset and has approved 528 new gene
symbols, representing an increase of 11% for these genes. Note that the HGNC only provides one name per lncRNA
gene without naming separate non-coding isoforms. Where
possible, lncRNA genes have been named based on func-
tional data from publications. Recent examples include
CHASERR (39), MYOPARR (40) and CEROX1 (41,42). Where no published data are available, the HGNC prior-
itizes naming lncRNA genes that have been manually an-
notated by both the RefSeq and Ensembl-Havana projects. These lncRNA genes are named based on genomic context
using a systematic schema, as outlined in (43). The HGNC
has also increased its small nuclear RNA dataset by 13%
and its transfer RNA dataset by 2.5%. The RNAcentral website has been continuously updated
with new features, such as the inclusion of the information
about paralogs and orthologs from the Ensembl Compara
pipeline (5). To increase discoverability with search engines,
automatically generated summaries have been added for all
sequences. The RNAcentral users can also display the miR-
Base word clouds (17) based on literature mining, which al-
lows the users to see related terms at a glance. For example,
microRNA mir-100 (URS000054969A 9606) is associated
with cancer, with this term prominently featured in the word
cloud. Following user requests, RNAcentral now hosts a public
Postgres database that provides the same data as the RNA-
central website. The database is meant to help users who
would like to access RNAcentral programmatically or are
interested in tasks that are not yet supported by the web-
site. The connection details, example queries and a sample
Python script can be found in (46) and at https://rnacentral. org/help/public-database. With the most recent release of lncRNA database LNCi-
pedia (version 5.2), significant efforts have been made to ex-
pand the functional annotation of lncRNAs in the database
(44). By combining manual and programmatical curation
of thousands of lncRNA papers in PubMed, 2482 PubMed
articles were associated with lncRNAs in LNCipedia. As a
result, LNCipedia currently contains 1555 unique lncRNA
genes with at least one published article. In addition, im- Significant data updates How-
ever, ncRNA annotations for other Drosophila species are
still available in RNAcentral as they are imported directly
from NCBI/RefSeq and the ENA. REFERENCES 1. RNAcentral Consortium (2019) RNAcentral: a hub of information
for non-coding RNA sequences. Nucleic Acids Res., 47, D221–D229. 1. RNAcentral Consortium (2019) RNAcentral: a hub of information
for non-coding RNA sequences. Nucleic Acids Res., 47, D221–D229. 2. Karagkouni,D., Paraskevopoulou,M.D., Chatzopoulos,S.,
Vlachos,I.S., Tastsoglou,S., Kanellos,I., Papadimitriou,D.,
Kavakiotis,I., Maniou,S., Skoufos,G. et al. (2018) DIANA-TarBase
v8: a decade-long collection of experimentally supported
miRNA–gene interactions. Nucleic Acids Res., 46, D239–D245. 3. Paraskevopoulou,M.D., Vlachos,I.S., Karagkouni,D.,
Georgakilas,G., Kanellos,I., Vergoulis,T., Zagganas,K., Tsanakas,P.,
Floros,E., Dalamagas,T. et al. (2016) DIANA-LncBase v2: indexing
microRNA targets on non-coding transcripts. Nucleic Acids Res., 44,
D231–D238. 4. Binns,D., Dimmer,E., Huntley,R., Barrell,D., O’Donovan,C. and
Apweiler,R. (2009) QuickGO: a web-based tool for Gene Ontology
searching. Bioinformatics, 25, 3045–3046. CONCLUSIONS The RNAcentral database continues to grow in size and
increase its utility. The addition of the 2D structure for a
wide range of RNAs fills an important gap, as the users Nucleic Acids Research, 2021, Vol. 49, Database issue D219 This paper is linked to: doi:10.1093/nar/gkaa1047. This paper is linked to: doi:10.1093/nar/gkaa1047. DATA AVAILABILITY 5. Pignatelli,M., Vilella,A.J., Muffato,M., Gordon,L., White,S.,
Flicek,P. and Herrero,J. (2016) ncRNA orthologies in the vertebrate
lineage. Database:J. Biol. Database. Curat., 2016, bav127. All data are freely available at https://rnacentral.org. The
data can be accessed in the FTP archive, as well as
through an API and a public Postgres database (see https:
//rnacentral.org/help for instructions). The code is available
at https://github.com/rnacentral under the Apache 2.0 li-
cense. 6. Kalvari,I., Argasinska,J., Quinones-Olvera,N., Nawrocki,E.P.,
Rivas,E., Eddy,S.R., Bateman,A., Finn,R.D. and Petrov,A.I. (2017)
Rfam 13.0: shifting to a genome-centric resource for non-coding
RNA families. Nucleic Acids Res., 46, D335–D342. 7. Chan,P.P. and Lowe,T.M. (2016) GtRNAdb 2.0: an expanded
database of transfer RNA genes identified in complete and draft
genomes. Nucleic Acids Res., 44, D184–D189. ACKNOWLEDGEMENTS 8. Cannone,J.J., Subramanian,S., Schnare,M.N., Collett,J.R.,
D’Souza,L.M., Du,Y., Feng,B., Lin,N., Madabusi,L.V., M¨uller,K.M. et al. (2002) The Comparative RNA Web (CRW) Site: an online
database of comparative sequence and structure information for
ribosomal, intron, and other RNAs. BMC Bioinformatics, 3, 2. RNAcentral has been prepared by Blake A. Sweeney, An-
ton I. Petrov, Carlos E. Ribas, Robert D. Finn, Alex
Bateman (RNAcentral), Maciej Szymanski, Wojciech M. Karlowski (5SrRNAdb), Stefan E. Seemann, Jan Gorod-
kin (CRS), Jamie J. Cannone, Robin R. Gutell (CRW),
Simon Kay (ENA), Steven Marygold, Gil dos Santos
(FlyBase), Adam Frankish, Jonathan M Mudge (GEN-
CODE), Ruth Barshir, Simon Fishilevich (GeneCards),
Patricia P. Chan (GtRNAdb), Todd M. Lowe (GtR-
NAdb and snoRNA Database), Ruth Seal, Elspeth Bru-
ford (HGNC), Simona Panni, Pablo Porras (IntAct), Dim-
itra Karagkouni, Artemis G. Hatzigeorgiou (LncBase),
Lina Ma, Zhang Zhang (LncBook), Pieter-Jan Volders,
Pieter Mestdagh (LNCipedia), Sam Griffiths-Jones (miR-
Base), Bastian Fromm, Kevin J. Peterson (MirGeneDB),
Ioanna Kalvari, Eric P. Nawrocki (Rfam), Anton S. Petrov (RiboVision), Shuai Weng (SGD), Philia Bouchard-
Bourelle, Michelle Scott (snoDB), Lauren M. Lui (snoRNA
Database), David Hoksza (Traveler), Ruth C. Lovering,
Barbara Kramarz (UCL), Prita Mani, Sridhar Ramachan-
dran (ZFIN), Zasha Weinberg (ZWD). bosomal, intron, and other RNAs. BMC Bioinformatics, 3, 9. Holley,R.W., Apgar,J., Everett,G.A., Madison,J.T., Marquisee,M.,
Merrill,S.H., Penswick,J.R. and Zamir,A. (1965) STRUCTURE OF
A RIBONUCLEIC ACID. Science, 147, 1462–1465. 10. Sweeney,B.A., Hoksza,D., Nawrocki,E.P., Ribas,C.E., Madeira,F.,
Cannone,J.J., Gutell,R.R., Maddala,A., Meade,C., Williams,L.D. et al. (2020) R2DT: computational framework for template-based
RNA secondary structure visualisation across non-coding RNA
types. bioRxiv doi: https://doi.org/10.1101/2020.09.10.290924, 11
September 20220, preprint: not peer reviewed. 11. Chan,P.P., Lin,B.Y., Mak,A.J. and Lowe,T.M. (2019) tRNAscan-SE
2.0: Improved Detection and Functional Classification of Transfer
RNA Genes. bioRxiv doi: https://doi.org/10.1101/614032, 30 April
2019, preprint: not peer reviewed. 12. Elias,R. and Hoksza,D. (2017) TRAVeLer: a tool for template-based
RNA secondary structure visualization. BMC Bioinformatics, 18,
487. 13. Bernier,C.R., Petrov,A.S., Waterbury,C.C., Jett,J., Li,F., Freil,L.E.,
Xiong,X., Wang,L., Migliozzi,B.L.R., Hershkovits,E. et al. (2014)
RiboVision suite for visualization and analysis of ribosomes. Farada
Discuss., 169, 195–207. 14. Wheeler,T.J. and Eddy,S.R. (2013) nhmmer: DNA homology search
with profile HMMs. Bioinformatics, 29, 2487–2489. 15. Nawrocki,E.P. and Eddy,S.R. (2013) Infernal 1.1: 100-fold faster
RNA homology searches. Bioinformatics, 29, 2933–2935. 16. Gardner,P.P., Daub,J., Tate,J., Moore,B.L., Osuch,I.H., 16. Gardner,P.P., Daub,J., Tate,J., Moore,B.L., Osuch,I.H.,
Griffiths-Jones,S., Finn,R.D., Nawrocki,E.P., Kolbe,D.L., Eddy,S.R. et al. (2011) Rfam: Wikipedia, clans and the ‘decimal’ release. Nucleic
Acids Res., 39, D141–D145.i Conflict of interest statement. None declared. are now able to access not only the primary sequences but
also the base pairing information and the 2D structure vi-
sualizations. The improved sequence search is faster and
more user-friendly, and the embeddable search component
is available for use on any website, enabling an ecosystem
of RNAcentral member databases to reuse the resources
in a cost-efficient way. The SO integration enables more
granular annotation of ncRNAs and powers new ways of
discovering the data using text search. The development
of the next versions of RNAcentral is underway, focus-
ing on the gene-centric organization of ncRNA transcripts
and automatic incorporation of the latest scientific litera-
ture using text mining. We aim to continue integrating ad-
ditional member databases, with 12 databases pending im-
port, and we invite the developers of RNA databases wish-
ing to join the RNAcentral Consortium to get in touch at
https://rnacentral.org/contact. FUNDING Braschi,B., Denny,P., Gray,K., Jones,T., Seal,R., Tweedie,S., Yates,B. and Bruford,E. (2019) Genenames.org: the HGNC and VGNC
resources in 2019. Nucleic Acids Res., 47, D786–D792. 39. Rom,A., Melamed,L., Gil,N., Goldrich,M.J., Kadir,R., Golan,M.,
Biton,I., Perry,R.B.-T. and Ulitsky,I. (2019) Regulation of CHD2
expression by the Chaserr long noncoding RNA gene is essential for
viability. Nat. Commun., 10, 5092. 25. Ruzicka,L., Howe,D.G., Ramachandran,S., Toro,S., Van Slyke,C.E.,
Bradford,Y.M., Eagle,A., Fashena,D., Frazer,K., Kalita,P. et al. (2019) The Zebrafish Information Network: new support for
non-coding genes, richer Gene Ontology annotations and the
Alliance of Genome Resources. Nucleic Acids Res., 47, D867–D873. y
40. Hitachi,K., Nakatani,M., Takasaki,A., Ouchi,Y., Uezumi,A.,
Ageta,H., Inagaki,H., Kurahashi,H. and Tsuchida,K. (2019)
Myogenin promoter-associated lncRNA Myoparr is essential for
myogenic differentiation. EMBO Rep., 20. 26. Howe,K.L., Contreras-Moreira,B., De Silva,N., Maslen,G.,
Akanni,W., Allen,J., Alvarez-Jarreta,J., Barba,M., Bolser,D.M.,
Cambell,L. et al. (2020) Ensembl Genomes 2020––enabling
non-vertebrate genomic research. Nucleic Acids Res., 48, D689–D695. 41. Sirey,T.M., Roberts,K., Haerty,W., Bedoya-Reina,O.,
Rogatti-Granados,S., Tan,J.Y., Li,N., Heather,L.C., Carter,R.N.,
Cooper,S. et al. (2019) The long non-coding RNA Cerox1 is a post
transcriptional regulator of mitochondrial complex I catalytic
activity. Elife, 8, e45051. 27. Orchard,S., Ammari,M., Aranda,B., Breuza,L., Briganti,L.,
Broackes-Carter,F., Campbell,N.H., Chavali,G., Chen,C.,
del-Toro,N. et al. (2014) The MIntAct project–IntAct as a common
curation platform for 11 molecular interaction databases. Nucleic
Acids Res., 42, D358–D363. 42. Sirey,T.M., Roberts,K., Haerty,W., Bedoya-Reina,O.,
Rogatti-Granados,S., Tan,J.Y., Li,N., Heather,L.C., Carter,R.N.,
Cooper,S. et al. (2019) Correction: The long non-coding RNA
Cerox1 is a post transcriptional regulator of mitochondrial complex I
catalytic activity. Elife, 8, e50980.i 28. Huntley,R.P., Kramarz,B., Sawford,T., Umrao,Z., Kalea,A.,
Acquaah,V., Martin,M.J., Mayr,M. and Lovering,R.C. (2018)
Expanding the horizons of microRNA bioinformatics. RNA, 24,
1005–1017. 29. Kramarz,B., Huntley,R.P., Rodr´ıguez-L´opez,M., Roncaglia,P.,
Saverimuttu,S.C.C., Parkinson,H., Bandopadhyay,R., Martin,M.-J.,
Orchard,S., Hooper,N.M. et al. (2020) Gene ontology curation of
neuroinflammation biology improves the interpretation of
Alzheimer’s disease gene expression data. J. Alzheimers. Dis., 75,
1417–1435. 43. Seal,R.L., Chen,L.-L., Griffiths-Jones,S., Lowe,T.M., Mathews,M.B.,
O’Reilly,D., Pierce,A.J., Stadler,P.F., Ulitsky,I., Wolin,S.L. et al. (2020) A guide to naming human non-coding RNA genes. EMBO J.,
39, e103777. 44. Volders,P.-J., Anckaert,J., Verheggen,K., Nuytens,J., Martens,L.,
Mestdagh,P. and Vandesompele,J. (2019) LNCipedia 5: towards a
reference set of human long non-coding RNAs. Nucleic Acids Res.,
47, D135–D139. 30. Szymanski,M., Zielezinski,A., Barciszewski,J., Erdmann,V.A. and
Karlowski,W.M. (2016) 5SRNAdb: an information resource for 5S
ribosomal RNAs. Nucleic Acids Res., 44, D180–D183. 45. Yates,A.D., Achuthan,P., Akanni,W., Allen,J., Allen,J.,
Alvarez-Jarreta,J., Amode,M.R., Armean,I.M., Azov,A.G.,
Bennett,R. et al. (2020) Ensembl 2020. Nucleic Acids Res., 48,
D682–D688. 31. Lui,L.M., Uzilov,A.V., Bernick,D.L., Corredor,A., Lowe,T.M. and
Dennis,P.P. FUNDING Biotechnology
and
Biological
Sciences
Research
Council
(BBSRC)
[BB/N019199/1];
Wellcome
Trust
[218302/Z/19/Z, 208349/Z/17/Z]; National Institutes of
Health [U24HG003345, U41HG000739]; Charles Univer-
sity [SVV 260588]. This research was supported by the
Intramural Research Program of the National Library of
Medicine at the NIH. Funding for open access charge:
Wellcome Trust [218302/Z/19/Z]. 17. Kozomara,A., Birgaoanu,M. and Griffiths-Jones,S. (2019) miRBase:
from microRNA sequences to function. Nucleic Acids Res., 47,
D155–D162. 18. Bouchard-Bourelle,P., Desjardins-Henri,C., Mathurin-St-Pierre,D.,
Deschamps-Francoeur,G., Fafard-Couture, ´E., Garant,J.-M.,
Elela,S.A. and Scott,M.S. (2020) snoDB: an interactive database of
human snoRNA sequences, abundance and interactions. Nucleic
Acids Res., 48, D220–D225. D220 Nucleic Acids Research, 2021, Vol. 49, Database issue 33. Fromm,B., Domanska,D., Høye,E., Ovchinnikov,V., Kang,W.,
Aparicio-Puerta,E., Johansen,M., Flatmark,K., Mathelier,A.,
Hovig,E. et al. (2020) MirGeneDB 2.0: the metazoan microRNA
complement. Nucleic Acids Res., 48, D1172. 19. Eilbeck,K., Lewis,S.E., Mungall,C.J., Yandell,M., Stein,L.,
Durbin,R. and Ashburner,M. (2005) The Sequence Ontology: a tool
for the unification of genome annotations. Genome Biol., 6, R44. 20. Ma,L., Cao,J., Liu,L., Du,Q., Li,Z., Zou,D., Bajic,V.B. and Zhang,Z. (2019) LncBook: a curated knowledgebase of human long
non-coding RNAs. Nucleic Acids Res., 47, D128–D134. 34. Eckert,I. and Weinberg,Z. (2020) Discovery of 20 novel ribosomal
leader candidates in bacteria and archaea. BMC Microbiol., 20, 130. 21. Stelzer,G., Rosen,N., Plaschkes,I., Zimmerman,S., Twik,M.,
Fishilevich,S., Stein,T.I., Nudel,R., Lieder,I., Mazor,Y. et al. (2016)
The GeneCards Suite: From Gene Data Mining to Disease Genome
Sequence Analyses. Curr. Protoc. Bioinformatics, 54, 1.30.1–1.30.33. 35. Seemann,S.E., Mirza,A.H., Hansen,C., Bang-Berthelsen,C.H.,
Garde,C., Christensen-Dalsgaard,M., Torarinsson,E., Yao,Z.,
Workman,C.T., Pociot,F. et al. (2017) The identification and
functional annotation of RNA structures conserved in vertebrates. Genome Res., 27, 1371–1383. 22. Rappaport,N., Twik,M., Plaschkes,I., Nudel,R., Iny Stein,T.,
Levitt,J., Gershoni,M., Morrey,C.P., Safran,M. and Lancet,D. (2017)
MalaCards: an amalgamated human disease compendium with
diverse clinical and genetic annotation and structured search. Nucleic
Acids Res., 45, D877–D887. 36. Quast,C., Pruesse,E., Yilmaz,P., Gerken,J., Schweer,T., Yarza,P.,
Peplies,J. and Gl¨ockner,F.O. (2013) The SILVA ribosomal RNA gene
database project: improved data processing and web-based tools. Nucleic Acids Res., 41, D590–D596. 23. Lee,C.M., Barber,G.P., Casper,J., Clawson,H., Diekhans,M.,
Gonzalez,J.N., Hinrichs,A.S., Lee,B.T., Nassar,L.R., Powell,C.C. et al. (2020) UCSC Genome Browser enters 20th year. Nucleic Acids
Res., 48, D756–D761. 37. Thurmond,J., Goodman,J.L., Strelets,V.B., Attrill,H., Gramates,L.S.,
Marygold,S.J., Matthews,B.B., Millburn,G., Antonazzo,G.,
Trovisco,V. et al. (2019) FlyBase 2.0: the next generation. Nucleic
Acids Res., 47, D759–D765. 24. Alliance of Genome Resources Consortium (2019) The alliance of
genome Resources: Building a modern data ecosystem for model
organism databases. Genetics, 213, 1189–1196. 38. FUNDING (2018) Methylation guide RNA evolution in archaea:
structure, function and genomic organization of 110 C/D box sRNA
families across six Pyrobaculum species. Nucleic Acids Res., 46,
5678–5691. 46. Sweeney,B.A., Tagmazian,A.A., Ribas,C.E., Finn,R.D., Bateman,A. and Petrov,A.I. (2020) Exploring Non-Coding RNAs in RNAcentral. Curr. Protoc. Bioinformatics, 71, e104. 32. Omer,A.D., Lowe,T.M., Russell,A.G., Ebhardt,H., Eddy,S.R. and
Dennis,P.P. (2000) Homologs of small nucleolar RNAs in Archaea. Science, 288, 517–522.
|
https://openalex.org/W4323967093
|
http://journal.thamrin.ac.id/index.php/jkmp/article/download/1184/pdf
|
Indonesian
| null |
Uji Coba Pembuatan Kue Kering Sagu Dengan Penambahan Tepung Tulang Ikan Dan Daya Terimanya
|
Jurnal Kesehatan Masyarakat Perkotaan
| 2,021
|
cc-by
| 5,294
|
Abstrak Latar belakang : Salah satu jenis kue kering adalah Kue kering sagu yang dibuat dengan bahan
dasar tepung sagu. Penelitian ini dilakukan untuk memanfaatkan limbah tulang ikan yang masih
memiliki zat gizi khususnya kalsium yang cukup tinggi yang ditambahkan pada pembuatan kue
kering sagu Tujuan : Mengetahui pengaruh penambahan tepung tulang ikan tongkol terhadap sifat
organoleptik (warna, tekstur, aroma dan rasa), daya terima dan kalsium pada kue kering sagu. Metode : Penelitian ini bersifat eksperimental, dengan melakukan penambahan tepung tulang ikan
tongkol pada kue kering sagu 0%, 10%, 15% dan 20%. Penelitian ini menggunakan uji Analysis of variance dilanjutkan menggunakan uji Duncan bila p-
Value <0,005. Hasil : Ada pengaruh penambahan tepung tulang ikan tongkol terhadap mutu
hedonik aspek warna, aroma, rasa dan tingkat kesukaan kue kering sagu, tidak berpengaruh
terhadap mutu hedonik aspek tekstur kue kering sagu. Kue kering sagu terpilih yaitu T2 (10%
tepung tulang ikan tongkol) dengan nilai energi total yaitu 368 kkal, protein 10 gram, karbohidrat
68 gram, lemak total 6 gram dan kalsium sebesar 0.1 gram dengan menggunakan metode AAS. Kesimpulan : Penambahan tepung tulang ikan tongkol berpengaruh terhadap aspek warna, aroma,
rasa dan tingkat kesukaan secara keseluruhan. Penambahan tepung tulang ikan tongkol tidak
berpengaruh terhadap aspek tekstur kue kering sagu. Aktivitas uji kalsium sebesar 0.1 gram dalam
100 gram bahan. Saran: analisis kelayakan usaha pembuatan kue kering sagu dengan penambahan
tepung tulang ikan 10% dan dilakukan diuji daya terima untuk snack pasien ginjal. Kata Kunci : kue kering sagu, tepung tulang ikan tongkol, sifat organoleptik Jurnal Kesehatan Masyarakat Perkotaan
Volume 2, No.1; Maret 2022 Jurnal Kesehatan Masyarakat Perkotaan
Volume 2, No.1; Maret 2022 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 Uji Coba Pembuatan Kue Kering Sagu Dengan Penambahan Tepung
Tulang Ikan Dan Daya Terimanya 1)2)Program Studi Gizi, Fakultas Kesehatan, Universitas Mohammad Husni Thamrin
Correspondence Author: alfasiregar07@gmail.com
DOI: https://doi.org/10.37012/jkmp.v1i1.1184
Parlin Dwiyana1), Elfa Siregar2)*) Abstrak Open Journal System (OJS): journal.thamrin.ac.id
http://journal.thamrin.ac.id/index.php/jkmp/article/view/74 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 2͕ Conclusion: The addition of mackarel tuna bone meal has an effect on aspects of color, aroma,
taste, and overall level of preference. The addition of mackarel tuna bone meal did not affect the
texture aspect of sago cookies. Calcium test activity is 0.1 grams in 100 grams of material. Suggestion: business feasibility analysis of making sago cookies with the addition of 10% fish bone
meal and tested for acceptability of snacks for kidney patients. Keywords: sago pastries, tuna bone meal, organoleptic properties Open Journal System (OJS): journal.thamrin.ac.id Abstrack Background of the research: One type of pastry is sago cookies made with sago flour as the basic
ingredient. This research was conducted to utilize fish bone waste which still has nutrients,
especially calcium which is quite high and is added to the manufacture of sago pastries. Objective:
To determine the effect of adding mackarel tuna bone meal on organoleptic properties (color,
texture, aroma, and taste), acceptability, and calcium in sago pastries. Methods: This research is experimental, by adding mackarel tuna bone meal to sago pastries of
0%, 10%, 15%, and 20%. This study uses the Analysis of variance test followed by Duncan's test if
the p-Value <0.005. Result: There is an effect of adding mackarel tuna bone meal to the hedonic
quality of the color, aroma, taste, and preference level of sago pastries, does not affect the hedonic
quality of the texture aspect of sago pastries. The selected sago cookies were T2 (10% mackarel
tuna bone meal) with a total energy value of 368 kcal, 10 grams of protein, 68 grams of
carbohydrates, 6 grams of total fat, and 0.1 grams of calcium using the AAS method. 46 Open Journal System (OJS): journal.thamrin.ac.id
http://journal.thamrin.ac.id/index.php/jkmp/article/view/74 METODE PELAKSANAAN METODE PELAKSANAAN Penelitian ini merupakan penelitian eksperimental yang bertujuan untuk mengetahui
komposisi tepung tulang ikan terhadap sifat organoleptik kue kering. Variable bebas
dalam penelitian ini yaitu komposisi tepung tulang ikan tongkol. Sedangkan varabel terikat
dalam penelitian ini yaitu sifat organoleptik kue kering. Penelitian dilakukan di
laboratorium gizi universitas MH.Thamrin Jakarta pada bulan Juli-Agustus 2020. Rancangan percobaan yang digunakan untuk penelitian ini adalah Rancangan Acak
Lengkap (RAL) dengan 3 kali perlakuan diulang sebanyak 2 kali. Tujuan dilakukan 3
perlakuan yaitu untuk mendapatkan sifat organoleptik kue kering yang optimal. Perlakuan
1 yaitu dengan penambahan tepung tulang tongkol sebesar 10%. Perlakuan 2 yaitu dengan
penambahan tepung tulang tongkol 15%. Perlakuan 3 yaitu dengan penambahan tepung
tulang tongkol sebesar 20%. Data yang telah dikumpulkan kemudian dianalisis dengan software analisis data. Penelitian
ini menggunakan Rancangan Acak Lengkap (RAL). Dengan 2 kali ulangan. Uji
organoleptik dianalisis dengan sidik garam (ANOVA), menunjukkan pengaruh perlakuan
nyata, maka dilanjutkan dengan Duncan’s Multiple Range Test untuk mencari keberadaan
perbedaan dari perlakuan yang ada. PENDAHULUAN Tulang merupakan salah satu bagian dari tubuh makhluk hidup yang paling banyak
mengandung kalsium. Diperkirakan 99% kalsium terdapat di dalam jaringan keras yaitu
tulang dan gigi dan 1% kalsium terdapat pada darah dan jaringan lunak. Tanpa 1%
kalsium, otot akan mengalami gangguan kontraksi, darah akan sulit membeku, rangsangan
saraf akan terganggu dalam pengahantarnya. Untuk memenuhi kebutuhan 1% kalsium,,
tubuh mendapatkannya dari makanan yang dimakan atau dari tulang, karena mayoritas
mineral dan vitamin tidak dapat di produksi sendiri oleh tubuh. Kandungan kalsium dan fosfor pada tulang cukup tinggi sehingga berpotensi untuk
mencukupi asupan kalsium. Tulang ikan dalam bentuk tepung ikan memiliki kandungan
kalsium yang cukup tinggi, pada ikan manyung sebesar 12,8 mg/100 gram, pada ikan mata
besar 15,2 mg/100 gram (Iwansyah, dkk., 2008). Dalam Almatsier (2004), mengkonsumsi
ikan dengan tulangnya merupakan salah satu sumber kalsium yang baik, sehingga tulang
ikan mempunyai potensi sebagai alternatif bahan makanan kaya kalsium. Selama ini tulang
ikan hanya menjadi limbah perikanan dapat mencemari lingkungan. Tulang ikan dapat
dimanfaatkan setelah diolah menjadi tepung tulang ikan misalkan dibuat menjadi kue
kering salah satu nya adalah kue kering sagu. Kue kering sagu adalah jenis kue yang
memanfaatkan sagu atau tapioka sebagai salah satu komponen penyusun adonannya. Pemanfaatan tulang ikan menjadi tepung kemudian diolah lebih lanjut, dapat mengurangi
jumlah limbah perikanan dan secara tidak langsung menyumbang asupan kalsium untuk
masyarakat Indonesia. Berdasarkan hal tersebut, peneliti ingin melakukan penelitian
tentang Uji Coba Pembuatan Kue Kering Sagu dengan Penambahan Tepung Tulang Ikan
dan Daya Terimanya. Penelitian ini bertujuan untuk mengetahui pengaruh penambahan
tepung tulang ikan pada pembuatan kue kering sagu dan daya terima nya. 47 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Jurnal Kesehatan Masyarakat Perkotaan Jurnal Kesehatan Masyarakat Perkotaan
sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 HASIL DAN PEMBAHASAN Penelitian ini dilakukan 2 tahap yaitu tahap pertama penelitian pendahuluan dan tahap
kedua penelitian utama. Penelitian pendahuluan pembuatan kue kering sagu dilakukan
pada bulan Juli 2020 Penelitian ini betujuan untuk mengetahui persen penambahan tepung
tulang ikan sehingga menghasilkan kue kering sagu yang baik dan dapat diterima. Dalam
penelitian pendahuluan telah dibuat kue kering sagu dengan penambahan tepung tulang
ikan tongkol sebanyak 10% dan 50%, dilakukan uji daya terima dan organoleptik dengan
hasil sebagai berikut: Tabel 1 Karakteristik Kue Kering sagu
Kriteria
10%
50%
Rasa
Manis
Kurang manis
Aroma
Tidak beraroma ikan
Aroma ikan sangat kuat
Warna
Krem
Cokelat
Tekstur
Agak keras
Sangat keras Tabel 1 Karakteristik Kue Kering sagu 48 Open Journal System (OJS): journal.thamrin.ac.id ͬ74 ͬ74 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944
Tingkat kesukaan
Suka
Tidak suka
sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 Berdasarkan hasil penelitian pendahuluan maka dapat disimpulkan bahwa kue kering sagu
dengan penambahan tepung tulang ikan 10% hasilnya bisa diterima oleh panelis dari pada
kue kering dengan penambahan tepung tulang ikan 50%. pada penambahan 50% penilaian
panelis tidak suka sehingga dapat disimpulkan penambahan tepung tulang ikan kurang dari
50%. Penelitian utama merupakan lanjutan dari penelitian pendahuluan. Kue kering dibuat
dengan konsentrasi penambahan yang telah di identifikasi pada penelitian pendahuluan
untuk selanjutnya akan diuji mutu organoleptik dan daya terimanya. Berdasarkan hasil uji
pendahuluan konsentrasi yang dipakai untuk penelitian lanjutan adalah 10%, 15%, 20%. Penelitian ini menggunakan kontrol untuk membandingkan pengaruh percobaan terhadap
kue kering sagu yang diteliti. Proses penelitian ini meliputi penambahan tepung tulang ikan
tongkol terhadap kue kering sagu pada persentase 10 gram, 15 gram, 20 gram dan 0 gram
sebagai kontrol. Selanjutnya dilakukan uji organoleptik yaitu uji mutu hedonik dengan
kriteria uji warna, tekstur, aroma dan rasa. Prinsip uji mutu hedonik berdasarkan penilaian panelis terhadap sifat organoleptik dengan
penganalisaan tingkat kesan (skala mutu hedonik). Pada uji mutu hedonik produk kue
kering sagu ditentukan nilai 1 hingga nilai 5, dimana nilai paling rendah yang berarti
kualitas kue kering paling jelek, menaik hingga nilai 5 pada skala hedonik dimana kualitas
kue kering sagu semakin baik seiring menaiknya penilaian panelis. Uji mutu hedonik pada
penelitian kue kering sagu mencakup kriteria uji warna, tekstur, aroma dan rasa. Uji mutu
hedonik kue kering sagu dilakukan di ruang kelas yang dikondisikan sebagai laboratorium
uji. Pengujian dilakukan oleh panelis agak terlatih dari mahasiswa S1 Gizi semester 6
sebanyak 32 orang. HASIL DAN PEMBAHASAN Panelis melakukan uji mutu hedonik produk kue kering sagu 4
perlakuan 2 ulangan dengan konsentrasi tepung tulang ikan tongkol 0 gram, 10 gram, 15
gram dan 20 gram. Penelitian ini menggunakan Rancangan Acak Lengkap (RAL) dengan 2 kali ulangan. Uji
organoleptik dianalisis dengan sidik ragam (ANOVA), menunjukkan beda nyata pada taraf
5%. Jika ANOVA menunjukkan pengaruh perlakuan nyata, maka dilanjutkan dengan
Duncan’s Multiple Range Test untuk mencari keberadaan perbedaan dari perlakuan yang 49 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944
ada. Data yang didapatkan dari uji organoleptik untuk menentukan produk terpilih yang
kemudian akan diuji laboratorium yaitu uji aktivitas Proksimat dan Kalsium. sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22
Hal 46-60 Jurnal Kesehatan Masyarakat Perkotaan
sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 y
2Ϭ22 2͕ ada. Data yang didapatkan dari uji organoleptik untuk menentukan produk terpilih yang
kemudian akan diuji laboratorium yaitu uji aktivitas Proksimat dan Kalsium. ada. Data yang didapatkan dari uji organoleptik untuk menentukan produk terpilih yang
kemudian akan diuji laboratorium yaitu uji aktivitas Proksimat dan Kalsium. Warna Warna termasuk dalam penilaian uji organoleptik. Menurut Nurwantoro et al (2016),
warna merupakan faktor yang cukup penting untuk sebuah produk, karena dapat
mempengaruhi persepsi terhadap rasa dari produk dan menjadikan produk lebih menarik. Hasil uji organoleptik menunjukkan bahwa nilai rata-rata uji mutu hedonik aspek warna
pada T1 = 4.58 (Krem kecoklatan), T2 = 3.64 (Agak coklat), T3 = 4.34 (Krem ), T4= 3.63
(agak coklat) Hasil uji anova didapatkan bahwa terdapat pengaruh penambahan teung tulang ikan
tongkol terhadap sifat organoleptk mutu hedonik warna produk kue kering sagu karena
nilai p value < 0.05 yaitu sebesar 0.000 yang artinya H0 ditolak. Secara deskriptif terlihat
adanya pengaruh penambahan tepung tulang ikan tongkol yaitu produk kue kering sagu
cenderung agak coklat. Hal ini didukung dengan hasil analisis statistik menunjukkan ada perbedaan yang nyata
pada penambahan tulang ikan tongkol terhadap tingkat kesukaan aspek warna kue kering
sagu. Untuk mengetahui pengaruh tersebut maka dilakukan uji lanjut Duncan. Berdasarkan hasil uji Duncan terhadap mutu hedonik aspek warna terlihat bahwa T1-T2
dan T1-T3 menunjukkan perbedaan yang nyata. Urutan nilai terendah sampai tertinggi
terhadap aspek warna yaitu perlakuan penambahan 20 gram tepung tulang ikan tongkol
(T4) memperoleh nilai terendah dengan nilai 3.63 (agak coklat), kemudian dengan
tambahan 10 gram tepung tulang ikan tongkol (T2) dan yang tertinggi adalah dengan
tambahan 15 gram tepung tulang ikan tongkol (T3) dengan nilai 4.34 (cenderung krem). Sedangkan T2-T3 tidak menunjukkan ada perbedaan yang nyata. p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 Tabel 2. Data Hasil Uji Organoleptik Kriteria Tekstur
Konsentrasi
Kriteria Uji Tekstur
0 gram T11
2.84a
0 gram T12
2.91a
10 gram T21
3.16a
10 gram T22
3.03a
15 gram T31
2.97a
15 gram T32
2.84a
20 gram T41
3.06a
20 gram T42
2.94a Tabel 2. Data Hasil Uji Organoleptik Kriteria Tekstur Berdasarkan Tabel 2 hasil uji organoleptik tekstur menunjukkan bahwa nilai rata-rata uji
mutu hedonik aspek tekstur pada T1 = 2.87 (cenderung agak keras), T2 = 3.09 (cenderung
renyah), T3 = 2.90 (cenderung agak keras), T4=3 (cenderung renyah). Hal ini berhubungan
dengan penambahan tepung tulang ikan kedalam kue kering, semakin besar penambahan
tulang ikan akan menambah kerenyahan kue kering yang dihasilkan. Kerenyahan kue kering disebabkan tepung tulang ikan mengandung protein, dimana
protein memiliki gugus hidrofil (Pratiwi, 2008). Gugus hidrofil pada protein jauh lebih
banyak dibandingkan banyak dibandingkan pati, sehingga tekstur semakin bertambah gurih
dan renyah (Tababaka, 2004). Hasil uji anova didapatkan bahwa terdapat pengaruh penambahan tepung tulang ikan
tongkol terhadap sifat organoleptik tekstur produk kue kering sagu karena nilai p value >
0.05 yaitu sebesar 0.928 yang artinya H0 diterima. Secara deskriptif tidak adanya pengaruh
penambahan tepung tulang ikan tongkol terhadap tekstur produk kue kering sagu. Tekstur Tekstur merupakan kategori penilaian uji organoleptik mutu hedonik Tekstur kue kering
sagu umumnya keras. Penambahan tepung tulang ikan tongkol dengan konsentrasi berbeda
pada penelitian ini dilakukan untuk mengetahui pengaruh konsentrasi tepung tulang ikan
tongkol yang diberikan terhadap aspek tekstur kue kering sagu. Kategori penilaian tekstur
kue kering sagu meliputi keras sampai amat sangat renyah. Hasil uji organoleptik terhadap
aspek tekstur dapat dilihat pada Tabel 2. 50 Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Aroma Aroma termasuk dalam penilaian uji organoleptik. Penilaian aroma dilakukan dengan
indera penciuman. Aroma kue kering sagu berasal dari aroma khas kelapa sangrai. Penambahan tepung tulang ikan tongkol dengan konsentrasi berbeda pada penelitian ini
dilakukan untuk mengetahui pengaruh konsentrasi tepung tulang ikan yang diberikan
terhadap aroma. Kategori penilaian aroma kue kering sagu meliputi tidak beraroma ikan
sampai aroma ikan amat sangat kuat. Berdasarkan Tabel 3, hasil uji organoleptik
menunjukkan bahwa nilai rata-rata uji mutu hedonik aspek aroma pada T1 = 4.77 (agak
beraroma ikan), T2 = 3.92 (cenderung aroma ikan), T3 = 3.24 (cenderung aroma ikan),
T4=3.04 (cendrung aroma ikan). 51 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944
sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22
Hal 46-60 Jurnal Kesehatan Masyarakat Perkotaan
sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hasil uji anova didapatkan bahwa terdapat pengaruh penambahan tepung tulang ikan
tongkol terhadap sifat organoleptik aroma produk kue kering sagu karena nilai p value <
0.05 yaitu sebesar 0.000 yang artinya H0 ditolak. Secara deskriptif terlihat bahwa semakin
bertambah konsentrasi tepung tulang ikan, produk kue kering sagu cenderung beraroma
ikan agak kuat. sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 y
2Ϭ22 2͕ Hasil uji anova didapatkan bahwa terdapat pengaruh penambahan tepung tulang ikan
tongkol terhadap sifat organoleptik aroma produk kue kering sagu karena nilai p value <
0.05 yaitu sebesar 0.000 yang artinya H0 ditolak. Secara deskriptif terlihat bahwa semakin
bertambah konsentrasi tepung tulang ikan, produk kue kering sagu cenderung beraroma
ikan agak kuat. Hasil uji organoleptik terhadap aspek aroma dapat dilihat pada Tabel 3 Hasil uji organoleptik terhadap aspek aroma dapat dilihat pada Tabel 3 Tabel 3 Data Hasil Uji Organoleptik Kriteria Aroma
Konsentrasi
Kriteria Uji Aroma
0 gram T11
4.88c
0 gram T12
4.66c
10 gram T21
3.97b
10 gram T22
3.88b
15 gram T31
3.21a
15 gram T32
3.28a
20 gram T41
3.00a
20 gram T42
3.09a Tabel 3 Data Hasil Uji Organoleptik Kriteria Aroma Hasil analisis statistik memunjukkan ada perbedaan yang nyata pada penambahan tulang
ikan tongkol terhadap tingkat kesukaan aspek aroma kue kering sagu. Untuk mengetahui
pengaruh tersebut maka dilakukan uji lanjut Duncan. Berdasarkan hasil uji Duncan terhadap mutu hedonik aspek aroma terlihat bahwa T1-T2,
T1-T3, dan T2-T4 menunjukkan perbedaan yang nyata. Aroma Urutan nilai terendah sampai
tertinggi terhadap aspek warna yaitu perlakuan penambahan 20 gram tepung tulang ikan
(T4) memperoleh nilai terendah dengan nilai 3.00 (aroma ikan), kemudian dengan
tambahan tepung tulang ikan tongkol 10 gram (T2) dan yang tertinggi adalah dengan
tanpa tambahan (T1) dengan nilai 4.88 (cenderung agak beraroma ikan). Sedangkan T3-T4
tidak menunjukkan ada perbedaan yang nyata. Rasa Salah satu faktor yang menentukan kualitas makanan adalah kandungan senyawa citarasa. Menurut West, et al., dalam Margaretha dan Edwin (2012) titik perasa dari lidah adalah
kemampuan mendeteksi dasar yaitu manis, asam, asin, pahit, trigeminal (astringent, dingin,
panas) dan aroma setelah mengkonsumsi senyawa tersebut. Lewat proses pemberian aroma
pada suatu produk pangan, lidah dapat mengecap rasa lain sesuai aroma yang diberikan
(Midayanto dan Yuwono, 2014). Dalam pengujian ini dilakukan terhadap kriteria uji rasa 52 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944
sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22
Hal 46-60 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944
sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 y
ƚ2Ϭ22 2͕ untuk mutu hedonik yang menyatakan kekhasan rasa kue kering sagu. Hasil uji
organoleptik terhadap kriteria uji rasa dapat dilihat pada Tabel 4 untuk mutu hedonik yang menyatakan kekhasan rasa kue kering sagu. Hasil uji
organoleptik terhadap kriteria uji rasa dapat dilihat pada Tabel 4 Tabel 4 Data Hasil Uji Organoleptik Kriteria Rasa Tabel 4 Data Hasil Uji Organoleptik Kriteria Rasa Konsentrasi
Kriteria Uji Rasa
0 gram T11
3.94bc
0 gram T12
4.19c
10 gram T21
3.53abc
10 gram T22
3.63abc
15 gram T31
3.47abc
15 gram T32
3.09a
20 gram T41
3.22ab
20 gram T42
3.44ab Berdasarkan Tabel 4, hasil uji organoleptik menunjukkan bahwa nilai rata-rata uji mutu
hedonik aspek rasa pada T1 = 4.06 (cenderung gurih tidak manis), T2 = 3.58 (cenderung
gurih), T3 = 3.28 (cenderung gurih), T4=3.33 (cenderung gurih). Hasil uji anova didapatkan bahwa terdapat pengaruh penambahan tepung tulang ikan
tongkol terhadap sifat organoleptik mutu hedonik rasa produk kue kering sagu karena nilai
p value > 0.05 yaitu sebesar 0.025 yang artinya H0 ditolak. Secara deskriptif terlihat bahwa
penambahan tulang ikan tongkol berpengaruh nyata terhadap aspek rasa kue kering sagu. Hal ini didukung dengan hasil analisis statistik memangmenunjukkan ada perbedaan yang
nyata pada penambahan tepung tulang ikan terhadap tingkat kesukaan aspek rasa kue
kering sagu. Untuk mengetahui pengaruh tersebut maka dilakukan uji lanjut Duncan,
hasilnya terlihat bahwa seluruh perlakuan menunjukkan perbedaan yang tidak nyata, yaitu
T1-T2, T1-T3, T1-T4, T2-T3, T2-T4 dan T3-T4. Rasa Urutan nilai terendah sampai tertinggi
terhadap aspek tingkat kesukaan rasa yaitu perlakuan dengan tambahan Tepung tulang ikan
15 gram (T3) memperoleh nilai terendah dengan nilai 3.09 (cenderung gurih), kemudian
penambahan tepung tulang ikan tongkol 10 gram (P2) dan yang tertinggi adalah dengan
tambahan tepung tulang ikan tongkol (P1) dengan nilai 4.19 (cenderung gurih agak
manis). Open Journal System (OJS): journal.thamrin.ac.id Jurnal Kesehatan Masyarakat Perkotaan
sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 2͕ Tingkat kesukaan Tingkat kesukaan meliputi penilaian terhadap aspek penilaian aroma, warna, tekstur dan
rasa dari kue kering sagu dengan penambahan tepung tulang ikan tongkol. Oleh karena itu 53 Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944
Ğ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 y
ƚ2Ϭ22 2͕ dilakukan uji organoleptik untuk mengetahui tingkat kesukaan konsumen terhadap kue
kering sagu tersebut dan hasilnya dapat dilihat pada tabel 5 dilakukan uji organoleptik untuk mengetahui tingkat kesukaan konsumen terhadap kue
kering sagu tersebut dan hasilnya dapat dilihat pada tabel 5 Tabel 5. Data Hasil Uji Organoleptik Kriteria Tingkat Kesukaan Konsentrasi
Kriteria Uji tingkat
kesukaan
0 gram T11
2.84de
0 gram T12
3.00e
10 gram T21
2.47bcd
10 gram T22
2.59cde
15 gram T31
2.13abc
15 gram T32
1.88a
20 gram T41
1.69a
20 gram T42
1.97ab Berdasarkan Tabel 5, hasil uji organoleptik mutu hedonik menunjukkan bahwa nilai rata-
rata uji mutu hedonik aspek tingkat keseukaan secara keseluruhan pada T1 = 2.92
(cenderung suka), T2 = 2.53 (cenderung suka), T3 = 2.00 (cenderung agak suka),
T4=1.83( cenderung tidak suka). Hasil uji anova didapatkan bahwa terdapat pengaruh penambahan tepung tulang ikan
tongkol terhadap sifat organoleptik mutu hedonik tingkat kesukaan secara keseluruhan
produk kue kering sagu karena nilai p value > 0.05 yaitu sebesar 0.00 yang artinya H0
ditolak. Secara deskriptif terlihat bahwa penambahan tepung tulang ikan tongkol
berpengaruh nyata terhadap aspek tingkat kesukaan kue kering sagu. Hal ini didukung dengan analisis statistik memang ada perbedaan yang nyata pada
penambahan tepung tulang ikan tongkol terhadap tingkat kesukaan aspek tingkat kesukaan
secara kesluruhan. Untuk mengetahui pengaruh tersebut maka dilakukan uji lanjut Duncan,
terlihat bahwa seluruh perlakuan menunjukkan perbedaan yang tidak nyata, yaitu T1-T2,
T2-T3, T2-T4, dan T3-T4. Urutan nilai terendah sampai tertinggi terhadap aspek tingkat
kesukaan rasa yaitu perlakuan dengan tambahan tepung tulang ikan tongkol 20 gram (P4)
memperoleh nilai terendah dengan nilai 1.69 (cenderung agak suka), kemudian dengan
penambahan tepung tulang ikan tongkol (T2) dan yang tertinggi adalah dengan tanpa
tambahan tepung tulang ikan tongkol (T1) dengan nilai 3.00 (cenderung suka). 54 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 p-ISSN: 2776-0952 e-ISSN : 2776-0944 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 Penentuan Produk Terpilih Penentuan produk terpilih ditentukan berdasarkan hasil uji tingkat kesukaan (hedonik) dan
seberapa banyak aktivitas Proksimat dan Kalsium yang dapat disumbangkan oleh tepung
tulang ikan tongkol ke dalam Kue Kering Sagu. Uji hedonik kue kering sagu meliputi
warna, tekstur, aroma dan rasa. Parameter uji hedonik yang digunakan adalah 1 (tidak
suka) hingga 5 (amat sangat suka). Nilai rata-rata dari hasil uji tingkat kesukaan (hedonik)
tepung tulang ikan tongkol dapat dilihat pada Tabel 6 Tabel 6 Hasil Uji Mutu Hedonik Kue Kering Sagu Tabel 6 Hasil Uji Mutu Hedonik Kue Kering Sagu
Perlakuan
Kategori
Warna
Tekstur
Aroma
Rasa
Tingkat
kesukaan
0 gram T11
4.47cd
2.84a
4.88c
3.94bc
2.84de
0 gram T12
4.69d
2.91a
4.66c
4.19c
3.00e
10 gram T21
3.84ab
3.16a
3.97c
3.53abc
2.47bcd
10 gram T22
3.44a
3.03a
3.88b
3.63aabc
2.59cde
15 gram T31
4.38bcd
2.97a
3.31a
3.47abc
2.13abc
15 gram T32
4.31bcd
2.84a
3.28a
3.09a
1.88a
20 gram T41
3.88abc
3.06a
3.09a
3.44ab
1.69a
20 gram T41
3.38a
2.94a
3.00a
3.22ab
1.97ab
Ket : Huruf yang beda pada baris yang sama menunjukkan perbedaan yang nyata
(p<0,05) Ket : Huruf yang beda pada baris yang sama menunjukkan perbedaan yang nyata
(p<0,05) Berdasarkan Tabel 6 nilai tertinggi pada kategori penilaian warna adalah T12 (tanpa
penambahan tulang ikan tongkol) yaitu 4.69 (cenderung krem kecoklatan ). Nilai tertinggi
pada kategori tekstur adalah T21 (10 gram) yaitu 3.16 (cenderung renyah). Nilai tertinggi
pada kategori aroma adalah T12 (tanpa penambahan tulang ikan tongkol) yaitu 4.88
(cenderung agak beraroma ikan ). Nilai tertinggi pada kategori rasa adalah T21 (tanpa
penambahan tulang ikan tongkol) yaitu 4.19 (cenderung gurih agak manis). Nilai tertinggi
dari tingkat kesukaan adalah T12 (tanpa penambahan tepung tulang ikan tongkol) yaitu
3.00 (cenderung suka). Dapat disumpulkan berdasarkan hasil uji mutu hedonik, diketahui produk terpilih adalah
T2 yaitu perlakuan dengan penambahan tepung tulang ikan tongkol sebanyak 10% yang
memiliki nilai tertinggi dari tingkat kesukaan aspek tekstur dan rasa. Deskripsi sifat fisik
produk kue kering sagu terpilih dapat dilihat pada Tabel 7 55 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 Tabel 7 Deskripsi Sifat Fisik Kue Kering Sagu Produk Terpilih (T2)
Aspek
Mutu Hedonik
Warna
Cenderung krem kecoklatan
Tekstur
Cenderung renyah
Aroma
Cenderung agak aroma ikan
Rasa
Cenderung gurih tidak manis
Tingkat kesukaan
Cenderung suka Tabel 7 Deskripsi Sifat Fisik Kue Kering Sagu Produk Terpilih (T2) Analisis Zat Gizi Nilai gizi kue kering sagu tertera pada setiap perlakuan berdasarkan bahan dari resep dasar
dengan penambahn tepung tulang ikan tongkol. Kebutuhan bahan masing-masing kue
kering sagu sesuai perlakuan untuk satu resep dapat dilihat pada Tabel 8 Tabel 8 Kebutuhan Kue Kering Sagu Untuk 1 Resep Tabel 8 Kebutuhan Kue Kering Sagu Untuk 1 Resep Tabel 8 Kebutuhan Kue Kering Sagu Untuk 1 Resep
Komposisi
Jumlah bahan yang di gunakan untuk satu resep (gram)
T1
T2
T3
T4
Tepung sagu
150
150
150
150
Gula
37.5
37.5
37.5
37.5
Rombutter
15
15
15
15
Telur
15
15
15
15
Margarin
45
45
45
45
Kelapa
15
15
15
15
Garam
1.5
1.5
1.5
1.5
Tepung tulang ikan tongkol
0
15
22.5
30
Jumlah Produk
60keping
70 keping
75 keping
80 keping
Berat total bahan
279 gr
289 gr
294 gr
299 gr Berdasarkan Tabel 8 satu resep dihasilkan T1 70 keping, T2 81 keping T3 87 keping dan
T4 93 keping. Sehingga T1, T2, T3, dan T4 memerlukan 1.5 resep untuk mencapai target
panelis 30 panelis. Dengan berat 1 Resep kue kering sagu 240 gram dengan berat 1 keping
6 gram. Analisis zat gizi makro dapat dilihat pada tabel pada tabel 9 dihitung
menggunakan data sekunder berdasarkan TKPI tahun 2009. Tabel 9. Zat Gizi Makro dan Kalsium kue kering sagu menurut TKPI Kandungan gizi Per
Resep
% penambahan tepung tulang ikan
0%
10%
15%
20 %
Kalori
321
321
321
321
Karbohidrat
52,91
52,91
52,91
52,91
Protein
1.1
4,25
6,23
7,94
Lemak
13.98
14,54
14.82
15.1
Kalsium
-
1.19 mg
1.78 mg
2.38 mg 56 Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944
sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 y
ƚ2Ϭ22 2͕ Analisis zat Gizi pada penelitian ini untuk produk terpilih di dapatkan dari hasil uji
proksimat dan uji kalsium yang dilakukan di laboratorium VICMALAB. Hasil penelitian
dapat dilihat pada tabel 10 Tabel 10. Perbandingan nilai gizi makro dan kalsium dengan hasil LAB dan KPI Tabel 10. KESIMPULAN Berdasarkan hasil penelitian dapat disimpulkan bahwa Karakteristik tepung tulang ikan
adalah berwarna krem kecoklatan, beraroma ikan agak tajam (amis) dan ada pengaruh
penambahan tepung tulang ikan terhadap aspek aroma, warna, rasa dan tingkat kesukaan
secara keseluruhan. Berdasarkan hasil uji organoleptic mutu hedonik Warna kue kering
sagu (T1 -T4) adalah krem kecoklatan, Tekstur adalah keras cenderung renyah, terhadap
Aroma adalah cenderung beraroma ikan, Rasa kue kering sagu adalah gurih cenderung
tidak manis, Tingkat kesukaan secara keseluruhan (Aroma, Rasa, Tekstur,dan Warna) kue
kering sagu terpilih adalah cenderung suka. Adapun Produk terpilih dari penilaian
kesukaan secara keseluruhan (Aroma, Rasa, Tekstur,dan Warna) adalah T2 dengan
penambahan tepung tulang ikan 10% dengan nilai gizi produk terpilih adalah sebagai
berikut : Energi (368 Kkal), Protein (10 gram), Karbohidrat (68 gram), Lemak (6 gram),
dan Kalsium (0,1mg/100gram), dengan Harga jual produk sebesar Rp. 570/ ressp (240gr). Analisis Zat Gizi Perbandingan nilai gizi makro dan kalsium dengan hasil LAB dan KPI
Parameter
Satuan Unit
Result
TKPI
Kadar air
%
13.36
-
Kadar Abu
%
2.04
-
Lemak total
%
6
14.54
Protein
%
10
4.25
Karbohidrat
%
68
52.91
Kalsium
mg/100g
0.1
1.19
Energi total
Kcal/100g
368
321 Berdasarkan tabel 10 hasil penelitian didapatkan kadar air kue kering yang dihasilkan
sebesar 13.36 yang terdapat pada kue kering yang terdapat pada tulang ikan sehibgga kadar
air berkurang. Menurut mahani 1999, adanya penambahan tepung tulang ikan berarti
terjadi penambahan partikel kalsium yang akan mengikat partikel air yang merupakan
bagian dari unsur-unsur air sehingga kadar air berkurang seiring dengan penambahan
tepung tulang ikan. Kadar abu terhadap kue kering sebesar 2,04 dalam tulang ikan
terkandung sel-sel dalam bentuk garam mineral. Garam mineral merupakan komponen yang terdiri dari kalsium posfat sebanyak 80% dan
sisanya terdiri dari kalsium karbonat dan magnesium posfat (Tababaka, 2004), sehingga
unsur ini membuat kadar abu padsa kue kering meningkat. Peningkatan kadar abu terhadap
produk akibat tepung tulang ikan yang digunakan. Sedangkan, pada uji proksimat dan
kalsium yang dilakukan pada produk terpilih pada kue kering sagu dan menggunakan
TKPI tidak berbeda jauh. Tabel 11 Hasil Uji Zat Gizi Makro Dalam 30 Gram Kue Kering Sagu
Nama bahan
Kalori
Karbohidrat
Protein
Lemak
Tepung sagu
45.25
10.86
0.06
0.03
Gula
20.6
4.9
0
0
Roombutter
38.95
0.07
0.026
4.27
Margarin
37.8
0.02
0.03
4.25
Kelapa
0.35
0.72
0.05
0.04
Telur
8.07
0.04
0.65
0.56 Tabel 11 Hasil Uji Zat Gizi Makro Dalam 30 Gram Kue Kering Sagu 57 Open Journal System (OJS): journal.thamrin.ac.id ͬ74 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 Jurnal Kesehatan Masyarakat Perkotaan 2͕ REFERENSI Almatsier, Sunita. 2009. Prinsip dasar ilmu gizi. Gramedia, Jakarta. Almatsier, Sunita. 2009. Prinsip dasar ilmu gizi. Gramedia, Jakarta. Anni Faridah. 2009. Patiseri .Jakarta: Direktorat pembinaan sekolah menengah kejuruan. Anni Faridah. 2009. Patiseri .Jakarta: Direktorat pembinaan sekolah menengah kejuruan. Asni, Yuli. 2004. Studi pembuatan biskuit dengan penambahan tepung tulang ikan patin. Skripsi. Fakultas perikanan dan ilmu kelautan. Institut Pertanian Bogor. Bogor. Darwin Philips.2013. Menikmati gula tanpa rasa takut. perpustakaan nasional:sinar ilmu. Darwin Philips.2013. Menikmati gula tanpa rasa takut. perpustakaan nasional:sinar ilmu. arwin Philips.2013. Menikmati gula tanpa rasa takut. perpustakaan nasional:sinar ilmu. Esti s p.2012. pemamfaatan tepung ubi kayu dan tepung biji kecipir sebagai subsitusi
terigu dalam pembuatan cookies.skripsi. diterbitkan Surakarta : fakultas pertanian. Universitas sebelas maret. Faole, M. 2003. The coconut odyssey:the bounteous possibilities of the tree of life. Autralian centre for international agricultural research, Canberra. Ibrahim.SM.2009. evaluation of production and quality of salt-biscuit supplemented with
fish protein concentrate. World journal of diary and food science 4(1):28-31. 58 Open Journal System (OJS): journal.thamrin.ac.id ͬ74 p-ISSN: 2776-0952 e-ISSN : 2776-0944 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944
Hal 46-60 Iwansyah, A.C.,dkk. 2008.Pengaruh Penambahan Tepung Tulang Ikan sebagai Sumber
Kalsium terhadap Mutu Kimia Kerupuk Ikan. Prosiding. Universitas lampung
sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22
Hal 46-60 Iwansyah, A.C.,dkk. 2008.Pengaruh Penambahan Tepung Tulang Ikan sebagai Sumber
Kalsium terhadap Mutu Kimia Kerupuk Ikan. Prosiding. Universitas lampung Kusumawardani, N.M. 2014. Kajian stok sumber daya ikan tongkol euthynnus affinis di
perairan selat sunda yang didaratkan di PPP Labuan, pandeglang, banten. skripsi. institut
pertanian bogor. Bogor.28 halm. Kusumawardani, N.M. 2014. Kajian stok sumber daya ikan tongkol euthynnus affinis di
perairan selat sunda yang didaratkan di PPP Labuan, pandeglang, banten. skripsi. institut
pertanian bogor. Bogor.28 halm. Lestari. S.2001. pemamfaatan limbah tulang ikan tuna menjadi tepung. Bogor. Fakultas
ilmu perikanan dan kelautan IPB. Muchtadi, Derry. 2009. Pengantar Ilmu Gizi. Penerbit Alfabeta Bandung. Bogor Nasiru, M., 2011. effect of cooking time and potash on organoleptic properties of red and
white meat, dalam ayustaningwarno, fitriyono.,2014, teknologi pangan, teori praktis dan
aplikasi, graha ilmu,Yogyakarta. Nigita, ariyani. 2012,.pemamfaatan limbah tulang ikan kuniran(upeneus moluccennesis)
untuk fortifikasi kalsium(ca) pada susu kedelai. Skripsi. fakultas tarbiah .IAIN .walisongo
.semarang. Nigita, ariyani. 2012,.pemamfaatan limbah tulang ikan kuniran(upeneus moluccennesis)
untuk fortifikasi kalsium(ca) pada susu kedelai. Skripsi. fakultas tarbiah .IAIN .walisongo
.semarang. Puspitasari, D. 2015.karakteristik biskuit subsitusi tepung sukun (artocarpus communis
forst) yang diperkaya dengan tepung kedelai (glycine max (linn.) Merrill). Skripsi. Teknologi pangan, fakultas teknik, universitas pasundan.bandung. Puspitasari, D. 2015.karakteristik biskuit subsitusi tepung sukun (artocarpus communis
forst) yang diperkaya dengan tepung kedelai (glycine max (linn.) Merrill). Skripsi. Teknologi pangan, fakultas teknik, universitas pasundan.bandung. Reski A.M. 2011 .pemamfaatan ekstraksi kulit ari biji kakao (theobroma cacao. L) pada
produk cookies cokelat. Skripsi. diterbitkan.fakultas pertanian. Universitas hasanuddin. Setyabudi, A. (2013). Pengembangan Mi Glosor Instan dari Tepung Sagu Aren dengan
Substitusi Tepung Labu Kuning sebagai Alternatif untuk Diversifikasi Pangan. Retrieved Substitusi Tepung Labu Kuning sebagai Alternatif untuk Diversifikasi Pangan. Retrieved
Setyaningsih, Dwi, Anton Apriyanto, dan maya Puspitasari. 2010. Open Journal System (OJS): journal.thamrin.ac.id arwin Philips.2013. Menikmati gula tanpa rasa takut. perpustakaan nasional:sinar ilmu. Analisis sensori untuk
Industri Pangan dan Argo. Bogor: IPB Setyaningsih, Dwi, Anton Apriyanto, dan maya Puspitasari. 2010. Analisis sensori untuk
Industri Pangan dan Argo. Bogor: IPB Sutomo, Budi. 2012. Rahasia Sukses Membuat Cake, Roti, KueKering & Jajanan Pasar. nsbooks. 59 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Jurnal Kesehatan Masyarakat Perkotaan p ISSN: 2776 0952 e ISSN : 2776 0944
Trilaksani. W, salamah E, and nabil M. 2006.pemanfaatan limbah tepung tulang ikan tuna
(thunnus sp) sebagai sumber kalsium dengan metode hidrolisis protein. Bulletin teknologi
hasil perikanan 9(2):34-35. sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22
Hal 46-60 y
p
Trilaksani. W, salamah E, and nabil M. 2006.pemanfaatan limbah tepung tulang ikan tuna
(thunnus sp) sebagai sumber kalsium dengan metode hidrolisis protein. Bulletin teknologi
hasil perikanan 9(2):34-35. sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22
Hal 46-60 Yanica I.A. (2013). Indeks Glikemik dan Karakterisasi Kimia Beras Analog Berbahan
Dasar Jagung, Sorgum, dan Sagu Aren. Institut Pertanian Bogor, Bogor. [BSN] Badan Standardisasi Nasional. 1992. Tepung tulang untuk bahan baku makanan
ternak: SNI 01-315. Jakarta: Badan Standardisasi Nasional. [DKBM] Daftar Komposisi Bahan Makanan. 2009. Tabel Komposisi Pangan
Indonesia.Jakarta: PT Elex Media Komputindo. [ISA] International Seafood of Alaska. 2002. Analysis of Fish Meal. Alaska. USA. Inc. Kodiak. 60 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74
|
https://openalex.org/W2592697347
|
https://www.biorxiv.org/content/biorxiv/early/2017/02/07/106500.full.pdf
|
English
| null |
Optimal Response Vigor and Choice Under Non-stationary Outcome Values
|
bioRxiv (Cold Spring Harbor Laboratory)
| 2,017
|
cc-by
| 23,204
|
.
CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017.
;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint Bernard W. Balleine
School of Psychology, UNSW, NSW 2052, Australia Bernard W. Balleine
School of Psychology, UNSW, NSW 2052, Australia Richard Nock
Data61, CSIRO, NSW 2015, Australia .
CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017.
;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint Optimal Response Vigor and Choice Under Non-stationary
Outcome Values Amir Dezfouli
School of Psychology, UNSW, NSW 2052, Australia Affiliation: School of Psychology, UNSW
Email: a.dezfouli@unsw.edu.au
Address: School of Psychology, UNSW, NSW 2052, Australia Abstract Within a rational framework a decision-maker selects actions based on the
reward-maximisation principle which stipulates they acquire outcomes with the
highest values at the lowest cost. Action selection can be divided into two dimen-
sions: selecting an action from several alternatives, and choosing its vigor, i.e.,
how fast the selected action should be executed. Both of these dimensions de-
pend on the values of the outcomes, and these values are often affected as more
outcomes are consumed, and so are the actions. Despite this, previous works
have addressed the computational substrates of optimal actions only in the spe-
cific condition that the values of outcomes are constant, and it is still unknown
what the optimal actions are when the values of outcomes are non-stationary. Here, based on an optimal control framework, we derive a computational model
for optimal actions under non-stationary outcome values. The results imply that
even when the values of outcomes are changing, the optimal response rate is
constant rather than decreasing. This finding shows that, in contrast to previ-
ous theories, the commonly observed changes in the actions cannot be purely
attributed to the changes in the outcome values. We then prove that this obser-
vation can be explained based on the uncertainty about temporal horizons; e.g.,
in the case of experimental protocols, the session duration. We further show that
when multiple outcomes are available, the model explains probability match-
ing as well as maximisation choice strategies. The model provides, therefore, a
quantitative analysis of optimal actions and explicit predictions for future test-
ing. Keywords: choice, responser vigor, reward learning, optimal actions . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 2 Disclosures and Acknowledgments Contents Contents Contents
Introduction
4
Model Specification
5
The outcome space . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
The reward . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Cost . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Results
7
Optimal response vigor
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Response vigor when the session duration is known
. . . . . . . . . . . . . . . . . . 7
Response vigor when session duration is unknown . . . . . . . . . . . . . . . . . . . 8
Effect of experimental parameters: Relating the model to currently available data . 10
Optimal choice and response vigor
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Conservative reward field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Non-conservative reward field . . . . . . . Contents . . . . . . . . . . . . . . . . . . . . . . . . 15
Discussion
16
References
17
Appendix
19
Value in non-deterministic schedules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Optimal actions in one-dimensional outcome space . . . . . . . . . . . . . . . . . . . . . 21
Theorem 1: Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Theorem 2: Proof and simulation details . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Proof of Theorem 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Simulation details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Simulation details of Figures 2, 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Optimal actions in multi-dimensional outcome space . . . . . . . . . . . . . . . . . . . . 25
Theorem 3: Proof and simulation details . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Proof of Theorem 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosures and Acknowledgments A.D completed this work when was employed by CSIRO. B.W.B. was supported by a Senior
Principal Research Fellowship from the National Health & Medical Research Council of Australia,
GNT1079561. All authors contributed in a significant way to the manuscript and that all authors have
read and approved the final manuscript. The author declares that the research was conducted in the absence of any commercial or
financial relationships that could be construed as a potential conflict of interest. The author declares that the research was conducted in the absence of any commercial or
financial relationships that could be construed as a potential conflict of interest. We are grateful to Hadi Lookzadeh and Peter Dayan for helpful discussions. We are grateful to Hadi Lookzadeh and Peter Dayan for helpful discussions. . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 3 .
CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017.
;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint Introduction According to normative theories of decision-making, actions made by humans and ani-
mals are chosen with the aim of earning the maximum amount of future reward whilst incurring
the lowest cost (Marshall, 1890; von Neumann & Morgenstern, 1947). Within such theories indi-
viduals optimize their actions by learning about their surrounding environment so as to satisfy
their long-term objectives. The problem of finding the optimal action is, however, argued to
have two aspects: (1) choice, i.e., deciding which action to select from several alternatives; and
(2) vigor, i.e., deciding how fast the selected action should be executed. For a rat in a Skinner box,
for example, the problem of finding the optimal action involves selecting a lever (choice) and de-
ciding at what rate to respond on that lever (vigor). High response rates can have high costs (e.g.,
in terms of energy consumption), whereas a low response rate could have an opportunity cost if
the experimental session ends before the animal has earned sufficient reward. Optimal actions
provide the right balance between these two factors and, based on the reinforcement-learning
framework and methods from optimal control theory, the characteristics of optimal actions and
their consistency with various experimental studies have been previously elaborated (Dayan,
2012; Niv, Daw, Joel, & Dayan, 2007; Salimpour & Shadmehr, 2014). These previous models have assumed, however, that outcome values are stationary and do
not change on-line over the course of a decision-making session. To see the limitations of such
an assumption, imagine the rat is in a Skinner box and has started to earn outcomes (e.g., food
pellets) by taking actions. One can assume that, as a result of consuming rewards, the motivation
of the animal to earn more food outcomes will decrease (e.g., because of satiety) and, therefore,
over time, the outcomes earned will have a lower value. Such changes in value should affect both
optimal choice and vigor (Killeen, 1995) but have been largely ignored in the previous models. This is while in most of the experimental and real-world scenarios, outcome values are affected
by the history of outcome consumption, a phenomenon known as “law of diminishing marginal
utility”1 in the economics literature, and as “drive reduction theory” in psychological accounts of
motivation, which indicates that the drive for earning an outcome decreases as the consequence
of prior consumption of the outcomes (Hull, 1943; Keramati & Gutkin, 2014). OPTIMAL RESPONSE VIGOR AND CHOICE 4 1Also known as “First Law of Gossen” named for Hermann Heinrich Gossen (1810 – 1858). Contents It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint Contents . 27
Simulation details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Theorem A2: Definition, proof and simulation details
. . . . . . . . . . . . . . . . . . . . 28
Proof of Theorem A2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Simulation details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Theorem A3: Definition, proof and simulation details
. . . . . . . . . . . . . . . . . . . . 29
Proof of Theorem A3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Simulation details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Introduction Here, building on previous work, we introduce a new concept called reward field, which
captures non-stationary outcome values. Using this concept and methods from optimal control
theory, we derive the optimal response vigor and choice strategy without assuming that outcome
values are stationary. In particular, the results indicate that even when the values of outcomes
are changing, the optimal response rate in an instrumental conditioning experiment is a con-
stant response rate. This finding rules out previous suggestions that the commonly observed
decrease in within-session response rates is due to decreases in outcome value (Killeen, 1995). Instead, we show that decreases in within-session response rates can be explained by uncer-
tainty regarding session duration. This later analysis is possible because the session duration
is explicitly represented in the current model, which is another dimension in which the current
model extends previous work. The framework is then extended to choice situations and specific
predictions are made concerning conditions under which the optimal strategy is maximization
or probability matching. . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint The outcome space We define the outcome space as a coordinate space with n dimensions, where n is the
number of outcomes in the environment. For example, in a concurrent instrumental condition-
ing experiment in which the outcomes are water and food pellets, the outcome space will have
two dimensions corresponding to water and food pellets. Within the outcome space, the state
of the decision-maker at time t is defined by two factors: (i) the amount of earned outcome up
to time t, which is denoted by xt and can be thought of as the position of the decision-maker
in outcome space; e.g., in the above example, xt = [1,2] would indicate that one unit of water
and two units of food pellet have been gained up to time t; and (ii) the outcome rate at time t,
denoted by vt, which can be considered the velocity of the decision-maker in the outcome space
(vt = dxt/dt); e.g., if a rat is earning two units of water and one unit of food pellet per unit of
time, then vt = [2,1]. In general, we assume that the outcome rate cannot be negative (v ≥0),
which means that the cumulative number of earned outcomes cannot decrease with time. OPTIMAL RESPONSE VIGOR AND CHOICE 5 The reward We assume that there exists an n-dimensional reward field, denoted by Ax,t, where each
element of Ax,t represents the per unit value of each of the outcomes. For example, the element
of Ax,t corresponding to food pellets represents the value of one unit of food pellet at time t,
given that x units of outcome have been previously consumed. As such, Ax,t is a function of both
time and the amount of outcome earned. This represents the fact that (i) the reward value of
an outcome can change value as a result of consuming previous outcomes, e.g., due to satiety
(depending on x) and (ii) the reward value of an outcome can change purely with the passage
of time; e.g., an animal can get hungrier over time causing the reward value of food pellets to
increase (depending on t). In general, we assume that Ax,t has two properties: ∂Ax,t
∂x
≤0, ∂Ax,t
∂t
≥0,
(1) (1) which entail that (i) the outcome values decrease (or remain constant) as more outcomes are
earned, and (2) that outcome values do not decrease with the passage of time. Cost Within the context of instrumental conditioning experiments, previous studies have ex-
pressed the cost of earning outcomes as a function of the delay between consecutive responses
made to earn outcomes. For example, if a rat is required to make several lever presses to earn
outcomes, then the cost will be higher if the delay between lever presses is short. More precisely,
if the previous response has occurred τ time steps ago, then the cost of the current lever press
will be (Dayan, 2012; Niv et al., 2007): Cτ = a
τ +b,
(2) (2) where a and b are constants. b is the constant cost of each lever press, which is independent
of the delay between lever presses whereas the factor a controls the rate-dependent component . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 6 6 of the cost. Previous research has established that predictions derived from this definition of
cost are consistent with experimental data (Dayan, 2012; Niv et al., 2007). Note that costs such
as basal metabolic rate and the cost of operating the brain, although consuming a high portion
of energy produced by the body, are not included in the above definition because they are con-
stant and independent of response rate and, therefore, are not directly related to the analysis of
response vigor and choice. Here, we express cost as a function of outcome rate rather than the rate of action execu-
tion. We define the cost function Kv as the cost paid at each time step for earning outcomes at
rate v. In the specific case that the outcome space has one dimension (there is only one out-
come), and under ratio schedules of reinforcement (fixed-ratio, variable-ratio, random-ratio) in
which the decision-maker is required to perform either precisely or on average k responses to
earn one unit of outcome, the cost defined in equation 2 will be equivalent to: Kv = ak2v2 +kbv. (3) (3) Kv = ak2v2 +kbv. See Theorem A1 in Appendix for the proof. This definition of cost implies that it is only a func-
tion of outcome rate and is time-independent (∂Kv/∂t = 0). Although, in general, it may seem
reasonable to assume that, as time passes within a session, the cost of taking actions will in-
crease because of factors such as effector fatigue, here we made a time-independence assump-
tion based on previous studies showing that factors such as effector fatigue have a negligible
effect on response rate (McSweeney, Hinson, & Cannon, 1996). In general, we assume that at
least one response is required to earn an outcome (k > 0), and the cost of earning outcomes is
non-zero (a > 0). OPTIMAL RESPONSE VIGOR AND CHOICE 7 7 Value The reward earned in each time step can be calculated as the reward of one unit of each of
the outcomes (Ax,t) multiplied by the amount earned from each of the outcomes, which will be
v.Ax,t. The cost of earning this amount of reward is Kv, and therefore the net amount of reward
earned will be: (4) Lx,v,t = v.Ax,t −Kv. (4) A decision-making session starts at t = 0 and the total duration of that session is T . We denote
the total reward gained in this period as S0,T , which is the sum of the net rewards earned at each
point in time: S0,T =
ZT
0
Lx,v,tdt. (5) (5) The quantity S0,T is called the value function, and the goal of the decision-maker is to find the
optimal rate of earning outcomes that yields the highest value. The optimal rates that maximize
S0,T can be found using different variational calculus methods, such as the Euler-Lagrange equa-
tion or the Hamilton-Jacobi-Bellman equation (Liberzon, 2011). The results presented in the
next sections are derived using the Euler-Lagrange equation (see Appendix for details of value
function in non-deterministic schedules). . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint Response vigor when the session duration is known.
We maintain the following theo-
rem: Response vigor when the session duration is known. We maintain the following theo-
rem: Theorem 1 If the duration of the session is fixed and the time-dependent change in the reward
field is zero (∂Ax,t/∂t = 0), then the optimal outcome rate is constant (dv/dt = 0). If the time-
dependent change in the reward field is positive (∂Ax,t/∂t > 0), then the optimal outcome rate will
be accelerating (dv/dt > 0). See Appendix for a proof of this theorem. Note that the assumption ∂Ax,t/∂t = 0 implies that the
passage of time has no significant effect on the reward value of the outcome; e.g., a rat is not get-
ting hungrier during an instrumental conditioning session, which is a reasonable assumption
given the short duration of such experiments (typically less than an hour). Within this condi-
tion, the above theorem states that the optimal response rate is constant throughout the session,
even under conditions in which the reward value of the outcome decreases within the session
as a result of earning outcomes, e.g., because of satiety. As an intuitive explanation for why a
constant rate is optimal, imagine a decision-maker who chooses a non-constant outcome rate
that results in a total of xT outcomes during the session. If, instead of the non-constant rate,
the decision-maker selects a constant rate v = xT /T , then the total outcomes earned will be the
same as before; however, the cost will be lower because cost is a quadratic function of the out-
come rate and, therefore, it is better to earn outcomes at a constant rate. Nevertheless, although
this prediction is clear enough, it is not consistent with the experimental results, described next. Within-session pattern of responses. It has been established that in various schedules
of reinforcement, including variable-ratio (VR) schedules (McSweeney, Roll, & Weatherly, 1994),
the rate of responding within a session has a particular pattern: the response rate reaches its
maximum a short time after the session starts (warm-up period), and then gradually decreases
toward the end of the session (Figure 1:left panel). Killeen (Killeen, 1994) proposed a mathe-
matical description of this phenomenon, which can be expressed as follows (Killeen & Sitomer,
2003): β =
r
δr +1/α,
(6) (6) where β is the response rate, δ is the minimum delay between responses, r is the resulting out-
come rate, and α is called specific activation2. 2Note that in the original notation in (Killeen & Sitomer, 2003), α is denoted by a and β is denoted by b. Optimal response vigor In this section we use the model presented above to analyse optimal response vigor when
there is one outcome and one response available in the environment. The analysis is divided
into two sections. In the first section, we assume that the decision-maker is certain about session
duration, i.e., that the session will continue for T time units, and will extend this analysis in the
next section to a condition in which the decision-maker assumes a probabilistic distribution of
session lengths. OPTIMAL RESPONSE VIGOR AND CHOICE Right panel: The theoretical pattern of within-session
responses predicted by the model in different conditions. Please see the text for details
of each condition. Figure 1. The pattern of within-session response rates (responses per minute). Left panel: Ex-
perimental data. The rate of responding per minute during successive intervals (each interval is
5 minutes) in a variable-ratio (VR15) schedule (k = 15). The figure is adopted from (McSweeney
et al., 1994). Right panel: The theoretical pattern of within-session responses predicted by the
model in different conditions. Please see the text for details of each condition. response rate
time
10
20
30
40
50
60
0
20
40
60
time (min)
response rate
decreasing reward and unknown session duration
fixed reward (known or unknown session duration)
known session duration (fixed or decreasing reward) onstant (dv/dt = 0). ward field is positive
me rate will be acceler-
Note that the assump-
ssage of time has no
Fig. 1. The pattern of within-session response rates (responses per minute). Left
panel: Experimental data. The rate of responding per minute during successive
intervals (each interval is 5 minutes) in a variable-ratio (VR15) schedule (k = 15). The figure is adopted from [11]. Right panel: The theoretical pattern of within-session
responses predicted by the model in different conditions. Please see the text for details
of each condition. Figure 1. The pattern of within-session response rates (responses per minute). Left panel: Ex-
perimental data. The rate of responding per minute during successive intervals (each interval is
5 minutes) in a variable-ratio (VR15) schedule (k = 15). The figure is adopted from (McSweeney
et al., 1994). Right panel: The theoretical pattern of within-session responses predicted by the
model in different conditions. Please see the text for details of each condition. RAFT
;
g ,
ental conditioning ses-
ven the short duration
an hour). Within this
the optimal response
even under conditions
e decreases within the
.g., because of satiety. nstant rate is optimal,
non-constant outcome
mes during the session. decision-maker selects
outcomes earned will
will be lower because
me rate and therefore
that the cause of any decrease in the within-session response
rate cannot be due purely to a change in outcome value. OPTIMAL RESPONSE VIGOR AND CHOICE 8 cause a decrease in response rate3. Although this model has been shown to provide a quantita-
tive match to the experimental data, it is not consistent with Theorem 1 which posits that, even
under conditions in which outcome values are changing within a session, the optimal response
rate should not decrease during the session. As a consequence, the present model suggests that
the cause of any decrease in the within-session response rate cannot be due purely to a change
in outcome value. cause a decrease in response rate3. Although this model has been shown to provide a quantita-
tive match to the experimental data, it is not consistent with Theorem 1 which posits that, even
under conditions in which outcome values are changing within a session, the optimal response
rate should not decrease during the session. As a consequence, the present model suggests that
the cause of any decrease in the within-session response rate cannot be due purely to a change
in outcome value. Note, however, the optimal response rate advocated by Theorem 1 is not consistent with
reports of decreasing response rates across a session, which implies that some of the assump-
tions made to develop the model may not be accurate. Although the form of the cost and reward
functions is reasonably general, we assumed that the duration of the session, T , is fixed and
known by the decision-maker. In the next section we show that relaxing this assumption such
that the duration of the session is unknown results much closer concordance between predic-
tions from the model and the experimental data. n duration, i.e., that
nits, and will extend
ondition in which the
distribution of session
uration is known. We
ssion is fixed and the
ld is zero (ˆAx,t/ˆt =
constant (dv/dt = 0). eward field is positive
me rate will be acceler-
Note that the assump-
assage of time has no
response rate
time
10
20
30
40
50
60
0
20
40
60
time (min)
response rate
decreasing reward and unknown session duration
fixed reward (known or unknown session duration)
known session duration (fixed or decreasing reward)
Fig. 1. The pattern of within-session response rates (responses per minute). Left
panel: Experimental data. The rate of responding per minute during successive
intervals (each interval is 5 minutes) in a variable-ratio (VR15) schedule (k = 15). The figure is adopted from [11]. Response vigor when the session duration is known.
We maintain the following theo-
rem: The model suggests that as the decision-maker
earns outcomes during the session, the value of α gradually declines due to satiety, which will where β is the response rate, δ is the minimum delay between responses, r is the resulting out-
come rate, and α is called specific activation2. The model suggests that as the decision-maker
earns outcomes during the session, the value of α gradually declines due to satiety, which will . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint DR
en established that
uding variable-ratio
inaccuracies in interval timing in animals [15, 16]. In this
condition, a plausible way to calculate optimal response rates
is to set an expectation as to how long the session will last
3Here satiety refers to both post-ingestive factors (such as blood glucose level; (Killeen, 1995)) and/or pre-ingestiv
actors (for example sensory specific satiety; (McSweeney, 2004)). OPTIMAL RESPONSE VIGOR AND CHOICE Note, however, the optimal response rate advocated by
Theorem 1 is not consistent with reports of decreasing re-
sponse rates across a session, which implies that some of the
assumptions made to develop the model may not be accurate. Although the form of the cost and reward functions is reason-
ably general, we assumed that the duration of the session, T,
is fixed and known by the decision-maker. In the next section
we show that relaxing this assumption such that the duration
of the session is unknown results much closer concordance
between predictions from the model and the experimental
data. Response vigor when session duration is unknown. In this section we assume that the
decision-maker is uncertain about the session duration, which can be either because the ses-
sion duration is in fact non-deterministic, or because of inherent inaccuracies in interval timing
in animals (Gallistel & Gibbon, 2000; Gibbon, 1977). In this condition, a plausible way to cal-
culate optimal response rates is to set an expectation as to how long the session will last and to
calculate the optimal response rate based on that expectation. Based on this, if t′ time step has
passed since the beginning of the session, then the expected session duration is E[T |T > t′] and
therefore the value of the rest of the session will be St′,E[T |T >t′]. The following theorem maintains
that the optimal rate of outcome delivery that maximizes the value function is a decreasing func-
tion of the current time in the session t′, and therefore the optimal response rates will decrease
throughout the session. OPTIMAL RESPONSE VIGOR AND CHOICE OPTIMAL RESPONSE VIGOR AND CHOICE 9 is non-zero (p(T ) > 0), (iii) values of outcomes decrease as more are consumed (∂Ax,t/∂x < 0), then
the optimal rate of outcome delivery is a decreasing function of t′: is non-zero (p(T ) > 0), (iii) values of outcomes decrease as more are consumed (∂Ax,t/∂x < 0), then
the optimal rate of outcome delivery is a decreasing function of t′: dv∗
t′
dt′ < 0. (7) dv∗
t′
dt′ < 0. dv∗
t′
dt′ < 0. (7) See Appendix for the proof of this theorem. Theorem 2 stems from two bases: (i) the opti-
mal rate of outcome delivery is a decreasing function of session length, i.e., when the session
duration is long the decision-maker can afford to earn outcomes more slowly, and (ii) when
the session duration is unknown, expected session duration should increase with the passage
of time. This phenomenon, which has been elaborated within the context of delayed gratifica-
tion (McGuire & Kable, 2013; Rachlin, 2000), is more significant if the decision-maker assumes a
heavy-tail distribution over T . Putting (i) and (ii) together implies that the optimal response rate
will decrease with the passage of time. Importantly, this suggests, from a normative perspective,
uncertainty about the session duration is necessary in order to explain within-session decreases
in response rates. For simulation of the model we characterized the session duration using a Generalized
Pareto distribution following (McGuire & Kable, 2013). Simulations of the model are depicted
in Figure 1:right panel. Simulations show three different conditions. In condition (i) the ses-
sion duration is known, and as the figure shows irrespective of whether the reward of outcomes
is decreasing or fixed, the optimal response rate is constant. In condition (ii) session duration
is unknown, but the reward of outcomes is constant. Again in this condition the optimal re-
sponse rate is constant. In condition (iii) session duration is unknown and the reward decreases
as more outcomes are consumed. Only in this condition, consistent with experimental data, re-
sponse rates decrease as time passes (see Appendix for details of the simulations). Therefore, the
simulations confirm that decreases in the reward of outcomes alone are not sufficient to explain
within-session response rates, but by assuming uncertainty about session duration, the pattern
of responses will be consistent with the experimental data. RA
nt rate. Nevertheless,
h, it is not consistent
next.
p
g
section we assume that the decision-maker is uncertain about
the session duration, which can be either because the session
duration is in fact non-deterministic or because of inherent
Theorem 2 Assuming St′,E[T |T >t′] is the value function and that (i) the time dependent change in
the reward field is zero (∂Ax,t/∂t = 0), (ii) the probability that the session ends at each point in time RA
nt rate. Nevertheless,
h, it is not consistent
next. p
g
section we assume that the decision-maker is uncertain about
the session duration, which can be either because the session
duration is in fact non-deterministic, or because of inherent
Theorem 2 Assuming St′,E[T |T >t′] is the value function and that (i) the time dependent change in
the reward field is zero (∂Ax,t/∂t = 0), (ii) the probability that the session ends at each point in time DRA
been established that
cluding variable-ratio
h
h
inaccuracies in interval timing in animals [15, 16]. In this
condition, a plausible way to calculate optimal response rates
is to set an expectation as to how long the session will last
3Here satiety refers to both post-ingestive factors (such as blood glucose level; (Killeen, 1995)) and/or pre-ingestive
factors (for example sensory specific satiety; (McSweeney, 2004)). . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint 9 OPTIMAL RESPONSE VIGOR AND CHOICE 10 lasts exactly 30 minutes whereas for another group the session length is uncertain and can end
at any time (but ends on average after 30 minutes), then the model predicts that the response
rate in the second group will be higher at the start and decrease more sharply than in the first
group. This effect is not anticipated by the temporal discounting account of the effect. Effect of experimental parameters: Relating the model to currently available data. Op-
timal response rates predicted by the model are affected by experimental parameters (e.g., re-
ward magnitude), which can be compared against experimental data. In general, in an instru-
mental conditioning experiment, the duration of the session can be divided into three sections:
(i) outcome handling/consumption time, which refers to the time that an animal spends con-
suming the outcome, (ii) post-reinforcer pause, which refers to the pause that occurs after con-
suming the outcome and before starting to make the next response (e.g., lever press). Such a
pause is consistently reported in previous studies using an FR schedule, (iii) run time, which
refers to the time spent making responses (e..g, lever pressing). Experimental manipulations
have been shown to have different effects on the duration of these three sections of the session,
and whether each of these sections is included when calculating response rates can affect the re-
sults. The predictions of the current model are with regard to response rate; whether manipulat-
ing experimental parameters are expected to change the two other measures (outcome handling
and post-reinforcer pause) cannot be predicted by the current model. In the following sections,
we briefly review the currently available data from instrumental conditioning experiments and
their relationship to predictions of the model4. The effect of response cost (a and b). Experimental studies in rats working on a FR sched-
ule (Alling & Poling, 1995) indicate that increasing the force required to make responses causes
increases in both inter-response time and the post-reinforcement pause. The same trend has
been reported in Squirrel monkeys (Adair & Wright, 1976). Consistent with this observation, the
present model predicts that increases in the cost of responding, for example by increasing the
effort required to press the lever (increases in a and b), lead to a lower rate of earned outcomes
and a lower rate of responding (Figure 2). OPTIMAL RESPONSE VIGOR AND CHOICE CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint 10 4Note that, for simplicity, the simulations in this section are made under the assumption that the session duration
is fixed. OPTIMAL RESPONSE VIGOR AND CHOICE Note that a similar pattern will be
obtained using any other distribution that assigns a non-zero probability to positive values of T . Relationship to temporal discounting. There are, however, alternative explanations
available based on changes in outcome value. One candidate explanation is based on the tem-
poral discounting of outcome value according to which the value of future rewards is discounted
compared to more immediate rewards. Typically, the discount value due to delay is assumed to
be a function of the interaction of delay and outcome value. If, at the beginning of the session,
outcome values are large (e.g., because a rat is more hungry), then any discount produced by
selecting a slow response rate will be larger at that point than later in the session when the value
of the outcome is reduced (e.g., due to satiety) and so a delay will have less impact. It could be
argued, therefore, that it is less punitive to maintain a high response rate at the beginning of the
session to avoid delaying outcomes and then to decrease response rate as time passes within
the session. As such, temporal discounting predicts decreases in within-session response rates
consistent both with experimental observations and with the argument that outcome value de-
creases within the session (e.g., the satiety effect). Prediction. Although plausible, such explanations make very different predictions com-
pared to the model. The most direct prediction from the model is that introducing uncertainty
into the session duration without altering the average duration should nevertheless lead to a
sharper decline in response rate within the session; e.g., if for one group of subjects the session . CC-BY 4.0 International license
available under a
as not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
ioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . OPTIMAL RESPONSE VIGOR AND CHOICE The reason for this is that, by increasing the cost, the
response rate for each outcome should slow in order to compensate for the increase in the cost
and so maintain a reasonable gap between the reward and the cost of each outcome. The effect of ratio-requirement (k). Experimental studies mainly imply that the rate of
earned outcomes decreases with increases in the ratio-requirement (Aberman & Salamone, 1999;
Barofsky & Hurwitz, 1968), which is consistent with the general trend of the optimal rate of out-
come earning implied by the present model (see below). Experimental studies on the rate of responding on FR schedules indicate that the post-
reinforcer pause increases with increases in the ratio-requirement (Ferster & Skinner, 1957, Fig-
ure 23)(Felton & Lyon, 1966; Powell, 1968; Premack, Schaeffer, & Hundt, 1964). In terms of overall
response rates, some experiments report that response rates increase with increases in the ratio-
requirement up to a point beyond which response rates will start to decline, in rats (Barofsky &
Hurwitz, 1968; Kelsey & Allison, 1976; Mazur, 1982), pigeons (Baum, 1993) and mice (Greenwood,
Quartermain, Johnson, Cruce, & Hirsch, 1974), although other studies have reported inconsis-
tent results in pigeons (Powell, 1968), or a decreasing trend in response rate with increases in the
ratio-requirement (Felton & Lyon, 1966; Foster, Blackman, & Temple, 1997). The inconsistency
is partly due to the way that response rates are calculated in the different studies; for example . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. OPTIMAL RESPONSE VIGOR AND CHOICE ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 11 OPTIMAL RESPONSE VIGOR AND CHOICE
10
20
30
40
50
3
4
5
6
7
cost (a.u)
inter-response
interval
Figure 2. Effect of response cost on response rates. Left panel: Empirical data. Inter-response
intervals when the force required to make a response is manipulated. Figure is adopted from
Adair and Wright (1976). Right panel: Model prediction. Inter-response interval (equal to the
inverse of response rates) as a function of cost of responses (b). 10
20
30
40
50
3
4
5
6
7
cost (a.u)
inter-response
interval Figure 2. Effect of response cost on response rates. Left panel: Empirical data. Inter-response
intervals when the force required to make a response is manipulated. Figure is adopted from
Adair and Wright (1976). Right panel: Model prediction. Inter-response interval (equal to the
inverse of response rates) as a function of cost of responses (b). Figure 2. Effect of response cost on response rates. Left panel: Empirical data. Inter-response
intervals when the force required to make a response is manipulated. Figure is adopted from
Adair and Wright (1976). Right panel: Model prediction. Inter-response interval (equal to the
inverse of response rates) as a function of cost of responses (b). 2.5
5.0
7.5
0
25
50
75
100
ratio requirement (k)
response rate
0.5
1.0
1.5
2.0
25
50
75
100
deprivation level (a.u)
response rate
0
5
10
15
0.00
0.25
0.50
0.75
1.00
reward magnitude (a.u)
response rate
Figure 3. Left panel: The effect of ratio-requirement on the response rate. Middle panel: The
effect of the initial motivational drive on response rates. Right panel: The effect of the reward
magnitude on response rates. 2.5
5.0
7.5
0
25
50
75
100
ratio requirement (k)
response rate
0.5
1.0
1.5
2.0
25
50
75
100
deprivation level (a.u)
response rate 25
50
75
100
deprivation level (a.u) 0
5
10
15
0.00
0.25
0.50
0.75
1.00
reward magnitude (a.u)
response rate Figure 3. Left panel: The effect of ratio-requirement on the response rate. Middle panel: The
effect of the initial motivational drive on response rates. Right panel: The effect of the reward
magnitude on response rates. in some studies outcome handling and consumption time are not excluded when calculating re-
sponse rates (Barofsky & Hurwitz, 1968), in contrast to the other studies (Foster et al., 1997). OPTIMAL RESPONSE VIGOR AND CHOICE 12 pause (see (Pear, 2001, Page 289) for a review of other reinforcer schedules). The model predicts
that, with increases in deprivation, the rate of responding and of earned outcomes will increase
linearly (Figure 3: middle panel). The rate of increase is, however, negligible when the session
duration is long, in which case, even under high deprivation, sufficient time is available to earn
sufficient reward and become satiated. This provides a potential reason why the effect of depri-
vation on response rate has not previously been observed in experimental data. The effect of reward magnitude. Some studies show that post-reinforcement pauses in-
crease as the magnitude of the reward increases (Powell, 1969), whereas other studies suggest
that the post-reinforcement pause decreases (Lowe, Davey, & Harzem, 1974), although, in this
later study, the magnitude of the reward was manipulated within-session and a follow-up study
showed that, at a steady state, the post-reinforcement pause increases with increases in the mag-
nitude of the reward (Meunier & Starratt, 1979). Reward magnitude does not, however, have a
reliable effect on the overall response rate (Keesey & Kling, 1961; Lowe et al., 1974; Powell, 1969). Regarding predictions from the model, the effect of the reward magnitude on earned outcome
and response rates is, again, predicted go take an inverted U-shaped relationship (Figure 3: right
panel), and, therefore, depending on the value of the parameters, the predictions of the model
are consistent with the experimental data. The model makes a U-shaped prediction because,
when the reward magnitude is large then, given high response rates, the animals will become
satiated quickly and, therefore, the reward value of future outcomes will decrease substantially
if animal maintains this high response rate. As a consequence, under a high reward magnitude
condition, increase in reward will cause response rates to decrease. Under a low reward mag-
nitude condition, however, satiety has a negligible effect and a high response rate ensures that
sufficient reward can be earned before the session ends. OPTIMAL RESPONSE VIGOR AND CHOICE ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE As
a consequence, the experimental data regarding the relationship between response rate and the
ratio-requirement is inconclusive. in some studies outcome handling and consumption time are not excluded when calculating re-
sponse rates (Barofsky & Hurwitz, 1968), in contrast to the other studies (Foster et al., 1997). As
a consequence, the experimental data regarding the relationship between response rate and the
ratio-requirement is inconclusive. With regard to this issue, the present model predicts that the relationship between re-
sponse rate and the ratio-requirement is an inverted U-shaped pattern (Figure 3: left panel),
which is consistent with the studies mentioned previously depending on the value of other ex-
perimental parameters. The model makes an inverted U-shaped prediction because, under a low
ratio-requirement, the cost is generally low and, therefore, as the ratio-requirement increases,
the decision-maker will make more responses to compensate for the drop in the amount of re-
ward. In contrast, when the ratio-requirement is high, the cost of earning outcomes is high and
the margin between the cost and the reward of each outcome becomes significantly tighter as the
ratio-requirement increases. The margin can, however, be loosened by decreasing the response
rate. The Effect of deprivation level. Experimental studies that have used FR schedules sug-
gest that response rates increase with increases in deprivation (Ferster & Skinner, 1957, Chap-
ter 4)(Sidman & Stebbins, 1954). However, such increases are mainly due to decreases in the
post-reinforcement pause, and not due to the increases in the actual rate of responding after the . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. OPTIMAL RESPONSE VIGOR AND CHOICE consuming one of the outcomes reduces the amount of future reward produced by the other out-
come. Here, if the effect of the outcomes on each other is symmetrical, i.e., consuming outcome
O1, reduces the reward elicited by outcome O2 by the same amount that consuming outcome O2
reduces the reward elicited by outcome O1, then it will not matter which outcome is consumed
first and the total reward during the session will be independent of the chosen order. As such,
the reward field will be conservative. consuming one of the outcomes reduces the amount of future reward produced by the other out-
come. Here, if the effect of the outcomes on each other is symmetrical, i.e., consuming outcome
O1, reduces the reward elicited by outcome O2 by the same amount that consuming outcome O2
reduces the reward elicited by outcome O1, then it will not matter which outcome is consumed
first and the total reward during the session will be independent of the chosen order. As such,
the reward field will be conservative. More precisely, a reward field is conservative if there exists a scalar field Dx such that: Ax,t = −∂Dx
∂x . (8) (8) It can be shown that if a reward field satisfies equation 8 then the amount of reward experienced
in a session depends on the total number of earned outcomes. Under this condition the optimal
response rate will be constant for each outcome relative to the other. Intuitively, this is because,
in terms of the total rewards per session, the only thing that matters is the final number of earned
outcomes and, therefore, there is no reason why the relative allocation of responses to outcomes
should vary within the session. The actual response rate for each outcome will, however, depend
on whether the costs of the outcomes are independent, a point elaborated in the next section. Conservative reward field and independent response cost. The costs of various out-
comes are independent if the decision-maker can increase their work for one outcome without
affecting the cost of other outcomes. As an example, imagine a decision-maker that is using their
left hand to make responses that earn one outcome and their right-hand to make responses that
earn the second outcome. Optimal choice and response vigor ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint 13 13 Optimal choice and response vigor In this section we address the choice problem, i.e., the case where there are multiple out-
comes available in the environment and the decision-maker needs to make a decision about the
response rate along each outcome dimension. An example of this situation is a concurrent in-
strumental conditioning experiment in which two levers are available and pressing each lever
produces an outcome on a ratio schedule. Unlike the case with single outcome environments,
the optimal rate of earning outcomes is not necessarily constant and can take different forms
depending on whether the reward field is a conservative field or a non-conservative field, and
whether the costs of responses along the outcome dimensions are independent of each other. Below, we derive the optimal choice strategy in each condition. Conservative reward field. A reward field is conservative if the amount of reward expe-
rienced by consuming different outcomes does not depend on the order of consumption and
depends only on the number of each outcome earned by the end of the session. This property
holds in two conditions (i) when the value of each outcome is unrelated to the prior consumption
of other outcomes; and (ii) the consumption of an outcome affects the value of other outcomes
and this effect is symmetrical. As an example of condition (i), imagine an environment with two
outcomes in which one of the outcomes only satisfies thirst and the other only satisfies hunger. Here, consumption of one of the outcomes will not affect the amount of reward that will be ex-
perienced by consuming the other outcome and, therefore, the total reward during the session
does not depend on the order of choosing the outcomes. As an example of condition (ii), imag-
ine an environment with two outcomes in which both outcomes satisfy hunger and, therefore, . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. OPTIMAL RESPONSE VIGOR AND CHOICE 14 See Appendix for the proof and for the specification of optimal responses. As an example, imag-
ine a concurrent fixed-ratio (FR) schedule in which an animal needs to make k responses on the
left lever in order to earn O1, and lk responses on the right lever in order to earn O2, and both
outcomes have the same reward properties. According to Theorem 3, the optimal response rate
for O1 (the outcome with the lower ratio-requirement) is l times greater than the response rate
for the second outcome O2. Figure 4:left panel independent cost condition shows the simulation
of the model and the optimal trajectory in the outcome space. As the figure shows, the rate of
earning O1 is higher than O2, however, the relative portion of earned outcomes remains the same
throughout the session. The above results are generally in line with the probability matching notion, which states
that a decision-maker allocates responses to outcomes based on the ratio of responses required
for each outcome (Estes, 1950). Probability matching is often argued to violate rational choice
theory because, within this theory, it is expected that a decision-maker works exclusively for the
outcome with the higher probability (i.e., the lower ratio-requirement). However, based on the
model proposed here probability matching is the optimal strategy and therefore consistent with
rational decision-making. Conservative reward field and dependent response cost. In this section we assume that
the cost of responses for an outcome is affected by the rate of responses for earning other out-
comes. In other words, what determines the cost is the delay between subsequent responses
either for the same or for a different outcome; i.e., the cost is proportional to the rate of earning
all of the outcomes. In instrumental conditioning this assumption entails, for example, that the
cost of pressing, say, the right lever is determined by the time that has passed since the last press
on either the right or a left lever. In this condition the optimal strategy is maximisation; i.e., to
take the action with the higher reward (or lower ratio-requirement) and to stop taking the other
action (see Theorem A2 in Appendix). OPTIMAL RESPONSE VIGOR AND CHOICE It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE In this case, the independence assumption entails that the cost of
responding with one or other hand is determined by the response rate on that hand; e.g., the
decision-maker can increase or decrease rate of responding on the left hand without affecting
the cost of responses on the right hand. More precisely, the independence assumption entails
that the Hessian matrix of Kv is diagonal: ∂2Kv
∂vi∂v j
= 0,i ̸= j. (9) (9) In this situation even if some of the outcomes have a lower reward or a higher cost (inferior out-
comes) compared to other outcomes (superior outcomes), it is still optimal to allocate a portion
of responses to the inferior outcomes. This is because responding for inferior outcomes has no
effect on the net reward earned from superior outcomes and, therefore, as long as the response
rate for inferior outcomes is sufficiently low that the reward earned from them is more than the
cost paid, responding for them is justified. The portion of responses allocated to each outcome
depends, however, on the cost and reward of each outcome. We maintain the following theorem: In this situation even if some of the outcomes have a lower reward or a higher cost (inferior out-
comes) compared to other outcomes (superior outcomes), it is still optimal to allocate a portion
of responses to the inferior outcomes. This is because responding for inferior outcomes has no
effect on the net reward earned from superior outcomes and, therefore, as long as the response
rate for inferior outcomes is sufficiently low that the reward earned from them is more than the
cost paid, responding for them is justified. The portion of responses allocated to each outcome
depends, however, on the cost and reward of each outcome. We maintain the following theorem: Theorem 3 If (i) the reward field is conservative, i.e., there exists a scalar field Dx such that equa-
tion 8 is satisfied, (ii) the time-dependent term of the reward field is zero (∂Ax,t/∂t = 0), and (iii)
the cost function satisfies equation 9, then the optimal rate of earning outcome v∗will be constant
(dv/dt = 0) and satisfies the following equation: ∂Kv∗
∂v∗= AT v∗,T . (10) ∂Kv∗
∂v∗= AT v∗,T . (10) . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. OPTIMAL RESPONSE VIGOR AND CHOICE 15 he structure of such studies cannot be readily related to the model proposed here. the structure of such studies cannot be readily related to the model proposed here. Prediction. One way of testing the above explanation for maximization and matching
strategies is to compare the pattern of responses when two different effectors are used to make
responses for the outcomes vs. when a single effector is being used to earn both outcomes. In
the first condition the costs of the two outcomes are independent of each other whereas in the
second condition they are dependent on each other. As a consequence, the theory predicts that,
in the first condition, response rates will reflect probability matching whereas in the second con-
dition they will reflect the maximization strategy. 0
25
50
75
0
25
50
75
O2(a.u)
O1(a.u)
independent cost
dependent cost
0
30
60
90
0
30
60
90
O2(a.u)
O1(a.u)
short
medium
long
Fig. 2. Left panel: Optimal trajectory in a conservative reward field. Earning O1
requires k responses and earning O2 requires lk responses. Initially the amount of
earned outcome is zero (starting point is at point [0, 0]), and the graph shows the
trajectories that the decision-maker takes in two different conditions corresponding
to when the costs of outcomes are independent, and when the costs are dependent
on each other. Right panel: The optimal trajectories in the outcome space when
the reward field is non-conservative. The graph shows the optimal trajectory in the
conditions that the session duration is short (T = 7), medium (T = 15.75) and long
(T = 23). properties, e.g., consumption of one unit of either O1 or O2
d
h
g
b
it h
th
g
t
diff
and here we extended
solution to the proble
are two significant di
here and previous m
although the effect o
values on response v
[22], the effects of on
a session were not ad
of changes in outcom
has been addressed in
role in determining re
We address such limit
Secondly, previous
the task as a semi-M
optimal actions that m
time (average reward
to calculate the optim
earned within the ses
in the previous works
range of instrumental
Figure 4. Left panel: Optimal trajectory in a conservative reward field. Earning O1 requires k
responses and earning O2 requires lk responses. OPTIMAL RESPONSE VIGOR AND CHOICE Earning O1
requires k responses and earning O2 requires lk responses. Initially the amount of
earned outcome is zero (starting point is at point [0, 0]), and the graph shows the
trajectories that the decision-maker takes in two different conditions corresponding
to when the costs of outcomes are independent, and when the costs are dependent
on each other. Right panel: The optimal trajectories in the outcome space when
the reward field is non-conservative. The graph shows the optimal trajectory in the
conditions that the session duration is short (T = 7), medium (T = 15.75) and long
(T = 23). properties, e.g., consumption of one unit of either O1 or O2
has been addressed in
role in determining r
We address such limi
Secondly, previou
the task as a semi-M
optimal actions that
time (average reward
to calculate the opti
earned within the ses
in the previous work
f i
Figure 4. Left panel: Optimal trajectory in a conservative reward field. Earning O1 requires k
responses and earning O2 requires lk responses. Initially the amount of earned outcome is zero
(starting point is at point [0,0]), and the graph shows the trajectories that the decision-maker
takes in two different conditions corresponding to when the costs of outcomes are independent,
and when the costs are dependent on each other. Right panel: The optimal trajectories in the
outcome space when the reward field is non-conservative. The graph shows the optimal trajec-
tory in the conditions that the session duration is short (T = 7), medium (T = 15.75) and long
(T = 23). g
y
,
,
y g
ent amount of rewards, e.g., one unit of O1 generates more
reward than one unit of O2. Within such an environment,
if hunger is high then consuming O1 generates significantly
more reward than O2 and, therefore, early in the session it is
better to allocate more responses to O1, whereas later in the
session (when hunger is presumably lower and the difference
in the value of the outcomes is small) responses for O2 can
gradually increase. If we reverse this order, i.e., first O2 is
consumed and then O1, then early consumption of O2 will
cause satiety and the decision-maker will lose the chance to
experience high reward from O1 when hungry. OPTIMAL RESPONSE VIGOR AND CHOICE As such, the
amount of experienced reward depends on the order of con-
suming the outcomes and, based on the above explanation, a
larger amount of reward can be earned during the session if
more responses are allocated to the outcome with the higher
reward at the beginning of the session (see Theorem S3 in SI
text). Figure 2:right panel shows the simulations of the model
under different session durations. In each simulation, at the
beginning of the session the initially earned outcomes are zero
and each line in the figure shows the trajectory of the amount
earned from each outcome during the session. As the figure
shows, in all conditions the rate of earning O1 is higher than
O2 and this difference is more apparent under long session
durations, in which a large amount of reward can be gained
during the session and it makes a significant difference to earn
schedules, whereas th
to the case of interval
average reward (as ad
the maximization of
i.e., the session durat
used here explicitly
which, as we showed,
of responses. ACKNOWLEDGMENT
Peter Dayan for helpful
Principal Research Fell
Research Council of Au
1. von Neumann J, Morgenster
university press Princeton) V
2. Marshall A (1890) Principles
3. Niv Y, Daw ND, Joel D, Daya
response vigor. Psychophar
4. Dayan P (2012) Instrument
neuroscience 35(7):1152–68
5. Salimpour Y, Shadmehr R (
Journal of neuroscience 34(
6. Killeen PR (1995) Economic
conditions of varying motivat
431. 7. Keramati M, Gutkin BS (201
lection and physiological sta
8. Hull CL (1943) Principles of
9. McSweeney FK, Hinson JM
operant conditioning. Psych
10
Lib
D (2011) C l
l
Non-conservative reward field. A reward filed is non-conservative if the total amount
of reward experienced during the session depends on the order of the consumption of the out-
comes. Imagine an environment with two outcomes say O1 and O2, where both outcomes have
the same motivational properties, e.g., consumption of one unit of either O1 or O2 decreases
hunger by one unit, however, they generate different amount of rewards, e.g., one unit of O1
generates more reward than one unit of O2. OPTIMAL RESPONSE VIGOR AND CHOICE The reason is, unlike the case with independent costs,
allocating more responses to earn an inferior outcome will increase the cost of earning superior
outcomes and, therefore, it is better to pay the cost for the superior outcome only, which requires
fewer responses per unit of outcome. For example, under a concurrent FR schedule in which an animal needs to make k re-
sponses on the left lever to earn O1, and lk responses on the right lever to earn O2 (O1 and O2
have the same reward properties), the optimal response rate will be a constant response rate
on the left lever and a zero response rate on the right lever. Figure 4:left panel dependent cost
condition shows a simulation of the model and the optimal trajectory in outcome space, which
shows that the subject only earns O1. As such, whether the outcome rate reflects a probability matching or a maximization strat-
egy depends on the cost function and, in instrumental conditioning experiments, the cost that
reflects the maximization strategy is more readily applicable. Regarding the experimental data,
evidence from concurrent instrumental conditioning experiments in pigeons tested using VR
schedules (Herrnstein & Loveland, 1975) is in-line with the maximization strategy and consis-
tent with predictions from the model. Within the wider scope of decision-making tasks, some
studies are consistent with probability matching, whereas other studies provide evidence in-line
with the maximization strategy (see (Vulkan, 2000) for a review). In many of these latter studies,
however, the decision-making task involved making a single choice (e.g., single button press)
with immediate feedback (about whether the choice was rewarded) after which the next trial
was initiated and, because such disjointed actions are unlikely to convey a rate-dependent cost, . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE Initially the amount of earned outcome is zero
(starting point is at point [0,0]), and the graph shows the trajectories that the decision-maker
takes in two different conditions corresponding to when the costs of outcomes are independent,
and when the costs are dependent on each other. Right panel: The optimal trajectories in the
outcome space when the reward field is non-conservative. The graph shows the optimal trajec-
tory in the conditions that the session duration is short (T = 7), medium (T = 15.75) and long
(T = 23). 0
25
50
75
0
25
50
75
O2(a.u)
O1(a.u)
independent cost
dependent cost 0
30
60
90
0
30
60
90
O2(a.u)
O1(a.u)
short
medium
long Fig. 2. Left panel: Optimal trajectory in a conservative reward field. Earning O1
requires k responses and earning O2 requires lk responses. Initially the amount of
earned outcome is zero (starting point is at point [0, 0]), and the graph shows the
trajectories that the decision-maker takes in two different conditions corresponding
to when the costs of outcomes are independent, and when the costs are dependent
on each other. Right panel: The optimal trajectories in the outcome space when
the reward field is non-conservative. The graph shows the optimal trajectory in the
conditions that the session duration is short (T = 7), medium (T = 15.75) and long
(T = 23). properties, e.g., consumption of one unit of either O1 or O2
has been addressed in
role in determining r
We address such limi
Secondly, previou
the task as a semi-M
optimal actions that m
time (average reward
to calculate the opti
earned within the ses
in the previous work
Figure 4. Left panel: Optimal trajectory in a conservative reward field. Earning O1 requires k
responses and earning O2 requires lk responses. Initially the amount of earned outcome is zero
(starting point is at point [0,0]), and the graph shows the trajectories that the decision-maker
takes in two different conditions corresponding to when the costs of outcomes are independent,
and when the costs are dependent on each other. Right panel: The optimal trajectories in the
outcome space when the reward field is non-conservative. The graph shows the optimal trajec-
tory in the conditions that the session duration is short (T = 7), medium (T = 15.75) and long
(T = 23). Fig. 2. Left panel: Optimal trajectory in a conservative reward field. Discussion Computational models of action selection are essential for understanding decision-
making processes in humans and animals, and here we extended them by providing a general
analytical solution to the problem of response vigor and choice. There are two significant differ-
ences between the model proposed here and previous models of response vigor (Dayan, 2012;
Niv et al., 2007). Firstly, although the effect of between-session changes in outcome values on re-
sponse vigor was addressed in previous models (Niv, Joel, & Dayan, 2006), the effects of on-line
changes in outcome values within a session were not addressed. On the other hand, the effect
of changes in outcome value on the choice between actions has been addressed in some previ-
ous models (Keramati & Gutkin, 2014), however their role in determining response vigor has not
been investigated. We address such limitations directly in this model. Secondly, previous work conceptualized the structure of the task as a semi-Markov deci-
sion process and derived the optimal actions that maximize the average reward per unit of time
(average reward). Here, we used a variational analysis to calculate the optimal actions that max-
imize the reward earned within the session. One benefit of the approach taken in the previous
works is that it extends naturally to a wide range of instrumental conditioning schedules such
as interval schedules, whereas the extension of the model proposed here to the case of interval
schedules is not trivial. Optimizing the average reward (as adopted in previous work) is equiv-
alent to the maximization of reward in an infinite-horizon time scale; i.e., the session duration
is unlimited. In contrast, the model used here explicitly represents the duration of the session
which, as we showed, plays an important role in the pattern of responses. Relationship to principle of least action. A basic assumption that we made here is that
the decision-maker takes actions that yield the highest amount of reward. This reward max-
imization assumption has a parallel in physics literature known as the principle of least action,
which implies that among all trajectories that a system can take, the true trajectories are the ones
that minimize the action. Here action has a different meaning from that used in psychology lit-
erature, and it refers to the integral of the Lagrangian (L) along the trajectory. OPTIMAL RESPONSE VIGOR AND CHOICE it makes a significant difference to earn O1 first. Prediction. A test of the above prediction would involve an experiment in which the sub-
ject is responding for two food outcomes containing an equal number of calories (and therefore
having the same impact on motivation) but with different levels of the desirability (e.g., having
different flavors) and, therefore, having a different reward effect. Theorem A3 predicts that, if the
session duration is long enough, early in the session the response rate for the outcome with the
greater desirability will be higher whereas, later in the session, responses for the other outcome
will increase. OPTIMAL RESPONSE VIGOR AND CHOICE Within such an environment, if hunger is high then
consuming O1 generates significantly more reward than O2 and, therefore, early in the session
it is better to allocate more responses to O1, whereas later in the session (when hunger is pre-
sumably lower and the difference in the value of the outcomes is small) responses for O2 can
gradually increase. If we reverse this order, i.e., first O2 is consumed and then O1, then early con-
sumption of O2 will cause satiety and the decision-maker will lose the chance to experience high
reward from O1 when hungry. As such, the amount of experienced reward depends on the order
of consuming the outcomes and, based on the above explanation, a larger amount of reward can
be earned during the session if more responses are allocated to the outcome with the higher re-
ward at the beginning of the session (see Theorem A3 in Appendix). Figure 4:right panel shows
the simulations of the model under different session durations. In each simulation, at the begin-
ning of the session the initially earned outcomes are zero and each line in the figure shows the
trajectory of the amount earned from each outcome during the session. As the figure shows, in
all conditions the rate of earning O1 is higher than O2 and this difference is more apparent under
long session durations, in which a large amount of reward can be gained during the session and . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint 16 Discussion In the case of a
charged particle with charge q and mass m in a magnetic field B, the Lagrangian will be: L = 1
2mv2 + qv.A,
(11) (11) where A is the vector potential (B = ∇× A). By comparing equation 11 with equations 4 and
5, we can see that the reward field Ax,t corresponds to the vector potential, and the term Kv
corresponds to 1
2mv2 by assuming m = 2ak2, and b = 0. This parallel can provide some in-
sights into the properties of the response rates. For example, it can be shown that when the where A is the vector potential (B = ∇× A). By comparing equation 11 with equations 4 and
5, we can see that the reward field Ax,t corresponds to the vector potential, and the term Kv
corresponds to 1
2mv2 by assuming m = 2ak2, and b = 0. This parallel can provide some in-
sights into the properties of the response rates. For example, it can be shown that when the . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint 17 OPTIMAL RESPONSE VIGOR AND CHOICE 17 Lagrangian is not explicitly dependent on time (time-translational invariance), which here im-
plies that ∂Ax,t/∂t = 0, then the Hamiltonian (H , or energy) of the system is conserved. The
Hamiltonian in the case of the system defined in equation 4 (with single outcome) is: H = Kv −∂Kv
∂v v = −ak2v2
(using equation 3). = −ak2v2
(using equation 3). = −ak2v2
(using equation 3). Conservation of the Hamiltonian implies that ak2v2 (and therefore v) is constant (response-rate
is constant), as stated by Theorem 1. Further exploration of this parallel can be an interesting
future direction. References CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 18 Killeen, P. R. (1995, nov). Economics, ecologics, and mechanics: The dynamics of responding under
conditions of varying motivation. Journal of the Experimental Analysis of Behavior, 64(3), 405–431. Killeen P R & Sitomer M T (2003 apr) MPR Behavioural Processes 62(1 3) 49 64 Killeen, P. R. (1995, nov). Economics, ecologics, and mechanics: The dynamics of responding under
conditions of varying motivation. Journal of the Experimental Analysis of Behavior, 64(3), 405–431. Killeen, P. R., & Sitomer, M. T. (2003, apr). MPR. Behavioural Processes, 62(1-3), 49–64. y
g
f
p
y
f
Killeen, P. R., & Sitomer, M. T. (2003, apr). MPR. Behavioural Processes, 62(1-3), 49–64. Killeen, P. R., & Sitomer, M. T. (2003, apr). MPR. Beh Liberzon, D. (2011). Calculus of Variations and Optimal Control Theory: A Concise Introduction. Princeton
University Press. Lowe, C. F., Davey, G. C. L., & Harzem, P. (1974). Effects of reinforcement magnitude on interval and rati
schedules. Journal of the Experimental analysis of behavior, 22(3), 553–560. Marshall, A. (1890). Principles of Economics. London: Macmillan and Co., Ltd. Mazur, J. E. (1982). A molecular approach to ratio schedule performance. In M. L. Commons, R. J. Her-
rnstein, & H. Rachlin (Eds.), Quantitative analyses of behavior. vol. 2: Matching and maximizing
accounts. Ballinger. McGuire, J. T., & Kable, J. W. (2013, apr). Rational temporal predictions can underlie apparent failures to
delay gratification. Psychological review, 120(2), 395–410. McSweeney, F. K. (2004). Dynamic changes in reinforcer effectiveness: Satiation and habituation have
different implications for theory and practice. The Behavior Analyst, 27(2), 171–188. McSweeney, F. K., Hinson, J. M., & Cannon, C. B. (1996). Sensitization–habituation may occur during
operant conditioning. Psychological Bulletin, 120(2), 256. McSweeney, F. K., Roll, J. M., & Weatherly, J. N. (1994, jul). Within-session changes in responding durin
several simple schedules. Journal of the Experimental Analysis of Behavior, 62(1), 109–132. Meunier, G. F., & Starratt, C. (1979). On the magnitude of reinforcement and fixed-ratio behavior. Bulleti
of the Psychonomic Society, 13(6), 355–356. Niv, Y., Daw, N. D., Joel, D., & Dayan, P. (2007, apr). Tonic dopamine: opportunity costs and the control of
response vigor. Psychopharmacology, 191(3), 507–20. Niv, Y., Joel, D., & Dayan, P. (2006, aug). A normative perspective on motivation. Trends in cognitive
sciences, 10(8), 375–81. Pear, J. (2001). The science of learning. Psychology Press. Pear, J. (2001). The science of learning. Psychology Press. Powell, R. W. (1968). References Aberman, J. E., & Salamone, J. D. (1999). Nucleus accumbens dopamine depletions make rats more
sensitive to high ratio requirements but do not impair primary food reinforcement. Neuroscience,
92(2), 545–52. Adair, E. R., & Wright, B. A. (1976). Behavioral thermoregulation in the squirrel monkey when response
effort is varied. Journal of Comparative and Physiological Psychology, 90(2), 179. Alling, K., & Poling, A. (1995, may). The effects of differing response-force requirements on fixed-ratio
responding of rats. Journal of the experimental analysis of behavior, 63(3), 331–46. Barofsky, I., & Hurwitz, D. (1968). Within ratio responding during fixed ratio performance. Psychonomic
Science, 11(7), 263–264. Baum, W. M. (1993). Performances on ratio and interval schedules of reinforcement: Data and theory. Journal of the Experimental Analysis of Behavior, 59(2), 245. Dayan, P. (2012, apr). Instrumental vigour in punishment and reward. The European journal of neuro
science, 35(7), 1152–68. Estes, W. K. (1950). Toward a statistical theory of learning. Psychological review, 57(2), 94. Felton, M., & Lyon, D. O. (1966). The post-reinforcement pause. Journal of the Experimental Analysis of
Behavior, 9(2), 131–134. Ferster, C. B., & Skinner, B. F. (1957). Schedules of reinforcement. Prentice-hall inc. Foster, M., Blackman, K., & Temple, W. (1997). Open versus closed economies: performance of domestic
hens under fixed ratio schedules. Journal of the Experimental Analysis of Behavior, 67(1), 67. Gallistel, C. R., & Gibbon, J. (2000). Time, rate, and conditioning. Psychological review, 107(2), 289. Gibbon, J. (1977). Scalar expectancy theory and Weber’s law in animal timing. Psychological review, 84(3
279. Greenwood, M. R., Quartermain, D., Johnson, P. R., Cruce, J. A., & Hirsch, J. (1974, nov). Food motivated
behavior in genetically obese and hypothalamic-hyperphagic rats and mice. Physiology & behavior,
13(5), 687–92. Herrnstein, R. J., & Loveland, D. H. (1975). Maximizing and matching on concurrent ratio schedule
Journal of the experimental analysis of behavior, 24(1), 107. Hull, C. L. (1943). Principles of Behavior. New York: Appleton-Century. Keesey, R. E., & Kling, J. W. (1961). Amount of reinforcement and free-operant responding. Journal of the
Experimental analysis of behavior, 4(2), 125–132. Kelsey, J. E., & Allison, J. (1976). Fixed-ratio lever pressing by VMH rats: Work vs accessibility of sucrose
reward. Physiology & behavior, 17(5), 749–754. y
gy
Keramati, M., & Gutkin, B. S. (2014). Homeostatic reinforcement learning for integrating reward collectio
and physiological stability. eLife, 3. Killeen, P. R. (1994, feb). Mathematical principles of reinforcement. Behavioral and Brain Sciences, 17,
105–172. . .
CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017.
;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint Value in non-deterministic schedules The value of a trajectory in the outcome space is the sum of the net amount of rewards
that can be earned during a session. However, the amount of reward earned during a session can
be non-deterministic, as for example in the case of VR and RR schedules of reinforcement, ac-
tions lead to outcomes probabilistically. Similarly, the cost of earning outcomes will also be non-
deterministic, since the number of responses required to earn outcomes is non-deterministic. Let’s denote the cost of earning outcomes under such non-deterministic schedules by K ′
v. Using
this, we define the value function as the sum of the expected net amount of rewards that will be
earned during a session: S0,T =
ZT
0
E[v.Ax,t −K ′
v]dt =
ZT
0
Lx,v,tdt,
(A12) (A12) where the expectation is w.r.t the number of earned outcomes along each outcome dimension
during dt time step. Following the above definition, we have: Lx,v,t = E[v.Ax,t −K ′
v],
(A13) (A13) where Lx,v,t is the expected net reward earned in dt time step. In the main text and in the follow-
ing sections, E[v] is denoted by v for simplicity of notation. By replacing v by E[v] in equation 4
we get: (A14) Lx,v,t = E[v].Ax,t −KE[v]. (A14) In the main text, equation A14 (equation 4 in the main text) was used instead of equation A13,
and the aim of this section is to show that equation A14 and equation A13 are equivalent. We first
consider environments with one-dimensional outcome spaces, and then we extend it to the case
of environments with multi-dimensional outcome spaces. We maintain the following theorem: Theorem A1 Assume that the cost of one response, given that the delay since the last response is τ,
is as follows: Cτ = a/τ+b. (A15) (A15) Furthermore, assume that on average, or exactly, k responses are required to earn one unit of the
outcome, and r is the number of outcomes earned. Then we have: Lx,v,t = Er [v]Ax,t −KEr [v],
(A16)
Kv = vk(kav +b). (A17) Lx,v,t = Er [v]Ax,t −KEr [v],
(A16) (A16) where Kv = vk(kav +b). (A17) (A17) Proof. We first provide an intuitive explanation for why the cost defined in equation A15
is the same as the cost defined in equation A17 in the case of FR schedules of reinforcement (i.e.,
exactly k responses are required to earn an outcome). OPTIMAL RESPONSE VIGOR AND CHOICE 19 OPTIMAL RESPONSE VIGOR AND CHOICE The effect of small sequential changes in fixed-ratio size upon the post-reinforcemen
pause. Journal of the Experimental Analysis of Behavior, 11(5), 589–593. Powell, R. W. (1969). The effect of reinforcement magnitude upon responding under fixed-ratio schedules. Journal of the Experimental Analysis of Behavior, 12(4), 605–608. Premack, D., Schaeffer, R. W., & Hundt, A. (1964). Reinforcement of drinking by running: effect of fixed
ratio and reinforcement time. Journal of the experimental analysis of behavior, 7(1), 91–96. Rachlin, H. (2000). The Science of Self-Control. Harvard University Press. alimpour, Y., & Shadmehr, R. (2014, jan). Motor costs and the coordination of the two arms. The Journa
of neuroscience, 34(5), 1806–18. f
Sidman, M., & Stebbins, W. C. (1954). Satiation effects under fixed-ratio schedules of reinforcement. Journal of Comparative and Physiological Psychology, 47(2), 114. f
p
y
g
y
gy
von Neumann, J., & Morgenstern, O. (1947). Theory of games and economic behavior. Princeton university
press Princeton. Vulkan, N. (2000). An economist’s perspective on probability matching. Journal of economic surveys,
14(1), 101–118. . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint .
CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017.
;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint Value in non-deterministic schedules Earning the outcome at rate v implies that
the time it takes to earn the outcome is 1/v, and since k responses have been executed in this
period, the delay between responses is:
1 τ = 1
kv ,
(A18) τ = 1
kv , (A18) . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 20 and therefore using equation A15 (equation 2 in the main text), the cost of making one response
will be akv+b. Since k responses are required for earning each outcome, the total cost of earning
one unit of the outcome will be k times the cost of one response, which will be k(akv +b). Since
the total amount of outcome earned is vdt, the total cost per unit of time will be: Kv =k(akv +b)vdt
dt
=vk(akv +b),
(A19) (A19) which is equivalent to equation 3 and A17. which is equivalent to equation 3 and A17. We now show that equation A16 and equation A13 are equivalent in order to prove Theo-
rem A1. Equation A13 has two terms. As for the first term, Ax,t can be assumed to be constant in
dt time step, and therefore we have: Er [v Ax,t] = Er [v]Ax,t. (A20) (A20) As for the second term we maintain that: As for the second term we maintain that: As for the second term we maintain that: Er [K ′
v] = KEr [v]. (A21) (A21) To show the above relation, assume that r is the number of outcomes earned after making one
response. Since according to the definition of RR and VR schedules, out of N responses on av-
erage N/k will be rewarded, we have Er [r] = 1/k and the expected rate of outcome earning will
be: Er [v] = Er
hr
τ
i
= 1
kτ. (A22) (A22) Furthermore, according to equation A15 the cost of one response is a/τ + b, and therefore, the
cost per unit of time will be:
b Furthermore, according to equation A15 the cost of one response is a/τ + b, and therefore, the
cost per unit of time will be: K ′
v = a/τ+b
τ
. (A23) (A23) Therefore: OPTIMAL RESPONSE VIGOR AND CHOICE 21 which states that E[K ′
v] can be represented as a function of E[v]. To show this, assume ri is the
number of outcomes earned after making one response for outcome i, and τi is the delay be-
tween responses for outcome i. We have: which states that E[K ′
v] can be represented as a function of E[v]. To show this, assume ri is the
number of outcomes earned after making one response for outcome i, and τi is the delay be-
tween responses for outcome i. We have: E[vi] = E
·ri
τi
¸
= E[ri]
τi
,
(A26) (A26) and therefore τi can be expressed as a function of E[vi]. Next, assume that [Cτ]i is the cost of
making one response for outcome i with delay τi between the responses, and τ is a vector con-
taining the delay between responses for each outcome (τ = [τ1 ...τn]). In dt time step, dt/τi re-
sponses for outcome i are made, and therefore the total cost in dt time period will be [Cτ]idt/τi,
which implies that the cost for outcome i per unit of time is [Cτ]i/τi. Given this, the total cost
paid for all the outcomes per unit of time will be: E[K ′
v] =
X
i
[Cτ]i
τi
=
X
i
[Cτ]i
E[vi]
E[ri] (using equation A26)
= KE[v], E[K ′
v] =
X
i
[Cτ]i
τi
=
X
i
[Cτ]i
E[vi]
E[ri] (using equation A26)
= KE[v], = KE[v], where we used the fact that τ in Cτ can be expressed using E[v] (using equation A26), and there-
fore E[K ′
v] can be expressed as a function of E[v], which is denoted by KE[v] (as noted in equa-
tion A25). Substituting equation A25,A24 in equation A13 yields equation A14. where we used the fact that τ in Cτ can be expressed using E[v] (using equation A26), and there-
fore E[K ′
v] can be expressed as a function of E[v], which is denoted by KE[v] (as noted in equa-
tion A25). Substituting equation A25,A24 in equation A13 yields equation A14. Therefore: Er [K ′
v] =a/τ+b
τ
=Er [v]k(akEr [v]+b) (using equation A22)
=KEr [v]
(using equation A17), which proves equation A21. Substituting equations A21, A20 in equation A13 yields equa-
tion A16, which proves the theorem. which proves equation A21. Substituting equations A21, A20 in equation A13 yields equa-
tion A16, which proves the theorem. We now turn to the case of multi-dimensional outcome spaces. The aim is to show equa-
tion A13 is equivalent to equation A14. To show this, we first maintain that: E[v.Ax,t] = E[v].Ax,t,
(A24) (A24) which holds since Ax,t can be assumed to be constant during dt time step. Next, we show that: E[K ′
v] = KE[v],
(A25) (A25) E[K ′
v] = KE[v], . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 22 which implies:
∂Kv
∂v
¯¯¯¯
t=T
= Ax,t
¯¯
t=T ,
(A31) which implies: which implies: which implies:
∂Kv
∂v
¯¯¯¯
t=T
= Ax,t
¯¯
t=T ,
(A31) ∂Kv
∂v
¯¯¯¯
t=T
= Ax,t
¯¯
t=T ,
(A31) (A31) where as mentioned T is the total session duration. re as mentioned T is the total session duration. where as mentioned T is the total session duration. ubstituting equation A27 in equation A29 we will have: By substituting equation A27 in equation A29 we will have: By substituting equation A27 in equation A29 we will have: d
dt
µ
−∂Kv
∂v + Ax,t
¶
= v d Ax,t
dx . (A32) (A32) The term d Ax,t/dt has two components: the first component is the change in Ax,t due to the
change in x and the second component is due to the time-dependent changes in Ax,t: The term d Ax,t/dt has two components: the first component is the change in Ax,t due to the
change in x and the second component is due to the time-dependent changes in Ax,t: d Ax,t
dt
=dx
dt
∂Ax,t
∂x
+ ∂Ax,t
∂t
=v ∂Ax,t
∂x
+ ∂Ax,t
∂t
. (A33) (A33) Furthermore we have: Furthermore we have: Furthermore we have:
d
dt
µ∂Kv
∂v
¶
= dv
dt
∂2Kv
∂v2 . (A34) d
dt
µ∂Kv
∂v
¶
= dv
dt
∂2Kv
∂v2 . (A34) (A34) Substituting equations A33, A34 in equation A32 yields: Substituting equations A33, A34 in equation A32 yields: Substituting equations A33, A34 in equation A32 yields: dv
dt
µ∂2Kv
∂v2
¶
= ∂Ax,t
∂t
. (A35) (A35) In the condition that the rate of outcome earning is constant (dv/dt = 0), we have xT = vT ,
which by substituting in equation A31 yields: In the condition that the rate of outcome earning is constant (dv/dt = 0), we have xT = vT ,
which by substituting in equation A31 yields: ∂Kv∗
∂v∗= AT v∗,T . (A36) (A36) The above equation will be used in order to calculate the optimal rate of outcome earning. The above equation will be used in order to calculate the optimal rate of outcome earning. Th
1 P
f Optimal actions in one-dimensional outcome space The aim is to derive optimal actions when the outcome space has one dimension. Given
the reward field Ax,t, the reward of gaining dx units of outcome will be Ax,tdx, and since dx =
vdt, the reward earned in each time step is v Ax,t. Given that Kv is the cost function (the cost
paid in each time step), the net reward in each time step can be written as: Lx,v,t = v Ax,t −Kv,
(A27) (A27) and based on this, the value, which is the sum of net rewards in each time step, will be: and based on this, the value, which is the sum of net rewards in each time step, will be: S0,T =
ZT
0
Lx,v,tdt. (A28) (A28) The optimal rates that maximize S0,T can be found using different variational calculus
methods such as the Euler-Lagrange equation, or the Hamilton-Jacobi-Bellman equation (Liber-
zon, 2011). Here we use the Euler-Lagrange form, which sets a necessary condition for δS = 0: d
dt
∂L
∂v = ∂L
∂x . (A29) (A29) Furthermore, since the end-point of the trajectory is free (the amount of outcomes that can be
gained during a session is not limited, but the duration of the session is limited to T ), the optimal
trajectory will also satisfy the transversality conditions: Furthermore, since the end-point of the trajectory is free (the amount of outcomes that can be
gained during a session is not limited, but the duration of the session is limited to T ), the optimal
trajectory will also satisfy the transversality conditions: ∂L
∂v
¯¯¯¯
t=T
= 0,
(A30) (A30) . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE
22 OPTIMAL RESPONSE VIGOR AND CHOICE OPTIMAL RESPONSE VIGOR AND CHOICE 23 Theorem 1: Proof The cost function Kv defined in equation 3 satisfies the following relation: ∂2Kv
∂v2 > 0,
(A37) (A37) which holds as long as at least one response is required to earn an outcome (k > 0), and the cost
of earning outcomes is non-zero (a > 0). which holds as long as at least one response is required to earn an outcome (k > 0), and the cost
of earning outcomes is non-zero (a > 0). Assuming that ∂Ax,t/∂t = 0, and given equation A37, the only admissible solution to equa-
tion A35 is:
dv Assuming that ∂Ax,t/∂t = 0, and given equation A37, the only admissible solution to equa-
tion A35 is:
dv dv
dt = 0. (A38) (A38) thermore, assuming ∂Ax,t/∂t > 0, and given equation A37, the only admissible solution to
ation A35 is: dv
dt > 0,
(A39) dv
dt > 0, (A39) which proves Theorem 1. which proves Theorem 1. . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint .
CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017.
;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 24 24 Simulation details. The simulation of the model depicted in Figure 1:right panel re-
quires defining (i) the reward field, (ii) the cost function, and (iii) a probability distribution
over the session duration. As for the probability distribution of the session duration, following
McGuire et al (McGuire & Kable, 2013), we assumed that T follows a Generalized Pareto distri-
bution:
/k F(T ) = 1−
µ
1+ kt
σ
¶−1/k
,
(A47) (A47) where k is a shape parameter (note that k is not the ratio-requirement here) and σ is a scale
parameter, and the third parameter (location µ) was assumed to be zero. Furthermore we have: where k is a shape parameter (note that k is not the ratio-requirement here) and σ is a scale
parameter, and the third parameter (location µ) was assumed to be zero. Furthermore we have: F(T |T > t′) = 1−
µ
1+
kt
σ+kt′
¶−1/k
,
(A48) (A48) which has the following expected value: which has the following expected value: E[T |T > t′] = σ+kt′
1−k ,
(A49) (A49) which as we expect is an increasing function of t′. For the simulation of the model we assumed
that k = 0.9 and σ = 6, which represents that the initial expectation for the session duration is 60
minutes. For the cost function, in all the simulations the cost defined in equation 3 was used, which
is equivalent to the cost function used in the previous works (Dayan, 2012; Niv et al., 2007). For the definition of the reward field, we used the framework provided by Keramati et al
(Keramati & Gutkin, 2014), which provides a computational model for how the values of out-
comes change with the consumptions of the outcomes. They suggested that the dependency of
the reward field on the amount of outcome earned is indirect and it is through the motivational
drive. They conceptualized the motivational drive as the deviations of the internal states of a
decision-maker from their homeostatic set-points. For example, let’s assume that there is only
one internal state, say hunger, where H denotes its homeostatic set-point (which corresponds
to the deprivation level, assuming that initial value of x is zero), and there is an outcome which
consuming each unit of it satisfies l units of the internal state. Theorem 2: Proof and simulation details ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint Theorem 2: Proof and simulation details Proof of Theorem 2. In order to prove the theorem, we first provide a lemma. Assuming
that the total session duration (T ) has the probability density function f (T ) and that f (T ) > 0,
here we show that the expectation of the total session duration never decreases as time passes
throughout the session. Lemma 1 Let’s denote the expectation of the session duration at time t′ with T ′ T ′ = E[T |T > t′],
(A40) (A40) and assume T has the following probability density function: and assume T has the following probability density function: T ∼f (T ), f (T ) > 0. (A41) (A41) f ( ), f ( )
(
)
Then:
∂T ′
∂t′ > 0. (A42) Then:
∂T ′
> 0
(A42) Then: Then: ∂T ′
∂t′ > 0. (A42) ∂T ′
∂t′ > 0. (A42) (A42) Proof. We have:
∂T ′
∂t′ = ∂E[T |T > t′]
∂t′
=
f (T )
1−F(T )
¡
E[T |T > t′]−t′¢
> 0,
(A4 Proof. We have: Proof. We have: ∂T ′
∂t′ = ∂E[T |T > t′]
∂t′
=
f (T )
1−F(T )
¡
E[T |T > t′]−t′¢
> 0,
(A43) (A43) where F(T ) is the cumulative distribution function of T . Based on the above lemma, we show that the optimal response rate is a decreasing func-
tion of t′. Based on equation A31, the optimal response rate satisfies the following equation: ∂Kv
∂v
¯¯¯¯
t=T ′ = Ax,t
¯¯
t=T ′ . (A44) (A44) Taking the derivative w.r.t to t′ we get: Taking the derivative w.r.t to t′ we get: Taking the derivative w.r.t to t′ we get: Taking the derivative w.r.t to t′ we get: dv
dt′
µ∂2Kv
∂v2
¶
= ∂T ′
∂t′
µ
v ∂Ax,t
∂x
+ ∂Ax,t
∂T ′
¶
. (A45) (A45) Theorem 2 assumes that ∂Ax,t/∂x < 0 and ∂Ax,t/∂T ′ = 0, which given equations A42,A37, and
that v > 0 yields: dv∗
dt′ < 0,
(A46) (A46) which implies that the rate of earning outcomes decreases as time passes within a session. . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. OPTIMAL RESPONSE VIGOR AND CHOICE 25 Equation A52 was used in the simulations of the “decreasing reward and unknown session dura-
tion” condition in Figure 1:right panel. The simulation of this condition was done using param-
eters k = 15, l = 0.1, a = 0.002, b = 0.1, H = 80. As for T , in each time t′ within the session, the
expected session duration (E[T |T > t′]) was calculated using equation A49, and was used as T in
equation A52. Equation A52 was used in the simulations of the “decreasing reward and unknown session dura-
tion” condition in Figure 1:right panel. The simulation of this condition was done using param-
eters k = 15, l = 0.1, a = 0.002, b = 0.1, H = 80. As for T , in each time t′ within the session, the
expected session duration (E[T |T > t′]) was calculated using equation A49, and was used as T in
equation A52. For the “known session duration (fixed or decreasing reward)” condition in Figure 1:right
panel, the same parameters as the previous condition were used, but the session duration was
fixed to T = 60. For the “fixed reward (known or unknown session duration)” condition, we
assumed that the reward field is independent of the amount of reward earned: Ax,t = lH. (A53) (A53) Given the above reward field, the optimal rate of outcome earning is: Given the above reward field, the optimal rate of outcome earning is: v∗= Hl −bk
2ak2
. (A54) (A54) The simulation of this condition was done using parameters k = 15, l = 0.1, a = 0.002, b = 0.1,
H = 40. Note that in this condition the response rate was independent of the session duration. The response rates in all the conditions were obtained by multiplying the outcome rates by k
(since k responses are required to earn one unit of outcome). The simulation of this condition was done using parameters k = 15, l = 0.1, a = 0.002, b = 0.1,
H = 40. Note that in this condition the response rate was independent of the session duration. The response rates in all the conditions were obtained by multiplying the outcome rates by k
(since k responses are required to earn one unit of outcome). Simulation details of Figures 2, 3. OPTIMAL RESPONSE VIGOR AND CHOICE In this condition, the motivational
drive at point x, denoted by Dx, will be: Dx = 1
2(H −lx)2. (A50) (A50) Keramati et al (Keramati & Gutkin, 2014) showed that such a definition of the motivational drive
has implications that are consistent with the behavioral evidence. According to the framework,
the reward generated by earning δx units of the outcome is proportional to the change in the
motivational drive, which can be expressed as: Ax,t = −∂Dx
∂x = l(H −lx). (A51) (A51) As equation A51 suggests, with earning more outcomes (increase in x) Ax,t decreases. Given the
above reward field, the optimal response rate of outcome earning, obtained by equation A36, will
b As equation A51 suggests, with earning more outcomes (increase in x) Ax,t decreases. Given the
above reward field, the optimal response rate of outcome earning, obtained by equation A36, will
b be: v∗=
Hl −bk
T l2 +2ak2 . (A52) (A52) . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE The simulation depicted in Figure 2 and Figure 3 are
using equation A52 with the following parameters (note that the optimal response rates were
obtained by multiplying v∗by k). For Figure 2:right panel simulation parameters are T = 50,
k = 1, l = 1, a = 1, H = 8. Parameter b is varied between 3 to 7 in order to generate the plot. In Figure 3:left panel simulation parameters are T = 50, l = 1, a = 0.3, b = 0.05, H = 100. Parameter k was varied between 1 to 100 in order to generate the plot. In Figure 3:middle panel simulation parameters are T = 50, k = 1, l = 1, a = 0.3, b = 0.05. Parameter H was varied between 10 to 100 in order to generate the plot. In Figure 3:right panel simulation parameters are T = 50, k = 1, a = 0.1, b = 0.1, H = 100. Parameter l was varied between 0 to 1 in order to generate the plot. Optimal actions in multi-dimensional outcome space The aim of this section is to derive the optimal actions in the condition that the outcome
space is multi-dimensional. Optimal trajectory will satisfy the Euler-Lagrange equation along
each outcome dimension:
d ∂
∂ d
dt
∂L
∂v = ∂L
∂x ,
(A55) (A55) where: Lx,v,t = Ax,t.v−Kv. (A56) (A56) Furthermore since the end point of the trajectory is free (the total amount of outcomes is not
fixed) we have:
∂L ¯ Furthermore since the end point of the trajectory is free (the total amount of outcomes is not
fixed) we have:
∂L ¯ ∂L
∂v
¯¯¯¯
t=T
= 0. (A57) (A57) Using equation A55, A56 we have: d
dt
µ d
dv(−Kv +v.Ax,t)
¶
= d(v.Ax,t)
dx
. (A58) (A58) . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 26 For the right hand side of the above equation we have: For the right hand side of the above equation we have: d(v.Ax,t)
dx
= v⊺∂Ax,t
∂x . (A59)
We also have:
d Ax,t
dt
= ∂Ax,t
∂t
+ ∂Ax,t
∂x v,
(A60) d(v.Ax,t)
dx
= v⊺∂Ax,t
∂x . (A59) d(v.Ax,t)
dx
= v⊺∂Ax,t
∂x . ( (A59) dx
∂x
We also have:
d Ax,t
dt
= ∂Ax,t
∂t
+ ∂Ax,t
∂x v,
(A60) We also have:
d Ax,t
dt
= ∂Ax,t
∂t
+ ∂Ax,t
∂x v,
(A60) (A60) which by substitution into equation A58 yields: which by substitution into equation A58 yields: which by substitution into equation A58 yields: d
dt
∂Kv
∂v = ∂Ax,t
∂t
+
Ã
∂Ax,t
∂x
−
∂A⊺
x,t
∂x
! v. (A61) (A61) We now provide two lemmas, which will be used in the proof of the following theorems. We now provide two lemmas, which will be used in the proof of the following theorems. where: Lemma 2 Assume that H is the Hessian matrix of Kv, i.e., [H]i,j =
K 2
v
∂vi∂v j
,
(A62) [H]i,j =
K 2
v
∂vi∂v j
, [H]i,j =
K 2
v
∂vi∂v j
,
(A62 (A62) and furthermore assume that the cost of earning outcomes along each dimension is independent
of the outcome rate on the other dimensions, i.e., and furthermore assume that the cost of earning outcomes along each dimension is independent
of the outcome rate on the other dimensions, i.e., and furthermore assume that the cost of earning outcomes along each dimension is independent
of the outcome rate on the other dimensions, i.e., Hi,j = 0,i ̸= j. (A63) Hi,j = 0,i ̸= j. (A63) (A63) Then: Proof. Using equation A66 we get: [M]i,j = ∂[Ax,t]i
∂x j
−
∂[Ax,t]j
∂xi
= −∂2Dx
∂x j∂xi
+ ∂2Dx
∂xi∂x j
= −∂2Dx
∂xi∂x j
+ ∂2Dx
∂xi∂x j
(using Schwarz’s theorem)
= 0 = 0. Note that the use of Schwarz’s theorem is based on the assumption that Dx is twice differentiable,
which holds in the circumstances that we consider here. Note that the use of Schwarz’s theorem is based on the assumption that Dx is twice differentiable,
which holds in the circumstances that we consider here. Theorem 3: Proof and simulation details Proof of Theorem 3. Theorem 3 assumes that (i) the costs of earning outcomes are inde-
pendent (equation A63), (ii) the reward field is conservative (equation A66), and (iii) the reward
field is independent of time (∂Ax,t/∂t = 0). Based on Lemma 2, Lemma 3 and equation A61 we
have:
2 dv
dt ⊙
µ∂2Kv
∂v2
¶
= 0. (A68) (A68) Given that equation A37 holds along each outcome dimension (∂2Kv/∂v2 > 0), the only admis-
sible solution to equation A68 is dv/dt = 0, which shows that the optimal rate of earning out-
comes is constant. Since the optimal rate is constant, we have xT = T v∗, which by substituting
in boundary conditions implied by equation A57 yields equation 10: ∂Kv∗
∂v∗= AT v∗,T ,
(A69) (A69) Then: Then:
d
dt
∂Kv
∂v = dv
dt ⊙
µ∂2Kv
∂v2
¶
,
(A64) (A64) where ∂2Kv/∂v2 represents the diagonal terms of H, and ⊙is entrywise Hadamard product. where ∂Kv/∂v represents the diagonal terms of H, and ⊙is entrywise Hadamard product. Proof. Using equation A63 we have: Proof. Using equation A63 we have: Proof. Using equation A63 we have: d
dt
∂Kv
∂v = dv
dt H
= dv
dt ⊙
µ∂2Kv
∂v2
¶
, d
dt
∂Kv
∂v = dv
dt H
= dv
dt ⊙
µ∂2Kv
∂v2
¶
,
(A65) (A65) where the last equation comes from the fact that H is a diagonal matrix. Lemma 3 Assuming that the reward field is conservative, i.e., Ax,t = −∂Dx
∂x ,
(A66)
then:
M = ∂Ax,t
∂x
−
∂A⊺
x,t
∂x
= 0. (A67) then: M = ∂Ax,t
∂x
−
∂A⊺
x,t
∂x
= 0. (A67) (A67) . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE OPTIMAL RESPONSE VIGOR AND CHOICE 27 Proof. Using equation A66 we get: OPTIMAL RESPONSE VIGOR AND CHOICE 28 The above equation was used as the reward field in the simulations. As for the cost function,
earning one unit of O1 requires k responses on the left lever, and earning one unit of O2 requires
lk responses on the right lever. Based on this and using equation 3, the cost function will be: Kv = v1[ak2v1 +kb]+ v2[ak2l2v2 +klb],
(A72) (A72) where v1 is the rate of earning O1 and v2 is the rate of earning O2. Using Theorem 3, the optimal response rate will be: Using Theorem 3, the optimal response rate will be: Using Theorem 3, the optimal response rate will be: Using Theorem 3, the optimal response rate will be: response rate =
for left lever
z
}|
{
kl2H
T l2 +2ak2l2 +T ,
for right lever
z
}|
{
klH
T l2 +2ak2l2 +T
,
(A73) (A73) where as mentioned in the main text “left lever” is the response that should be taken for earning
O1, and “right lever” is the response that should be taken for earning O2. Parameters used for
simulations are k = 1, l = 2, a = 1, b = 0, H = 100. where as mentioned in the main text “left lever” is the response that should be taken for earning
O1, and “right lever” is the response that should be taken for earning O2. Parameters used for
simulations are k = 1, l = 2, a = 1, b = 0, H = 100. which completes the proof the theorem. which completes the proof the theorem. Simulation details. For the simulation of the model in Figure 4:left panel “independent
cost” condition, it is assumed that the two outcomes have the same reward effect, but earning the
second outcome requires l times more responses. Following Keramati et al (Keramati & Gutkin,
2014), since the two outcomes have the same reward properties we defined the motivational
drive as follows: Dx = 1
2(H −x1 −x2)2,
(A70) (A70) where as mentioned Dx is the motivational drive and it represents the deviations of the internal
state of the decision-maker from its homeostatic set-point (H). x1 is the amount of O1 earned
and x2 is the amount of O2 earned, and the current motivational drive for earning outcomes de-
pends on the difference between the total amount of earned outcomes (x1 +x2) and the homeo-
static set-point (H). Given the motivational drive, the amount of reward generated by consuming each out-
come will be equal to the amount of change in the motivational drive due to the consumption of
the outcomes (equation 8), and therefore, we have: Ax,t = −∂Dx
∂x = [H −x1 −x2,H −x1 −x2]. (A71) (A71) . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint Using the boundary condition mentioned in equation A57 we have: Parameters used for simulations are k = 1, l = 2, a = 1, b = 0, H = 100. Parameters used for simulations are k = 1, l = 2, a = 1, b = 0, H = 100. OPTIMAL RESPONSE VIGOR AND CHOICE OPTIMAL RESPONSE VIGOR AND CHOICE 29 Theorem A2: Definition, proof and simulation details Proof of Theorem A2. The aim of this section is to derive optimal actions in the condi-
tions that the costs of earning outcomes are dependent on each other. In this condition, one can
assume what determines the cost is the delay between subsequent responses, either for the same
or for a different outcome, i.e., the cost is proportional to the rate of earning all of the outcomes. In particular, if for earning O1, k responses are required and for earning O2, lk responses are
required (l ̸= 1), then the delay between subsequent responses (τ) will be 1/(kv1 +lkv2). Given
equation 2, the cost of earning one unit of O1 will be k[a(kv1+lkv2)+b], and the cost of earning
one unit of O2 will be kl[a(kv1 +lkv2)+b]. Such a cost function can be achieved by defining the
cost as follows: Kv = v1[ak(kv1 +lkv2)+kb]+ v2[akl(kv1 +lkv2)+klb]. (A74) (A74) In the following theorem, we maintain that given the above cost function, the optimal actions
are to make no response for O2, and to make responses for O1 at a constant rate. In the following theorem, we maintain that given the above cost function, the optimal actions
are to make no response for O2, and to make responses for O1 at a constant rate. Theorem A2 Given the cost function defined in equation A74 and assuming that the two out-
comes have the same reward properties, i.e.,: [Ax,t]1 = [Ax,t]2. (A75) (A75) Then the optimal actions satisfy the following equations: Then the optimal actions satisfy the following equations: Then the optimal actions satisfy the following equations: p
fy
f
g q
dv1
dt = 0,
v2 = 0. (A76) dv1
dt = 0,
v2 = 0. (A76) (A76) Proof. By substituting equation A74 in equation A56 we have: Proof. By substituting equation A74 in equation A56 we have: uting equation A74 in equation A56 we have: L = −v1 [ak(kv1 +lkv2)+kb)]−v2 [akl(kv1 +lkv2)+klb]+
v1[Ax,t]1 + v2[Ax,t]2. (A77) (A77) . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint Using the boundary condition mentioned in equation A57 we have: Using the boundary condition mentioned in equation A57 we have: Using the boundary condition mentioned in equation A57 we have: [AxT ,T ]1 −2ak2lv2 −2ak2v1 −bk = 0,
[AxT ,T ]2 −2ak2l2v2 −2ak2lv1 −bkl = 0. (A78) (A78) v1 = −lv2 −b/(2ak),
(A79) (A79) which is not admissible given constraints v1 ≥0 and v2 ≥0, and therefore we assume either v1
or v2 will be equal to zero. The trajectory will have a higher value by setting v2 to zero since O2
has a higher cost, and therefore the optimal solution will be v2 = 0. Since v2 = 0 the problem de-
generates to a one-dimensional problem, in which according to Theorem 1 the optimal response
rate is constant, and therefore the rate of responding for O1 will be constant, which proves the
theorem. Simulation details. For the simulation of the model in Figure 4:left panel “dependent
cost” condition, it is assumed that k responses on the left lever are required to earn O1 and lk
response are required on the right lever to earn O2. Similar to the “independent cost” condition
mentioned in the previous section, the reward field was assumed as follows: Ax,t = −∂Dx
∂x = [H −x1 −x2,(H −x1 −x2)]. (A80) (A80) Since the response rate for one of the outcomes will be zero (according to Theorem A2), the
problem degenerates to an environment with one action and one outcome. Using Theorem 1,
and equation A36 the optimal response rate will be: Since the response rate for one of the outcomes will be zero (according to Theorem A2), the
problem degenerates to an environment with one action and one outcome. Using Theorem 1,
and equation A36 the optimal response rate will be: response rate =
k H −bk
T +2ak2
|
{z
}
for left lever
,
0
|{z}
for right lever
. (A81)
Parameters used for simulations are k = 1, l = 2, a = 1, b = 0, H = 100. response rate =
k H −bk
T +2ak2
|
{z
}
for left lever
,
0
|{z}
for right lever
. (A81) (A81) Parameters used for simulations are k = 1, l = 2, a = 1, b = 0, H = 100. Parameters used for simulations are k = 1, l = 2, a = 1, b = 0, H = 100. OPTIMAL RESPONSE VIGOR AND CHOICE 30 In this condition, ∂[Ax,t]1/∂x2 = −1 and ∂[Ax,t]2/∂x1 = −l, and therefore the reward of the second
outcome due to the consumption of the first outcome decreases more sharply than the reward
of the first outcome would, due to the consumption of the second outcome. We have: M = ∂Ax,t
∂x
−
∂A⊺
x,t
∂x
=
· 0
l −1
1−l
0
¸
,
(A84) (A84) and as long as l ̸= 1 then M ̸= 0, and therefore the reward field is non-conservative, because if it
was conservative then according to Lemma 3 we should have M = 0. If the reward field is non-conservative, i.e., there does not exist a scalar field Dx such that
Ax,t satisfies equation 8, then the optimal response rates are as follows: early in the session the
decision-maker exclusively works for the outcome with the higher reward value (O1) and, when
the time remaining in the session is less than the threshold (Tc), the decision-maker then gradu-
ally starts working for the outcome with the lower reward value (O2). More precisely we maintain
the following theorem: Theorem A3 If the reward field follows equation A83, ∂Ax,t/∂t = 0, and the cost is as follows: Kv = 1
2mv2
1 + 1
2mv2
2,
(A85) (A85) then the optimal trajectory in the outcome space will be: then the optimal trajectory in the outcome space will be: n the optimal trajectory in the outcome space will be: [v1,v2] =
(h
0, H(l−1)
T l−Tc
i
,
T −t > Tc
arc of a circle
T −t ≤Tc
,
(A86) (A86) where Tc = m arctan(1/l)
l −1
,
m = 2ak2. (A87) Tc = m arctan(1/l)
l −1
,
m = 2ak2. (A87) (A87) Proof. We have:
∂Ax,t
∂x
−
∂A⊺
x,t
∂x
=
· 0
l −1
1−l
0
¸
,
(A88) (A88) and based on equations A63, A85, A61 we get: dv1
dt = l −1
m v2,
dv2
dt = −l −1
m v1. (A89) (A89) Defining w = (l −1)/m, the solution to the above set of differential equations has the form: x =
£
q1 +r/w sin(wt +α),q2 +r/w cos(wt +α)
¤
,
(A90) (A90) which is an arc of a circle centered at [q1,q2], and r and α are free parameters. Theorem A3: Definition, proof and simulation details Proof of Theorem A3. The aim of Theorem A3 is to derive optimal actions when the re-
ward field is non-conservative and the costs of actions are independent. An example of a non-
conservative reward field is when the amount of reward that consuming an outcome produces
is greater or smaller than the change in the motivational drive. For example, assume that there
are two outcomes available, and the consumption of both outcomes has a similar effect on the
motivational drive:
1 Dx = 1
2(H −x1 −x2)2,
(A82) (A82) but the reward that the second outcome generates is l times larger (l ̸= 1) than the change it
creates in the motivational drive: but the reward that the second outcome generates is l times larger (l ̸= 1) than the change it
creates in the motivational drive: Ax,t =
·
−∂Dx
∂x1
,−l ∂Dx
∂x2
¸
= [H −x1 −x2,l(H −x1 −x2)]. (A83) (A83) . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint where Tc = m arctan(1/l)
l −1
,
(A93) (A93) and therefore Tc is independent of H (the initial motivational drive). As such if T ≤Tc (equa-
tion A92) then the optimal trajectory will be an arc of a circle starting from the origin. Otherwise,
if T > Tc, the optimal trajectory will be composed of two segments. In the first segment, v1 will
take the boundary condition v1 = 0 and the decision-maker earns only the second outcome (the
outcome with the higher reward effect). The first segment continues until the remaining time
in the session satisfies equation A92 (the remaining time is less than Tc), after which the second
segment starts, which is an arc of a circle defined by equation A90. The rate of earning the second
outcome, v2, in the first segment of the trajectory (when v1 = 0) can be obtained by calculating
the rates at the beginning of the circular segment. The initial rate at the start of the circular
segment is as follows: r = H(l −1)
T l −Tc
, r = H(l −1)
T l −Tc
,
(A94) (A94) which implies that at the first segment of the trajectory we have: [v1,v2] =
·
0, H(l −1)
T l −Tc
¸
,
(A95) (A95) which completes the proof of Theorem A3. which completes the proof of Theorem A3. It is interesting to mention that there is a parallel between the trajectory that a decision-
maker takes in the outcome space, and the motion of a charged particle in a magnetic field. In
the case that the outcome space is three dimensional, using equation A61 the optimal path in
the outcome space satisfies the following properties: m dv
dt =
Ã
∂Ax,t
∂x
−
∂A⊺
x,t
∂x
! v
= −v×B,
(A96) (A96) = −v×B, where × is the cross product, B is the curl of the reward field (B = curlAx,t), and m = 2ak2. The
equation A96 in fact lays out the motion of a unit charged particle (negatively charged) with mass
m in a magnetic field with magnitude B. where × is the cross product, B is the curl of the reward field (B = curlAx,t), and m = 2ak2. The
equation A96 in fact lays out the motion of a unit charged particle (negatively charged) with mass
m in a magnetic field with magnitude B. Simulation details. OPTIMAL RESPONSE VIGOR AND CHOICE The parameters
can be determined using the boundary condition imposed by equation A57, and also assuming
that the initial position is x = 0. The boundary condition in equation A57 implies: mv = Ax,t
¯¯
t=T =
hp
2Dx,l
p
2Dx
i
,
(A91) (A91) . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which
this version posted February 7, 2017. ;
https://doi.org/10.1101/106500
doi:
bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 31 which implies that at the end of the trajectory the rate of earning the second outcome is l times
larger than the first outcome. Therefore, the general form of the trajectory will be an arc starting
from the origin and ending along the above direction. Given the constraint that v ≥0 only the
solutions in which q2 ≤0 are acceptable ones (i.e., the center of the circle is below the x-axis). Solving equation A90 for q2 ≤0 we get: (A92) T ≤Tc,
(A92) T ≤Tc, where Simulations shown in Figure 4:right panel are based on Theorem A3,
and the parameters used are k = 1, l = 1.1, a = 1, b = 0, H = 100, m = 2ak2.
|
https://openalex.org/W2920840000
|
https://digital.csic.es/bitstream/10261/176567/1/Scientific%20Reports_Uluseker_2019.pdf
|
English
| null |
Quantifying dynamic mechanisms of auto-regulation in Escherichia coli with synthetic promoter in response to varying external phosphate levels
|
Scientific reports
| 2,019
|
cc-by
| 12,853
|
Quantifying dynamic mechanisms
of auto-regulation in Escherichia
coli with synthetic promoter in
response to varying external
phosphate levels
Cansu Uluşeker1,2, Jesús Torres-Bacete3, José L. García4,5, Martin M. Hanczyc 1,6,
Juan Nogales3 & Ozan Kahramanoğulları7 Received: 2 May 2018
Accepted: 13 December 2018
Published: xx xx xxxx Escherichia coli have developed one of the most efficient regulatory response mechanisms to phosphate
starvation. The machinery involves a cascade with a two-component system (TCS) that relays the
external signal to the genetic circuit, resulting in a feedback response. Achieving a quantitative
understanding of this system has implications in synthetic biology and biotechnology, for example,
in applications for wastewater treatment. To this aim, we present a computational model and
experimental results with a detailed description of the TCS, consisting of PhoR and PhoB, together
with the mechanisms of gene expression. The model is parameterised within the feasible range, and
fitted to the dynamic response of our experimental data on PhoB as well as PhoA, the product of this
network that is used in alkaline phosphatase production. Deterministic and stochastic simulations
with our model predict the regulation dynamics in higher external phosphate concentrations while
reproducing the experimental observations. In a cycle of simulations and experimental verification, our
model predicts and explores phenotypes with various synthetic promoter designs that can optimise the
inorganic phosphate intake in E. coli. Sensitivity analysis demonstrates that the Pho-controlled genes
have a significant influence over the phosphate response. Together with experimental findings, our
model should thus provide insights for the investigations on engineering new sensors and regulators for
living technologies. Mechanisms of inorganic phosphate intake within the context of cellular processes is a topic of extensive research
effort, also due to potential applications in enhanced biological phosphorus removal (EBPR) from wastewater. Phosphorous, which is one of the major causes of water quality problems, occurs in wastewater almost solely in
the form of phosphates such as inorganic phosphate (Pi). Microorganisms, which are key players in bioremedia-
tion, have potential to treat large amounts of the pollutants and hold promise for renewable sources1. An in-depth
understanding of the mechanisms controlling such processes should pave the way for metabolic engineering to
lead to improvements in wastewater treatment.h p
The physiological characteristics of Pi transport in E. coli have been extensively studied2–6. Nonetheless, the
regulatory interactions that control the Pi transport are complex, and they involve two major phosphate transport
systems, depending on external Pi levels. The low affinity phosphate inorganic transport (Pit) system depends on
the proton motive force; it is a coupled transporter of two different ions through the membrane2,7. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 2 May 2018
Accepted: 13 December 2018
Published: xx xx xxxx Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w www.nature.com/scientificreports/ Consequently, the tanscription factor
PhoB does not become phosphorylated by PhoR. Right: Due to the ABC transporter activity, the external Pi
binds to the PstS component of the ABC transporter. It is then translocated to the inner membrane domain of
the transporter through PstC and PstA. Following this, PstB changes its conformation by consuming ATP. Pi
is internalised and released to the cytosol. When the Pi influx through the ABC transporter decreases, PhoU
does not stabilise PhoR, which is depicted with the red bar next to PhoU. As a result of this, PhoR becomes
free to perform its auto-kinase-phosphotransferase activity, whereby it phosphorylates PhoB. Phosphorylated
PhoB then forms a dimer to act as a transcription factor for the operons, resulting in PhoA, PhoB and PhoR
expression. specific transport (Pst) system, on the other hand, is Pi-repressible, and it is induced when the external Pi concen-
tration is depleted8–10. p
Mechanistically, Pi signalling associated with the Pst system in E. coli, is a negative process, that is, excessive Pi
is required for turning the system off. Activation is the default state and occurs in conditions of Pi limitation2,8,11. Signal transduction by environmental Pi requires seven proteins, which are thought to interact in a membrane
associated signalling complex. These Pi signalling proteins include2,8. •
Four components of the ABC transporter Pst (PstSCAB), which consist of an extracellular binding protein
(PstS), two transmembrane proteins (PstC, PstA) that form the transmembrane domain (TMD), and a dimer
of cytosolic peripheral proteins (PstB), i.e., the nucleotide-binding domain; •
Four components of the ABC transporter Pst (PstSCAB), which consist of an extracellular binding protein
(PstS), two transmembrane proteins (PstC, PstA) that form the transmembrane domain (TMD), and a dimer
of cytosolic peripheral proteins (PstB), i.e., the nucleotide-binding domain; y
g
•
Two that are members of the large family of two component systems (TCS) that perform both positive and
negative regulation, a sensor histidine kinase PhoR and a transcriptional response regulator PhoB;
The chaperone like PhoR/PhoB inhibitor protein PhoU •
Two that are members of the large family of two component systems (TCS) that perform both positive and
negative regulation, a sensor histidine kinase PhoR and a transcriptional response regulator PhoB;
The chaperone like PhoR/PhoB inhibitory protein PhoU g
g
p
p
g
•
The chaperone-like PhoR/PhoB inhibitory protein PhoU. g
g
p
p
g
The chaperone-like PhoR/PhoB inhibitory protein PhoU. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. The transmembrane signal transduction due to external Pi levels and the Pho regulon activity when
extracellular inorganic phosphate (Pi) is in excess (left) and when it is depleted (right). The ABC transporter
consists of the extracellular domain PstS, transmembrane domains PstA and PstC, and intracellular domains
PstB (see the main text for the description). Left: when external Pi is in excess, the ABC transporter activity is
limited as the cell does not consume ATP for the Pi transport. According to the current model in the literature,
PhoR assesses Pi availability by monitoring the activity of Pst transporter, and relays the signal from PstB via
PhoU to PhoR. When the Pi influx is increased, PhoU stabilises PhoR, which is depicted with the green bars
around PhoU. This prevents PhoR dimers from autphosphorylating (red). Consequently, the tanscription factor
PhoB does not become phosphorylated by PhoR. Right: Due to the ABC transporter activity, the external Pi
binds to the PstS component of the ABC transporter. It is then translocated to the inner membrane domain of
the transporter through PstC and PstA. Following this, PstB changes its conformation by consuming ATP. Pi
is internalised and released to the cytosol. When the Pi influx through the ABC transporter decreases, PhoU
does not stabilise PhoR, which is depicted with the red bar next to PhoU. As a result of this, PhoR becomes
free to perform its auto-kinase-phosphotransferase activity, whereby it phosphorylates PhoB. Phosphorylated
PhoB then forms a dimer to act as a transcription factor for the operons, resulting in PhoA, PhoB and PhoR
expression. Figure 1. The transmembrane signal transduction due to external Pi levels and the Pho regulon activity when
extracellular inorganic phosphate (Pi) is in excess (left) and when it is depleted (right). The ABC transporter
consists of the extracellular domain PstS, transmembrane domains PstA and PstC, and intracellular domains
PstB (see the main text for the description). Left: when external Pi is in excess, the ABC transporter activity is
limited as the cell does not consume ATP for the Pi transport. According to the current model in the literature,
PhoR assesses Pi availability by monitoring the activity of Pst transporter, and relays the signal from PstB via
PhoU to PhoR. When the Pi influx is increased, PhoU stabilises PhoR, which is depicted with the green bars
around PhoU. This prevents PhoR dimers from autphosphorylating (red). Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w Quantifying dynamic mechanisms
of auto-regulation in Escherichia
coli with synthetic promoter in
response to varying external
phosphate levels
Cansu Uluşeker1,2, Jesús Torres-Bacete3, José L. García4,5, Martin M. Hanczyc 1,6,
Juan Nogales3 & Ozan Kahramanoğulları7 The phosphate 1University of Trento, Centre for Integrative Biology, Trento, 38123, Italy. 2The Microsoft Research – University of
Trento Centre for Computational and Systems Biology, Rovereto, 38068, Italy. 3Centro Nacional de Biotecnología
(CNB-CSIC), Systems Biology Department, Madrid, 28049, Spain. 4Centro de Investigaciones Biológicas (CIB-CSIC),
Microbial and Plant Biotechnology Department, Madrid, 28040, Spain. 5Institute for Integrative Systems Biology
(I2Sysbio-CSIC-UV), Applied Systems Biology and Synthetic Biology Department, Paterna, 46980, Spain. 6Chemical
and Biological Engineering, University of New Mexico, Albuquerque, NM, 87131, USA. 7University of Trento,
Department of Mathematics, Trento, 38123, Italy. Correspondence and requests for materials should be addressed
to O.K. (email: ozan.kahramanogullari@unitn.it) 1 Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w www.nature.com/scientificreports/ Previous studies have shown that the TCS plays a central role in sensing the Pi level in the environ-
ment and regulating the expression of genes that are directly involved in the intake2,12. g
g
p
g
y
Although much is known about the molecular aspects of this signal transduction pathway, a comprehensive
and structured mechanistic model of the Pho regulon is currently unavailable3. Here, we investigate the relation-
ship between Pi-starvation, the TCS signalling, and promoter activity by using a combination of wet lab exper-
iments and dynamic modelling. This work introduces the proteins involved in phosphate starvation response
mechanism. Our model describes the phosphate starvation response at the genetic level by considering all system
variables that affect the phosphate starvation response. We consider components where phosphate starvation
response of TCS model is described in terms of their interactions. The model is composed of a set of ordinary
differential equations and the corresponding stochastic model that are derived from a chemical reaction network. The model provides a quantitative description of how different processes interact to form a positively-regulated
biological control system. The mechanism is based on an E. coli Pi-starvation signalling system. Our model dis-
plays how specific TCS proteins work together to provide gene expression and increase in Pi intake, and provides
a set up for identifying a wide family of promoter mechanisms that potentially have synthetic applications.i p
y
g
y
p
p
y
y
pp
In recent years, significant advances have been made in understanding the role and the structure of TCS and
signalling mechanisms. It is known that most bacteria species have more than 10 different TCS13. Moreover,
in E. coli, 30 sensor kinases and 32 response regulators have been found14. Although there is large qualitative
knowledge, especially for E. coli, quantitative research is still scarce. A model for PhoR/PhoB signal transduction
was set up by Van Dien and Keasling earlier3, whereby the authors have determined a model for the induction of
PhoA. Models for other signal transduction systems in E. coli are described by Wong et al., Fisher et al., and Koh
et al.15–17. Recently, research groups have only just begun to use modelling in combination with experimental data
to analyse the TCS especially PhoR and PhoB4,18. Gao et al. built quantitative analyses of PhoR and PhoB protein
concentrations and activities. They showed that the experimental data and protein expression levels of TCSs
matched. www.nature.com/scientificreports/ Figure 1 displays a schematic representation of the system in the starvation condition. When Pi is limited in
quantity outside the cell, PstS binds to the Pi following its diffusion to the cell surface2,8,12. The transmembrane
domain of the ABC transporter, that is, PstC and PstA are integral membrane proteins that span the entirety of the
membrane. They regulate the translocation of Pi from PstS to the inner membrane2,3,8,12. Pi intake happens with
the conformational changes in the PstB as a result of ATP binding, also known as ATP-switch model. This way, the
ABC transporter provides the required increase in the amount of phosphate in the cell2,8.h p
p
q
p
p
The low activity of the ABC transporter PstSCAB causes, in a number of steps, the autophosphorylation of
the sensor kinase PhoR, which relays the signal to the transcription factor PhoB. The current evidence suggests
that PhoR and eventually PhoB assess Pi availability by monitoring the activity of Pst transporter via PhoU11,12. In mechanistic terms, when there is sufficient Pi flux, PhoU stabilizes PhoR. The resulting stable conformation
prevents PhoR from auto-phosphorylation. This, in return, prevents the down-stream signalling that would oth-
erwise result in the expression of the genes that feedback to the Pst system for further Pi intake. In fact, when
PhoU is deleted, PstB does not only continue to spend ATP and transport Pi, but PhoR acts as a constitutive PhoB
kinase, leading to high expression of the Pho regulon genes2,8, and thereby, activating the expression of the Pst sys-
tem. On the other hand, when external Pi is limited, PhoU does not stabilise PhoR. Consequently, PhoR is freed to
bind phosphoryl groups and passes to the active state. Active PhoR phosphorylates PhoB. Phosphorylated PhoB
then acts as a transcription factor for the operon. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 2 www.nature.com/scientificreports/ All experimental evidence indicates that Pho regulon is controlled by external phosphate limitation rather
than internal3. When the surrounding environment has abundant phosphate, E. coli uses as few resources as
possible to facilitate the phosphate intake. However, when Pi becomes low outside the cell, it turns into a growth
limiting factor and the cell spends energy to up-regulate the expression of target genes that are used to acquire
phosphate. www.nature.com/scientificreports/ Recently, Gao and Stock et al.19 built a quantitative analyses of TCS switch off mechanism. However, to
the best of our knowledge, there has been no focus on developing genetically encoded signal transduction path-
ways leading to transcription factors. y
g
p
In this work, we have developed an approach combining wet-lab experiments and modelling to show that
rates of histidine kinase and promoter activities can be used to tune TCS detection thresholds. We have described
the steps in the design of a synthetic biological system based on the use of TCS. This should constitute a contri-
bution to the research on genetically modified bacteria that detect environmental changes and respond to higher
inorganic phosphate levels. In order to better understand the quantitative analyses of protein concentrations in
response to modifications and activities in the environment, we have focused on the activation response of the
TCS components PhoB and PhoR. Our model should thus help to better understand the dynamic behaviour
of system activation, and to quantitatively evaluate the role of phosphatase activity under varying external Pi
conditions. The model includes TCS members and activation of the Pho regulon promoters pPhoB and pPhoA. Experimental data is used to fit the parameters to the feasible physiological range given in the literature, and to
determine the relative sensitivity to the parameters. The simulations with our model provide a dynamic descrip-
tion of the mechanisms. With a combination of wet-lab experiments and computer simulations, we use our model
to quantify dynamic mechanisms of auto-regulation in E. coli in response to varying external phosphate levels,
and explore and verify emerging phenotypes with synthetic promoters. Simulations with the model do not only
reproduce our experimental measurements, but also predict phenotypes with various synthetic promoter designs
that can optimise the Pi intake in E. coli. Sensitivity analysis on the parameters demonstrate the influence on the
expression of Pho-controlled genes and the gain of the system under variations in transcription efficiency in
response to external phosphate concentration. The model can thus serve as a virtual lab, and can be used to test
various promoter designs for enhanced Pi intake in biotechnology applications for phosphate sequestration. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w Results
Ph
h The
deterministic ODE and stochastic simulations are performed by applying the standard translation from CRNs
based on stoichiometry. The blue color denotes the proteins, orange denotes the promoters in their active and
inactive forms, and purple denotes the mRNA molecules. Filled arrowheads denote the reversible reactions. The red arrows denote degradation reactions, the green arrows denote complexations, the black arrows
denote phosphorylation and dephosphorylation, and the blue arrows denote the transcription and translation
reactions. The model species that are plotted in C and D are distinguished with frames. (C) The dynamics of
the highlighted species of the control model in panel B as a result of the fitting procedure together with the
experimental data, as described in Methods, are plotted. In the first of the two steps, the blue curves are obtained
by using only the experimental values of PhoA levels. In the second step, that delivers the control model, the
red curves are obtained by using the experimental values of both PhoA and PhoB levels. Inclusion of the PhoB
data highlights the contribution of the feedback mechanism to the response dynamics, which is otherwise not
represented. The stochastic dynamics, plotted in grey, display the fluctuations in the control model that are due
to small molecule numbers and are not observable in the deterministic simulations. (D) The response of the
control model to variations in the external Pi levels, which are represented as fold change factors, applied to the
autophosphorylation propensities of PhoR. A higher external Pi concentration corresponds to a smaller factor
and vice versa. Figure 2. Schematic representation of the experimental data and the control model, and its dynamics in
response to varying external Pi concentrations. (A) The data for the PhoA and PhoB expression are obtained
using PCR amplified DNA from E. coli MG1655 genome and transcriptionally fused to the translational coupler
BCD2 and the fluorescent ms-fgfp gene. (B) The control model has been obtained from the experimental
data and the chemical reaction network (CRN) described in Methods by applying a fitting procedure with
the physiological ranges obtained from the literature in Table 1, and verified by sensitivity analysis. The
deterministic ODE and stochastic simulations are performed by applying the standard translation from CRNs
based on stoichiometry. The blue color denotes the proteins, orange denotes the promoters in their active and
inactive forms, and purple denotes the mRNA molecules. Filled arrowheads denote the reversible reactions. Results
Ph
h The red arrows denote degradation reactions, the green arrows denote complexations, the black arrows
denote phosphorylation and dephosphorylation, and the blue arrows denote the transcription and translation
reactions. The model species that are plotted in C and D are distinguished with frames. (C) The dynamics of
the highlighted species of the control model in panel B as a result of the fitting procedure together with the
experimental data, as described in Methods, are plotted. In the first of the two steps, the blue curves are obtained
by using only the experimental values of PhoA levels. In the second step, that delivers the control model, the
red curves are obtained by using the experimental values of both PhoA and PhoB levels. Inclusion of the PhoB
data highlights the contribution of the feedback mechanism to the response dynamics, which is otherwise not
represented. The stochastic dynamics, plotted in grey, display the fluctuations in the control model that are due
to small molecule numbers and are not observable in the deterministic simulations. (D) The response of the
control model to variations in the external Pi levels, which are represented as fold change factors, applied to the
autophosphorylation propensities of PhoR. A higher external Pi concentration corresponds to a smaller factor
and vice versa. procedure. The rates in Table 1 are obtained as a result of this fitting procedure that delivered the control model
parameters, whereby we have enforced the displayed physiologically boundaries. The difference in the phenotype
between red and blue curves in Fig. 2C should thus highlight the role of the feedback of the PhoB and PhoR
expression to the system.i procedure. The rates in Table 1 are obtained as a result of this fitting procedure that delivered the control model
parameters, whereby we have enforced the displayed physiologically boundaries. The difference in the phenotype
between red and blue curves in Fig. 2C should thus highlight the role of the feedback of the PhoB and PhoR
expression to the system.i We have performed a large scale analysis of the system dynamics of the control model in terms of the fitted
parameter values within a broader range. We have first analysed the broader effect of the TCS disassociation rates
(r3r, r5r, r7r, r8r) on the dynamics and the PhoB transcription and translation parameters that deliver the expres-
sion of PhoB (r14, r15), which is a transcription factor. Results
Ph
h Phosphate intake at starvation requires rapid activation of PhoB dimers. Figure 2 provides a
schematic representation of the control model set up with the experimental data and the formal model. The
control model is set up with respect to the initial phosphate starvation in accordance with the experimental data
and the parametrisation and fitting procedure described in Methods. In first of the two steps, the blue curves in
Figs 2C and S2 are obtained by using only the experimental values of PhoA levels. When external Pi is abundant,
the Pst system inhibits the activation of TCS, and consequently the Pi intake; PhoA is then expressed at a basal
level. However, when Pi is limiting, inhibition of TCS is relieved, resulting in the activation of PhoA transcription. The alterations of PhoA expression can thus be interpreted to an extent as an indicator for the changes in external
phosphate level and Pi intake. In these simulations, in response to external Pi level at 0 μM, the system initiates the activation of PhoR, given
by the autophosphorylation of both of the monomers in the stable dimer. This results in the subsequent transfer
of phosphoryl groups. As a consequence, the response regulator PhoB rapidly becomes active and dimerises to
form active transcription factors. The resulting rapid increase in the promoter activity delivers the mRNA tran-
scription, and the consequent experimentally observed levels of PhoA. At a second step in our analysis, to highlight and contrast the role of PhoB dynamics in the feedback mecha-
nism, we have refined the model to include the experimental data on PhoB expression. The resulting red curves
in Figs 2C and S2 are obtained by using the relative experimental levels of both PhoA and PhoB in the fitting Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223 3 www.nature.com/scientificreports/ Figure 2. Schematic representation of the experimental data and the control model, and its dynamics in
response to varying external Pi concentrations. (A) The data for the PhoA and PhoB expression are obtained
using PCR amplified DNA from E. coli MG1655 genome and transcriptionally fused to the translational coupler
BCD2 and the fluorescent ms-fgfp gene. (B) The control model has been obtained from the experimental
data and the chemical reaction network (CRN) described in Methods by applying a fitting procedure with
the physiological ranges obtained from the literature in Table 1, and verified by sensitivity analysis. Results
Ph
h On the other hand, the dissociation rate of
the inactive PhoR from the active PhoB, given by r8r, affects the activation of PhoB as a transcription factor in a
proportional way, yet its effect on promoter activity is negligible (Fig. S8). As it can be seen from Table 1, there are
also other parameters that have an effect on both system dynamics and PhoB activity. Nonetheless, these are not
suitable candidates for modification at this first analysis due to their physiologic ranges, and they are discussed
below. transcription and translation of PhoB and none on PhoA (Fig. S7). On the other hand, the dissociation rate of
the inactive PhoR from the active PhoB, given by r8r, affects the activation of PhoB as a transcription factor in a
proportional way, yet its effect on promoter activity is negligible (Fig. S8). As it can be seen from Table 1, there are
also other parameters that have an effect on both system dynamics and PhoB activity. Nonetheless, these are not
suitable candidates for modification at this first analysis due to their physiologic ranges, and they are discussed
below. As it can be observed in Fig. 2, and supported by the analysis above, the simulations that include the PhoB
data for parametrisation result in faster response dynamics, measured in terms of the time required to reach a
peak state. This can be explained by the self-feeding role of the TCS, and the resulting increased requirement for
the active transcription factors to sustain the experimentally observed protein levels due to their feedback to the
network: because PhoR and PhoB are encoded by the same operon, not only PhoB levels, but also PhoR levels
increase as a result of the changes in the reaction rates. This causes the cell to increase the sensor histidine kinase
levels, resulting in a more immediate response. The response time thus decreases with an increase in the amount
of sensor histidine kinase as well as an increase in its activity.h y
The deterministic ODE simulations lead to observations that describe the average dynamic behaviour of the
variable concentrations for the simulated 4.5 hours. To observe the possible fluctuations in the system, we have
performed stochastic simulations. This way, we have been able to compare the mean behaviour with the regula-
tory dynamics that incorporates the noise due to smaller molecular numbers. Results
Ph
h The cumulative output of different simulations with fold
changes that cover the physiological intervals for r14 and r15 as well as a broad range for r3r, r5r, r7r, and r8r are
depicted in Figs S3, S4, S5, S6, S7 and S8, respectively.f In these simulations, the dissociation rates of PhoR and PhoB, that is, r3r and r5r, have transient effects on
the steady state concentrations of the complexes formed by these molecules. These rates do not modify the
PhoB activity or the levels of PhoA and PhoB as shown in Figs S5 and S6. Variations in the dissociation rate of
PhoB, given by r7r, affect the activity of PhoB as a transcription factor, however this has a minor effect on the Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 4 www.nature.com/scientificreports/ Reaction Number
Rate Symbol
Fit Value
Literature Value
References
1
r1
25.3658
10–100 s−1
5
1 reverse
r1r
8.1165
−
s
10
1
5
2
r2
25.3658
10–100 s−1
5
2 reverse
r2r
8.1165
−
s
10
1
5
3
r3
100
100 μM−1 s−1
48
3 reverse
r3r
44.9411
N.A. N.A. 4
r4
21.3718
17–23 s−1
5
5
r5
100
100 μM−1 s−1
48
5 reverse
r5r
94.9411
N.A. N.A. 6
r6
21.3718
17–23 s−1
5
7
r7
100
100 μM−1 s−1
48
7 reverse
r7r
24.9411
N.A. N.A. 8
r8
100
100 μM−1 s−1
48
8 reverse
r8r
34.9411
N.A. N.A. 9
r9
12.95
−
s
17
1
5
10
r10
10000
10000 μM−1 s−1
6
10 reverse
r10r
1000
N.A. 6
11
r11
10000
10000 μM−1 s−1
6
11 reverse
11r
1000
N.A. 6
12
r12
0.0540
0.0025–0.2 s−1
47,49
13
r13
0.0302
0.0006–0.05 s−1
47,50
14
r14
0.130
0.0025–0.2 s−1
47,49
15
r15
0.035
0.0006–0.05 s−1
47,50
16
r16
0.0302
0.0006–0.05 s−1
47,50
17
r17
0.0001
0.000096–0.00079 s−1
47,49,50
18
r18
0.0001
0.000096–0.00079 s−1
47,49,50
19
r19
0.0001
0.000096–0.00079 s−1
47,49,50
20
r20
0.0055
0.0055 s−1
47
21
r21
0.0055
0.0055 s−1
47
Table 1. Reactions and deterministic rates obtained from the physiological ranges. Table 1. Reactions and deterministic rates obtained from the physiological ranges. transcription and translation of PhoB and none on PhoA (Fig. S7). Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w www.nature.com/scientificreports/ Figure 3. The variations in the mRNA and active promoter levels due to the unbinding rate of the promoter and
the active transcription factor. An unbinding rate of 100/sec (A) results in much less spread in the steady state
distributions in comparison to unbinding rates of 1000/sec (B) and 5000/sec (C). The variations are quantified as
the ratio of the standard deviation and the mean. Figure 3. The variations in the mRNA and active promoter levels due to the unbinding rate of the promoter and
the active transcription factor. An unbinding rate of 100/sec (A) results in much less spread in the steady state
distributions in comparison to unbinding rates of 1000/sec (B) and 5000/sec (C). The variations are quantified as
the ratio of the standard deviation and the mean. To this end, we have experimented in stochastic simulations with different DNA unbinding rates, that is, r10r
and r11r, from 100/s up to 5000/s. In accordance with the common practice, we have used stochasticity to quan-
tify the noise that arises from the binding of a regulatory protein to a promoter21. The resulting amplification in
oscillations in stochastic simulations, shown in Fig. 3 as well as Figs S9, S10 and S11, are due to the increase in the
promoter unbinding rates. In Fig. 3, we have quantified the decrease in noise in the steady state distribution of
mRNA and active promoter levels in terms of the ratio of the standard deviation over the mean. In this respect,
the deterministic simulations display how DNA binding rates affect the mean behaviour, while the stochastic sim-
ulations bring about the loss of coherence due to noise in gene expression. Figure 3B displays the simulations with
the rates of the control model. In Fig. 3A, DNA unbinding rate is decreased by an order of magnitude, whereas in
Fig. 3C, the effect of a higher DNA unbinding rate of 5000/s is depicted. These results indicate that lower unbind-
ing rates, as observed in saturation conditions, are required for stable gene regulation that is not affected by noise. This also reflects how both genetics and noise due to environmental factors can affect the development of targeted
pathway interventions for faster Pi accumulation.hf The results above show that our control model provides detailed predictions about the complex effects of pro-
duction pathways of the Pi accumulation system. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w Results
Ph
h Figure 2 as well as Figure S2 include
a comparison of the deterministic and the stochastic simulations. The stochastic simulations are performed by
applying the standard conversion to obtain molecule numbers from the concentrations. The grey fluctuating lines
show the stochastic results at a single representative simulation; the simulation shows the expected fluctuations in
the model species with smaller numbers such as mRNA molecules as well as the qualitative agreement between
the stochastic and the deterministic simulations. Binding and unbinding of transcription regulators are a primary mechanism for gene regulation, whereby
transcription factors operate at a fast time-scale. While the rate of binding of transcription regulators are known
in many cells, little is known about how cells can modulate their unbinding for regulation20. The unbinding rate
of an active transcription factor can thus vary over many orders of magnitude6,20. Therefore, in the initial analysis,
we have fixed the transcription factor unbinding rates (r10r, r11r) to 1000 s−1 as shown in Table 1, and analysed
the system behaviour with respect to variations. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ The simulations are in good agreement with the experimental
data and the general concepts described in the literature for the functionality of Pho regulon2,3. The control model
proposed can thus serve as a virtual lab, which can be used to test and justify the theoretical approaches on the Pi
intake system. PhoR tunes the Pi intake both up and down. Besides the chemical properties of the proteins in the reg-
ulatory system4, also the activity of the TCS proteins influence the Pi intake. Moreover, as displayed in Fig. 2C, the
feedback mechanism due to the increased expression of sensor histidine kinase PhoR and the response regulator
PhoB introduces a speed-up of an hour in comparison to the simulations, where this feedback mechanism is not
taken into consideration. This indicates that a faster response in terms of Pi intake is delivered by an increase in
the histidine kinase levels as well as the increase of its activity due to signalling. A notable feature of the Pi response system is that the sensor histidine kinase is bifunctional: it participates in
both phosphorylation and dephosphorylation of its cognate response regulator. In this respect, the TCS autoreg-
ulatory design is a distinct mechanism from the conventional positive feedback loops. The bifunctional PhoR
component is an autokinase with concomitant opposing phosphatase activity22–24.h The dual role of PhoR is a mechanism that enhances signal robustness18,25. Moreover, it has also been shown
that the phosphatase activity in TCS provides a rapid dephosphorylation mechanism that shuts off the system,
and thereby restores it to the original state19. Such a dynamics can be triggered, for example, by an increase in the
external Pi concentration and the consequent decrease in the autophosphorylation activity of PhoR. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 6 www.nature.com/scientificreports/ We have analysed the effect of the changes in autophosphorylation rates to the system behaviour. Because
autophosphrylation becomes possible when the starvation signal prevents PhoU from inhibiting PhoR, the pro-
pensity of autophosphorylation depends on the incoming signal, which is a function of the external Pi levels. By decreasing the autophosphorylation propensity by applying various fold changes, we can thus see the effect
of an increase in external Pi concentration on the system as depicted in Figs 2D and S12. www.nature.com/scientificreports/ In these simulations,
a decrease in PhoR activity due to increased external Pi concentration results in a proportional decrease in the
active PhoB dimers, and a decrease in the promoter activity as well as the PhoR activity. p
y
y
A complementary realisation of this mechanism is given by the association of PhoB to PhoR, that is, r8. Although the physiological range for this parameter is narrow, as a result of a hypothetical increase in the asso-
ciation rate of PhoB and PhoR, the PhoB concentration stays low for a longer time period and the levels of active
PhoB dimers decrease proportionally as displayed in the fold change experiments in Fig. S13. p
p
y
p y
g
p
g
In our model, we assume that the growth in the cell culture within the considered time interval is negligible. However, due to cell cycle, which has a time scale in the order of an hour, the protein concentrations can be
subject to dilution besides the active degradation of the molecules we have considered. To this end, Figs S14, S15
and S16 explore the effect of higher degradation rates due to dilution in growth conditions, (r17, r18, r19, r20,
and r21), together with higher external Pi levels, given with a decrease in the rates r1 and r2. As it can be seen in
Fig. S12, a decrease in the autophosphorylation rates does not only lower the steady state levels, but also slows
down the activation of the transcription factor by preventing the formation of an initial peak in DiPhoBpp levels. A concomitant increase in the degradation and dilution rates delays reaching a steady state. However, this does
not drastically alter the eventual active DiPhoBpp concentrations. Starvation response can be obtained with synthetic promoters at higher external Pi concen-
trations. The simulation results in Figs 2D and S12 demonstrate the system’s adaptation to the stimuli due to
Pi concentration, whereby the autophosphorylation propensity of PhoR acts as a proxy for the external Pi levels. These simulations predict how changes in the external Pi concentration affect the Pho regulon, and in particular,
how the promoter activity decreases with an increase in the external Pi concentration. These results thus confirm
that the adaptation of gene expression is clearly dependent on the Pi response stimuli2,12. www.nature.com/scientificreports/ Accumulation of
ppGpp triggers the stringent response and a radical decrease in ribosome and protein synthesis, even leading to
growth arrest31. When the level of RpoS is higher, E. coli is more resistant to stress but grows more slowly under
a variety of conditions32,33.f We have further assessed our analysis to factor for the possible effects of stress response in Pi starvation con-
ditions. As depicted in Fig. S17, Pi limitation induces the general stress response regulated by RpoS, sigma fac-
tor29,30. Concomitantly, Pi limitation increases the intracellular level of guanosine tetraphosphate (ppGpp), which
is known as stringent stress response12,30,31. RpoS and ppGpp are thought to maintain a balance between vegetative
d
/
i
f E
li Th
G
l
id i d
R
S
l i
32 Th R
S
d and stress/starvation states of E. coli. The ppGpp nucleotide induces RpoS accumulation32. The RpoS responds to
stress and shifts transcription away from vegetative growth and towards stress resistance31–34. Accumulation of
ppGpp triggers the stringent response and a radical decrease in ribosome and protein synthesis, even leading to
growth arrest31. When the level of RpoS is higher, E. coli is more resistant to stress but grows more slowly under
a variety of conditions32,33.f y
It is known that RpoS and ppGpp direct RNA polymerase to promoters, and negatively affect the expression
of several PHO genes with varying effects18,35,36. We have assessed the effect of the RpoS and SpoT activity on
the system by applying sensitivity analysis on the transcription rate of PhoB, as depicted in Fig. S3 for r14. In the
light of this analysis, we have investigated the effect of stress response on the results depicted in Fig. 4, whereby
the model is calibrated under varying stress response conditions due to Pi starvation. By repeating the analysis
in Fig. 4 under these conditions, we have tested the activity of various promoter designs (Fig. S18). These results
confirm the previous observations, and indicate that stress response can have a modulating effect, which can
however be factored for by promoter design.h y p
g
These results indicate that the steady state promoter activity and the PhoA yield are highly correlated in all the
regimes and for all the promoter binding and unbinding rates. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w www.nature.com/scientificreports/ Moreover, the model
provides a mechanistic explanation for the interplay between the system components under the conditions of
varying external Pi concentrations, which result in variations in the promoter activity.li It is well established that the protein production rate is greatly influenced by the specific nucleotide sequence
of the promoter26–28. In this respect, synthetic biology and genetic engineering methods aim at synthesising pro-
moters with the desired strength. To this end, in order to observe the possible variations in gene expression due
to variations in promoter strength, we have performed a class of simulations. The results of these simulations in
Fig. 4 display measurements of the steady state levels of PhoA promoter activity (pPhoAa) as well as the PhoA
yield of the system as the resulting product in terms of the area under the curve (PhoA AUC). In these simulations, we have scanned regimes with varying external Pi concentrations simulated by applying
fold changes to the rates r1 and r2 as in the simulations in Figs 2D and S12. We have applied a fold change of 1 for
the control regime with 0 μM external Pi, and 0.5, 0.2 and 0.1 for increasing external Pi levels. For each external Pi
regime, we have scanned 100 different promoter designs by means of simulations that apply 10 fold change values
from 0 to 2.5 with steps of 0.25 to the promoter binding rates r10 and r11 as well as 10 such fold change values
to the promoter unbinding rates r10r and r11r. The heatmaps resulting from these 100 simulations are depicted
in Fig. 4, where the control values for 1 fold change for binding and unbinding rates are marked by dashed lines.f g
g
g
g
y
We have further assessed our analysis to factor for the possible effects of stress response in Pi starvation con-
ditions. As depicted in Fig. S17, Pi limitation induces the general stress response regulated by RpoS, sigma fac-
tor29,30. Concomitantly, Pi limitation increases the intracellular level of guanosine tetraphosphate (ppGpp), which
is known as stringent stress response12,30,31. RpoS and ppGpp are thought to maintain a balance between vegetative
and stress/starvation states of E. coli. The ppGpp nucleotide induces RpoS accumulation32. The RpoS responds to
stress and shifts transcription away from vegetative growth and towards stress resistance31–34. www.nature.com/scientificreports/ As expected, when the control system’s output in
Pi starvation condition is compared with the output in regimes with increased external Pi, we observe a decrease
in PhoA yield. Moreover, these results predict that in order to obtain the starvation response in the conditions
with higher external Pi concentration, promoter binding rates need to be increased and unbinding decreased. Promoters that provide the required strengths can be obtained by modifying the nucleotide sequences, for exam-
ple, as in28,37,38. p
We have tested the predictions of the model on experimental data obtained with the synthetic promoter under
starvation conditions with 0 μM external Pi concentration. Moreover, we have used the model to explore the
effect of such synthetic promoters under various external Pi concentration conditions. The experimental data and 7 www.nature.com/scientificreports/ Figure 4. Heatmaps for the activity of various promoter designs for pPhoA and pPhoB, and the resulting
PhoA expression (bottom-row) under different external Pi concentration conditions. The heatmaps are ordered
decreasingly from left to right according to the external Pi concentration given by the fold changes applied
to the PhoR autphosphorylation reactions r1 and r2. The left most column with 1 as the fold change value is
the starvation condition with 0 μM external Pi. Each heatmap scans 100 simulations by applying 10 different
fold change values to the promoter binding rates r10 and r11 as well as 10 different fold change values to the
promoter unbinding rates r10r and r11r. The upper row displays the resulting steady state levels of the active
promoter pPhoAa, whereas the lower row displays the yield of PhoA gene expression measured as the area
under the curve (AUC). The intersection of the dashed lines in the left column delivers the experimentally
observed regime observed in Fig. 2. The levels of this regime, that display the starvation response, are
highlighted in all the heatmaps. Figure 4. Heatmaps for the activity of various promoter designs for pPhoA and pPhoB, and the resulting
PhoA expression (bottom-row) under different external Pi concentration conditions. The heatmaps are ordered
decreasingly from left to right according to the external Pi concentration given by the fold changes applied
to the PhoR autphosphorylation reactions r1 and r2. The left most column with 1 as the fold change value is
the starvation condition with 0 μM external Pi. www.nature.com/scientificreports/ Each heatmap scans 100 simulations by applying 10 different
fold change values to the promoter binding rates r10 and r11 as well as 10 different fold change values to the
promoter unbinding rates r10r and r11r. The upper row displays the resulting steady state levels of the active
promoter pPhoAa, whereas the lower row displays the yield of PhoA gene expression measured as the area
under the curve (AUC). The intersection of the dashed lines in the left column delivers the experimentally
observed regime observed in Fig. 2. The levels of this regime, that display the starvation response, are
highlighted in all the heatmaps. Figure 5. Comparison of the experimental data on PhoA expression with the synthetic promoter together with
the simulation results with varying external Pi concentrations and promoter parameters that model synthetic
designs. The experimental data on the starvation response with the synthetic promoter is represented as hollow
circles. As in Fig. 4, the plots are ordered decreasingly from left to right according to the external Pi concentration
given by the fold changes applied to the PhoR autphosphorylation reactions r1 and r2. The left most column with
1 as the fold change value is the starvation condition with 0 μM external Pi. Each plot displays four simulations
with varying fold change values applied to promoter binding and unbinding rates that model various promoter
designs. A modified promoter (blue curve) can reproduce the starvation response in low as well as high external Pi
concentration, and reproduce the experimental data under the synthetic promoter starvation conditions. Figure 5. Comparison of the experimental data on PhoA expression with the synthetic promoter together with
the simulation results with varying external Pi concentrations and promoter parameters that model synthetic
designs. The experimental data on the starvation response with the synthetic promoter is represented as hollow
circles. As in Fig. 4, the plots are ordered decreasingly from left to right according to the external Pi concentration
given by the fold changes applied to the PhoR autphosphorylation reactions r1 and r2. The left most column with
1 as the fold change value is the starvation condition with 0 μM external Pi. Each plot displays four simulations
with varying fold change values applied to promoter binding and unbinding rates that model various promoter
designs. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. Heatmap displaying the results of the sensitivity analysis by considering the physiological interval in
Table 1. For each parameter, the maximum and minimum values within its physiological range are considered
for simulation, and the area under the curve (AUC) for each species is computed. The difference of the AUC for
the maximum and minimum parameter values are then normalised with the AUC of the control model. Red
represents the decreasing effect and green represents the increasing effect. Figure 6. Heatmap displaying the results of the sensitivity analysis by considering the physiological interval in
Table 1. For each parameter, the maximum and minimum values within its physiological range are considered
for simulation, and the area under the curve (AUC) for each species is computed. The difference of the AUC for
the maximum and minimum parameter values are then normalised with the AUC of the control model. Red
represents the decreasing effect and green represents the increasing effect. the simulation results with our model are depicted in Fig. 5. The selected synthetic promoter verifies the model
predictions as it has a similar behaviour as the control model for the pPhoA promoter in starvation conditions. Moreover, the model further predicts the synthetic promoters with increased strength, given with higher bind-
ing rates and lower unbinding rates, deliver responses similar to the starvation response, also in the presence of
higher external Pi concentrations. Within a modular framework, the simulation results mechanistically quantify
how changes in the genetic components affect the behaviour of the circuit. the simulation results with our model are depicted in Fig. 5. The selected synthetic promoter verifies the model
predictions as it has a similar behaviour as the control model for the pPhoA promoter in starvation conditions. Moreover, the model further predicts the synthetic promoters with increased strength, given with higher bind-
ing rates and lower unbinding rates, deliver responses similar to the starvation response, also in the presence of
higher external Pi concentrations. Within a modular framework, the simulation results mechanistically quantify
how changes in the genetic components affect the behaviour of the circuit. Sensitivity Analysis. To assess the sensitivity of our model to the parameters, we have performed a two-step
analysis. In the first step, we have considered the physiological interval of the parameters given in Table 1. www.nature.com/scientificreports/ For this
analysis, we have only included the rate parameters that have been taken from literature and have been estimated
within a given range. For each parameter, we have run simulations by instantiating the model with the maximum
and minimum values of its physiological range, and, for each species, we have computed the yield of the system
in terms of the area under the curve (AUC). Figure 6 displays the results obtained by taking the difference of the
AUC for the maximum and minimum parameter values, normalised with the AUC of the control model. The
resulting heatmap quantifies the impact of each parameter on the system dynamics with respect to plausible var-
iations within its physiological range. The results demonstrate that the changes in the translation parameters are
more pronounced than in the others. Moreover, in accordance with the results above, the autophosphorylation
rate of PhoR impacts the active transcription factor levels and the transient species that lead to it. p
p
p
In the second step, we have performed a sweeping analysis by considering all the model reactions and spe-
cies within a range of 3 orders of magnitude both up and down. That is, each reaction rate is multiplied with a
fold-change factor within a spectrum of 6 orders of magnitude, that is, from 10−3 to 103. We have then computed
the AUC for each species and normalised the outcome with the AUC of the control model. The heatmap depicted
in Fig. S19 quantifies the impact of these changes and predicts the system behaviour under hypothetical condi-
tions simulated by such variations in parameters. www.nature.com/scientificreports/ A modified promoter (blue curve) can reproduce the starvation response in low as well as high external Pi
concentration, and reproduce the experimental data under the synthetic promoter starvation conditions. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 8 Discussion We have
presented a recipe for the design process of such an application, which integrates gene expression data from E. coli into a computer model. p
Our model, which includes 29 reactions that describe the dynamic behaviour of the key regulatory network
components, provides a quantification of the phosphate starvation response by means of both deterministic and
stochastic simulations. The deterministic simulations allowed us to estimate the missing parameters of the model
and analyse the mean behaviour of the system dynamics. In particular, we have exposed the phosphorylation
cycle in TCS signalling and its role in the positive feedback mechanism in determining the network yield in
terms of PhoA. Moreover, the steady state properties of the system could be displayed by sensitivity analysis on
deterministic simulations.f In accordance with the common practice, we have employed stochastic simulations to highlight the effect of
noise in the system39. As expected, the stochastic simulations are consistent with the deterministic simulations,
which display the mean behaviour. Stochastic simulations, on the other hand, capture the noise due to small
species numbers and concomitant fast and slow reactions. As a result of this, stochastic simulations with our
model display the fluctuations observed in experimental observations, thereby exposing the fluctuations due to
binding and unbinding of the transcription factors with the promoters, which operate at a much faster time scale
in comparison to the preceding signalling cascade40–42. In these simulations, higher unbinding rates result in
greater fluctuations that correlate with the decrease in binding saturation, a requirement for a robust signal. While
confirming the notion that the complex networks of interacting molecules within cells should be robust43,44, these
results highlight the additional role of the transcription factor unbinding rate in tuning the protein synthesis. Our
stochastic simulation results, together with the experimental data on PhoB concentration in wild-type, suggest
that the noise at the level of promoters controls the phenotypic variability in the mean behaviour. Large levels of
noise at the level of PhoA and PhoB promoters are results of DNA unbinding rates. Moreover, our model shows
that the smaller DNA unbinding rates also reduce the response time.i g
p
Our results confirm that the dynamics of the TCS and its responsiveness to both genetic and environmental
perturbations play a key role in tuning the E. coli Pi response. Discussion We have presented a computational model and its experimental validation for quantifying dynamic mechanisms
of auto-regulation in Escherichia coli in response to external Pi levels. Our model provides a mechanistic expla-
nation of the interplay between transcriptional regulatory network, given by a two-component system (TCS)
and Pho regulon, and promoter efficiency under variations in external Pi concentrations. Being parameterised
within the physiological ranges of its components, the output of the model in terms of gene regulation delivers
the expected system dynamics. The results provided by the model are in good agreement with the theory and
the general concepts described in the literature for the functionality of Pho regulon2,3. The model provides pre-
dictions for the complex effects of TCS activity and consequent dynamics, including synthetic promoters with
varying affinities to their transcription factor. A direct validation of the predictions is provided by the good fit of
the experimental data. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 9 www.nature.com/scientificreports/ With these insights, this work offers a mechanistic understanding of inorganic phosphate intake, realised
by Pho regulon in a way that connects signalling with the genetic level. It provides a quantitative descrip-
tion of how different proteins interact to form a biological control system. It also describes the control of the
phosphate-starvation response at the genetic level. Our work provides measurements of protein, phosphoryla-
tion, and promoter activity levels that are fundamental to define features of TCS circuits. One of the major out-
comes is that our results do not only explain the observed experimental data, but also provide predictions on the
physiology of the Pho regulon and insights for the synthetic promoter design. Moreover, our work has implica-
tions for applications in artificial life and for others in biotechnology that exploit such mechanisms. As the model
architecture is intrinsically open to integrate supplementary layers, together with experimental findings, it should
provide insights in investigations on engineering new dynamic sensors and regulators for living technologies. p
g
g
g
g
y
g
g
g
Our model describes the responsive structure of the TCS system, which can be used to synthesize bacteria
that detect changes in their environment and respond by modulating the synthesis and intake. Applications of
such an engineering approach for bacteria should provide a basis for a new generation of bio-materials. Discussion In this respect, with parameter values obtained by
fitting the experimental data on PhoA and PhoB promoters, we have investigated and identified the response
dynamics of individual system species. Moreover, we have analysed synthetic promoter affinities that reproduce
the wild type response to Pi starvation. For this purpose, we have tested and verified the model with experimental
data for the response regulator binding affinity to infer the functional relation between the fraction of bound
response regulator and the transcriptional activity. We have then quantified the effect of each protein in the path-
way, through sensitivity analysis, singling out the main regulator mechanisms of the TCS. Our results indicate the extent to which synthetic promoters can be tuned for various TCS detection thresh-
olds such that the organism adapts to various environmental conditions. In this regard, because our model
includes mechanisms for quantifying the information E. coli has about its phosphate environment, the model
can be used to perform further experiments on the Pi intake system. Moreover, it can be used to explore and test
various promoter designs, for example, in biotechnology applications such as sensors for wastewater treatment or
detecting environmental pollutants to relocate towards them. g
p
In conclusion, our study reveals how the tight interplay between theory and wet-lab experiments greatly helps
to improve our understanding of bacterial sensing and signalling pathways. In particular, the TCS component
of our model, which relays the signal on environmental changes to the genetic components for tuning protein
expression, belongs to one of the largest and most diverse families of sensory components in biology. In this
respect, the TCS features examined in this work provide a template for the models of similar systems that regulate
the response for various external signals. An improved and quantitative understanding of such systems by formal
mechanistic models will likely contribute to our understanding of the engineering of biosensors for diverse syn-
thetic biology applications. www.nature.com/scientificreports/ www.nature.com/scientificreports/ When the external Pi concentration is limited, PhoR is free to bind ATP. This allows PhoR to autophosphoryl-
ate itself. PhoR is stable as a dimer, which is denoted by DiPhoR. Therefore, it is doubly phosphorylated. ↔
DiPhoR
DiPhoRp
(1)
↔
iPhoRp
DiPhoRpp
(2) (1) ↔
DiPhoR
DiPhoRp ↔
DiPhoRp
DiPhoRpp (2) PhoR is essential for the control of the activity of PhoB2,8,45. It phosphorylates PhoB through an autokinase/
phosphotransferase activity2,8. After autophosphorylating, PhoR relays the signal to the transcription factor PhoB. The bidirectional reactions 3 and 5 below model the association of phosphorylated PhoR dimer and PhoB, and
the unidirectional reactions 4 and 6 model the phosphotransferase. PhoB has been reported to exist primarily as
monomers and phosphorylation greatly enhances dimerisation of PhoB (DiPhoBpp), modelled by reaction 710. +
↔
−
DiPhoRpp
PhoB
DiPhoRpp
PhoB
(3)
−
→
+
DiPhoRpp PhoB
DiPhoRp
PhoBp
(4)
+
↔
−
DiPhoRp
PhoB
DiPhoRp
PhoB
(5)
−
→
+
DiPhoRp
PhoB
DiPhoR
PhoBp
(6)
+
↔
PhoBp
PhoBp
DiPhoBpp
(7) +
↔
−
DiPhoRpp
PhoB
DiPhoRpp
PhoB
(3)
−
→
+
DiPhoRpp PhoB
DiPhoRp
PhoBp
(4)
+
↔
−
DiPhoRp
PhoB
DiPhoRp
PhoB
(5)
−
→
+
DiPhoRp
PhoB
DiPhoR
PhoBp
(6)
+
↔
PhoBp
PhoBp
DiPhoBpp
(7) +
↔
−
DiPhoRpp
PhoB
DiPhoRpp
PhoB
(3) +
↔
−
DiPhoRpp
PhoB
DiPhoRpp
PhoB (3) (6) +
↔
PhoBp
PhoBp
DiPhoBpp +
↔
PhoBp
PhoBp
DiPhoBpp
(7) (7) In E. coli, the sensor histidine kinase PhoR is a bifunctional enzyme that paradoxically performs two opposed
tasks: in one direction, it catalyzes the phosphorylation of response regulator PhoB, and in the other, it also per-
forms the dephosphorylation of phosphorylated PhoB, which is PhoBp18,25. The association of PhoR dimers with
phosphorylated PhoB is modelled by the bidirectional reaction 8, whereas the phosphatase activity is given by
reaction 9. +
↔
−
DiPhoR
PhoBp
DiPhoR
PhoBp
(8)
−
→
+
DiPhoR
PhoBp
DiPhoR
PhoB
(9) +
↔
−
DiPhoR
PhoBp
DiPhoR
PhoBp
(8) +
↔
−
DiPhoR
PhoBp
DiPhoR
PhoBp (8) −
→
+
DiPhoR
PhoBp
DiPhoR
PhoB
(9) −
→
+
DiPhoR
PhoBp
DiPhoR
PhoB (9) Phosphorylated dimer structure PhoB (DiPhoBpp) is enabled for activating Pho regulon by binding to a con-
sensus promoter region. PhoB and PhoR in Pho regulon are encoded by the same operon, that is, the phoBR
operon. Methodsh The model is based on a mechanistic description of the system dynamics within a chemical reaction network rep-
resentation with respect to mass action kinetics. To build the simulation model, we have selected the part of the
system from the TCS to gene regulation, and used the promoter activity as well as the levels of active transcription
factor as indicators for quantifying the response to external Pi levels. q
y
g
In the following, to better illustrate the response of Pho regulon and TCS signalling to the Pi starvation,
we describe the network in terms of the interactions of system components as a chemical reaction network. Figure 2B provides an alternative representation of the regulatory system interactions given by the chemical
reaction network. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 10 Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w →∅
mRNAb →∅
mRNAb We have implemented the deterministic ordinary differential equation (ODE) model in Matlab by using the
standard translation from the chemical reaction network above, based on stoichiometries. The ODEs and detailed
descriptions of each system variable are listed in Fig. S1 and Table S1. We have also implemented a version of
the model for stochastic simulation, again based on mass action kinetics. The stochastic simulations capture
fluctuations due to small molecule numbers, which are not captured by the deterministic simulations. By using
the standard conversion factors for mass action kinetics, we could use the same rates for the deterministic and
stochastic simulations.h The initial concentrations of the model variables have been derived from the literature or obtained from our
experiments, described below. The control model has been calibrated for an initial culture containing 0 μM exter-
nal Pi. Prior to Pi starvation, the concentrations of proteins PhoR and PhoB are approximately 0.22 μM. The
concentrations of active PhoR and active PhoB are
μ
⋅
−
M
4
10 8
and
μ
⋅
−
M
6
10 8
, as determined by Keasling
et al.3. With a single plasmid, average mRNA number is 2–3 in E. coli47. Therefore, the initial states of mRNAa and
mRNAb are set to 0.00166 μM by taking E. coli volume as 1 μm3, and the initial promoters numbers are set to 10
for each. We assume that the ATP concentration stays constant throughout the considered time intervals.h The rates of chemical reactions are obtained in accordance with the variability of physiological ranges given in
the literature5,6,47–50. The model includes 29 reactions, including the reverse reactions. The control model has been
used to reproduce the data and the unknown parameters have been estimated by least square inference within
the plausible physiological ranges. When possible, parameter values are fixed or estimated by using experimental
measurements found in the literature. The parameter values taken from the literature and their physiological
ranges for the rates, if applicable, are listed in Table 1. The parameters without a range are fitted to the experimen-
tal data by using the deterministic model to reproduce the response curves. The parameter estimation procedure
has been carried out by using a multi-start approach. www.nature.com/scientificreports/ Thus, the synthesis of the regulatory proteins PhoB and PhoR is under Pho regulon control2,8,46. h
Based on experimental data, we consider the PhoA and PhoB promoters (pPhoA, pPhoB), whereby the PhoB
promoter provides feedback to the system as this results in the expression of both PhoB and PhoR. PhoR expres-
sion during Pi limitation is dependent on the upstream pPhoB; the operon structure indicates that PhoR gene
function requires expression from the pPhoB46. For this, phosphorylated PhoB dimers (DiPhoBpp) bind to the
promoter as active transcription factors. +
↔
DiPhoBpp
pPhoA
pPhoAa
(10)
+
↔
DiPhoBpp
pPhoB
pPhoBa
(11) +
↔
DiPhoBpp
pPhoA
pPhoAa
(10)
+
↔
DiPhoBpp
pPhoB
pPhoBa
(11) +
↔
DiPhoBpp
pPhoA
pPhoAa
(10) (10) +
↔
DiPhoBpp
pPhoB
pPhoBa +
↔
DiPhoBpp
pPhoB
pPhoBa
(11) (11) Active promoters pPhoAa and pPhoBa lead to the transcription of mRNA, which carry the information for
the subsequent translation, resulting in protein synthesis, which are PhoA, PhoB and DiPhoR2,8. →
+
pPhoAa
pPhoAa
mRNAa
(12)
→
+
mRNAa
PhoA
mRNAa
(13)
→
+
pPhoBa
pPhoBa
mRNAb
(14)
→
+
mRNAb
PhoB
mRNAb
(15)
→
+
mRNAb
DiPhoR
mRNAb
(16)
With the inclusion of the degradation/dilution terms, we obtain:
→∅
PhoA
(17)
→∅
PhoB
(18)
→∅
DiPhoR
(19) →
+
pPhoAa
pPhoAa
mRNAa
(12)
→
+
mRNAa
PhoA
mRNAa
(13)
→
+
pPhoBa
pPhoBa
mRNAb
(14)
→
+
mRNAb
PhoB
mRNAb
(15)
→
+
mRNAb
DiPhoR
mRNAb
(16)
dation/dilution terms, we obtain:
→∅
PhoA
(17)
→∅
PhoB
(18)
→∅
DiPhoR
(19) →
+
pPhoAa
pPhoAa
mRNAa →
+
mRNAb
DiPhoR
mRNAb With the inclusion of the degradation/dilution terms, we obtain: Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 11 www.nature.com/scientificreports/ (20) →∅
mRNAa →∅
mRNAa (21) →∅
mRNAb The rate values have been selected with respect to the best fit
to the physiological ranges, also listed in Table 1, and the dynamics in accordance with the experimental findings
in order to avoid discontinuities or states with unrealistic values. Th d
f
h
h
d
h
h
b
b
d
l fi d
f
l The data for the PhoA and PhoB expression have been obtained using PCR amplified DNA from E. coli
MG1655 genome and transcriptionally fused to the translational coupler BCD251 and the fluorescent ms-fgfp
gene. Subsequently the PphoA-BCD2-msfgpf and PphoB-BCD2-msfgfp fragments were cloned using the PacI/
HindIII restrictions sites in pSEVA234 plasmid (http://seva.cnb.csic.es/), generating the pSEVA237PphoA and
pSEVA237PphoB vectors as depicted in Fig. 2A.hi The synthetic promoter Pliar was obtained as in52,53 with modifications. Activities of the PphoA and PphoB
promoters have been determined as follows. Cells were maintained on LB rich medium. For Pi assays, an over-
night preculture of E. coli DH10B carrying pSEVA237PphoA, pSEVA237PphoB or pSEVA237PLiar00117 plas-
mids harbouring PhoA, PhoB or PLiar00117 promoters driving the expression of MsfGFP, respectively were
grown in 10 ml MOPS medium (pH = 7.2), supplemented with 0.02% casamino acids and 5 mM KH2PO4 at 37 °C
under constant shaking at 250 rpm. The activity of PhoB and PhoA promoters were analysed in resting cells, both
in presence or absence of 50 μg/ml antibiotic kanamycin, which did not have any impact on the promoter activity
as it can be seen in the Supplementary Figs S2 and S20. The bacterial precultures were used to inoculate 50 ml of
the same medium reaching an OD600 of 0.1 and grown until mild-late exponential phase (OD600 of 0.6–0.9). At
this point, the bacterial cells were pelleted at 1500 × g for 10 min at room temperature, and finally washed twice
in MOPS medium without
−
PO4
3 . Subsequently, the cells were suspended in 250 μl of MOPS to reach a final
OD600 of 2 with increasing concentrations of
−
PO4
3 (from 0 to 50 mM). The bacterial cell suspensions were
loaded in 96-well plates and the expression of MsfGFP (fluorescence) was measured at different times in a
SpectraMax i3x (Molecular Device) at 30 °C. The excitation wavelength was set to 485 nm and the fluorescence
emission was measured at different times at 525 nm. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w www.nature.com/scientificreports/ Biological insights from structures of two-component proteins. Annu Rev Microbiol. 63 (2009).h 46. Warner, B. & Chang, B. The phoBR operon in Escherichia coli K-12. Bacteriol 169, 5569–5574 (1987). h
47. OpenWetWare. Parameter estimation in E. coli. Available at http://www.openwetware.org/wiki/Computational_Biology/Gene_
Expression_modeling (2007). g
48. Dexter, J. & Gunawardena, J. Dimerization and bifunctionality confer robustness to the isocitrate dehydrogenase regulatory system
in Escherichia col. J Biol Chem. 288, 5770–5778 (2013).h J
(
)
49. Thattai, M. & van Oudenaarden, A. Intrinsic noise in gene regulatory networks. PNAS 98, 8614–8619 (2001). h
50. Milo, R., Phillips, R. & Orme, N. Cell Biology by the Numbers (Garland Science, 2015). 51. Mutalik, V. K. et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nature
Methods 10, 354–360 (2013). 51. Mutalik, V. K. et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nature
Methods 10, 354–360 (2013). 52. Uluşeker, C. et al. A dynamic model of the phosphate response system with synthetic promoters in Escherichia coli. Proc. of the
International Conference on Artificial Life (2017). 52. Uluşeker, C. et al. A dynamic model of the phosphate response system with synthetic promoters in Escherichia coli. Proc. of the
International Conference on Artificial Life (2017). f
fi
f (
)
3. Gilman, J. & Love, J. Synthetic promoter design for new microbial chassis. Biochem Soc Trans 44, 731–737 (2016). f
fi
f (
)
53. Gilman, J. & Love, J. Synthetic promoter design for new microbial chassis. Biochem Soc Trans 44, 731–737 (2016). www.nature.com/scientificreports/ www.nature.com/scientificreports/ 12. Lamarche, M., Wanner, B., Crepin, S. & Harel, J. The phosphate regulon and bacterial virulence: a regulatory network conne
phosphate homeostasis and pathogenesis. Federation of European Microbiological Societies 32 (2008). 13. McCleary, W. R. Molecular mechanisms of phosphate homeostasis in Escherichia coli. In Samie, A. (ed.) Escherichia coli, chap
https://doi.org/10.5772/67283 (IntechOpen, Rijeka, 2017). p
g
(
p
j
)
14. Kremlinga, A., Heermannb, R., Centlerc, F., Jungb, A. & Gillesa, E. D. Analysis of two-component signal transduction by
mathematical modeling using the KdpD/KdpE system of Escherichia coli. BioSystems 78, 23–37 (2004). g
g
p
p
y
y
15. Wong, P., Gladney, S., Centlerc, F. & Keasling, J. D. Mathematical model of the lac operon: indu
and diauxic growth on glucose and lactose. Biotechnol. Prog. 13, 132–143 (1997).i 5. Wong, P., Gladney, S., Centlerc, F. & Keasling, J. D. Mathematical model of the lac operon: inducer exclusion, catabolite repression
and diauxic growth on glucose and lactose. Biotechnol. Prog. 13, 132–143 (1997).i g
g
g
16. Fisher, S., Kim, S. K. K., Wanner, B. & Walsh, C. Kinetic comparison of the specifity of the vanomycin resistance kinase VanS for two
response regulators. VanR and PhoB. Biochemistry 35, 4732–4740 (1996). response regulators. VanR and PhoB. Biochemistry 35, 4732–4740 p
g
y
(
)
7. Koh, B., Tan, R. & Yap, M. Genetically structured mathematical modeling of trp attenuator mechanism. Biotechnol. Bioeng. 58
502–509 (1998). 18. Gao, R. & Stock, A. Probing kinase and phosphatase activities of two-component systems in vivo with concentration-depe
phosphorylation profiling. PNAS 110, 672–677 (2012).f p
p
y
pi
g
19. Gao, R. & Stock, A. Quantitative kinetic analyses of shutting off a two-component system. mBio 8, e00412–17 (2017). i
19. Gao, R. & Stock, A. Quantitative kinetic analyses of shutting of f
20. Chen, T. Y. et al. Concentration- and chromosome-organization-dependent regulator unbinding from DNA for trans
regulation in living cells. Nature Communications 6 (2015). 21. Karapetyan, S. & Buchler, N. E. Role of DNA binding sites and slow unbinding kinetics in titration-based oscillators. Physical review
E Statistical, nonlinear, and soft matter physics 92, 062712 (2015). ft
p y
22. Abreu, Rd. S., Penalva, L. O., Marcotte, E. M. & Vogel, C. Two-component signaling circuit structure and properties. Curr Opin
Microbiol 13, 184–189 (2010). 3. Miyashiro, T. & Goulian, M. High stimulus unmasks positive feedback in an autoregulated bacterial signaling circuit. PNAS 105
17457–17462 (2008). 24. References 1. Mosa, K., Saadoun, I., Kumar, K., Helmy, M. & Dhankher, O. Potential biotechnological strategies for the cleanup of heavy metal
and metalloids. Front Plant Sci. 7 (2016). 2. Wanner, B. Phosphorus assimilation and control of the phosphate regulon. In Neidhardt, F. (ed.) Escherichia coli and Salmonella
typhimurium cellular and molecular biology, 1357–1381 (ASM Press, Washington, DC, 1996).h yp
gy
g
3. Van Dien, S. J. & Keasling, J. D. A dynamic model of the Escherichia coli phosphate-starvation response. J. Theor. Biol 190, 37–49
(1997). 4. Gao, R. & Stock, A. Evolutionary tuning of protein expression levels of a positively autoregulated two-component system. PLoS
Genet 9, e1003927 (2013).hfl ,
(
)
5. Bloch, W. & Schlesinger, M. J. The phosphate content of Escherichia coli alkaline phosphatase and its effect on stopped flow kinetic
studies. JBC 248, 5794–5805 (1973). 6. Alon, U. An introduction to system biology: design principles of biological circuits (Chapman and Hall/CRC, 2007). bb
h
f
d
f
h
h
l ,
y
gy
g p
p
f
g
(
p
/ R ,
)
7. Harris, R. M., Webb, D. C., Howitt, S. M. & Cox, G. B. Characterization of PitA and PitB from Escherichia coli. J Bacteriol. 183,
5008–5014 (2001). 8. Wanner, B. L. et al. Are the multiple signal transduction pathways of the Pho regulon due to cross talk or cross regulation? In
Regulation of gene expression in Escherichia coli, 297–315 (R.G. Landes Company, Texas USA, 1995). 9. Chekabab, S. M., Harel, J. & Dozois, C. M. Interplay between genetic regulation of phosphate homeostasis and bacterial virulence. Virulence 5 (2014). 9. Chekabab, S. M., Harel, J. & Dozois, C. M. Interplay between genetic regulation of phosphate homeostasis and bacterial virulence. Virulence 5 (2014). (
)
0. Jansson, M. Phosphate uptake and utilization by bacteria and algae. Kluwer Academic Publishers 170, 177–189 (1988).h p
p
y
g
11. Gardner, S. G., Johns, K. D., Tanner, R. & McCleary, W. The PhoU protein from Escherichia coli interacts with PhoR, PstB, and metals
to form a phosphate-signaling complex at the membrane. J Bacteriol 196, 1741–1752 (2014). y
g
11. Gardner, S. G., Johns, K. D., Tanner, R. & McCleary, W. The PhoU protein from Escherichia coli interacts with PhoR, PstB, and metals
to form a phosphate-signaling complex at the membrane. J Bacteriol 196, 1741–1752 (2014). Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 12 www.nature.com/scientificreports/ Microbiology 57, 1–8 (2005)
4 Wang L et al Divergence involving global regulatory gene mutations in an Escherichia coli population evolving under phosphate 33. Ferenci, B. Maintaining a healthy SPANC balance through regulatory and mutational adaptation. Mol. Microbiology 57, 1–8 (2005). 34. Wang, L. et al. Divergence involving global regulatory gene mutations in an Escherichia coli population evolving under phosphate
limitation. Genome Biol. Evol. 2, 478–487 (2010).ff g
y
g
g
y
p
gy
(
)
4. Wang, L. et al. Divergence involving global regulatory gene mutations in an Escherichia coli population evolving under phosphate
limitation. Genome Biol. Evol. 2, 478–487 (2010).ff 5. Pasternak Taschner, N., Yagil, E. & Spira, B. A differential effect of σS on the expression of the PHO regulon genes of Escherichia coli
Microbiology 150, 2985–2992 (2004).i gy
6. Gao, R., Godfrey, K. A., Sufian, M. A. & Stock, A. Counterbalancing regulation in response memory of a positively autoregulated
two-component system. Journal of Bacteriology 119, e00390–17 (2017). p
y
f
gy
37. De Mey, M., Maertens, J., Lequeux, G. J., Soetaert, W. K. & Vandamme, E. J. Construction and model-based analysis of a promoter
library for E. coli: an indispensable tool for metabolic engineering. BMC Biotechnology 7 (2007).h 8. Li, J. & Zhang, Y. Relationship between promoter sequence and its strength in gene expression. The European Physical Journal E 37
(2014).h 39. Shahrezaei, V. & Swain, P. S. The stochastic nature of biochemical networks. Current Opinion in Biotechnology 19, 369–374 (20 h
, Levine, A. J., Siggia, E. D. & Swain, P. S. Stochastic gene expression h
Elowitz, M. B., Levine, A. J., Siggia, E. D. & Swain, P. S. Stochastic ge gg
g
p
g
41. Yu, J., Xiao, J., Ren, X., Lao, K. & Xie, X. S. Probing gene expression in live cells, one protein molecule at a time. Science
1600–1603 (2006). 42. Raj, A. & van Oudenaarden, A. Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135, 216
(2008). (
)
43. Williams, C. L. & Cotter, P. A. Autoregulation is essential for precise temporal and steady-state regulation by the Bordetella BvgAS
phosphorelay. J Bacteriol 189, 1974–1982 (2007). p
p
y
44. Mukhopadhyay, A., Gao, R. & Lynn, D. G. Integrating input from multiple signals: the VirA/VirG two-component syste
Agrobacterium tumefaciens. Chembiochem 5, 1535–1542 (2004). g
(
)
45. Gao, R. & Stock, A. www.nature.com/scientificreports/ Tiwari, A., Ray, J. C., Narula, J. & Igoshin, O. A. Bistable responses in bacterial genetic networks: designs and dynamical
consequences. Math Biosci 231, 76–89 (2011). 25. Shinar, G., Milo, R., Matinez, M. R. & Alon, U. Input output robustness in simple bacterial signaling systems. PNAS 104,
19931–19935 (2007). 6. Hawley, D. K. & McClure, W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Research 11
2237–2255 (1983).h 7. Jensen, P. R. & Hammer, K. The sequence of spacers between the consensus sequences modulates the strength of prokaryotic
promoters. Applied and Environmental Microbiology 64, 82–87 (1998).i p
pp
gy
8. Jensen, P. R. & Hammer, K. Artificial promoters for metabolic optimization. Biotechnology and Bioengineering 58, 191–195 (1998). 9 B
d
A Wi k
S & G
S M d l i
R B
i i
I P
l
l
f
S
bili
i E h i hi
li G 28. Jensen, P. R. & Hammer, K. Artificial promoters for metabolic optimization. Biotechnology and Bioengineering 58, 191 195 (1998). 29. Bougdour, A., Wickner, S. & Gottesman, S. Modulating RssB activity: IraP, a novel regulator of σS stability in Escherichia coli. Genes
Dev. 20, 884–897 (2006). 30. Merrikh, H., Ferrazzoli, A. E. & Lovett, S. Growth phase and (p)ppgpp control of IraD, a regulator of RpoS stability, in Escherichia
coli. Journal of Bacteriology 191, 7436–7446 (2009).h 1. Ferenci, T., Galbiati, H., Betteridge, T., Phan, K. & Spira, B. The constancy of global regulation across a species: the concentrations o
ppGpp and RpoS are strain-specific in Escherichia coli. BMC Microbiology 11, 62 (2011). 1. Ferenci, T., Galbiati, H., Betteridge, T., Phan, K. & Spira, B. The constancy of global regulation across a species: the concentrations o
ppGpp and RpoS are strain-specific in Escherichia coli. BMC Microbiology 11, 62 (2011). 2 Spira B Hu X & Ferenci T Strain variation in ppGpp concentration and RpoS levels in laboratory strains of Escherichia coli K 12 i
2. Spira, B., Hu, X. & Ferenci, T. Strain variation in ppGpp concentration and RpoS levels in laboratory strains of Escherichia coli K-12
Microbiology 154, 2887–2895 (2008). gy
. Ferenci, B. Maintaining a healthy SPANC balance through regula gy
3. Ferenci, B. Maintaining a healthy SPANC balance through regulatory and mutational adaptation. Mol. Author Contributions C.U. and O.K. designed the model and performed the simulations and the sensitivity analysis. J.T.-B., C.U., J.L.G. and J.N. designed and performed the experiments. C.U., O.K. and M.H. wrote and edited the manuscript. All the
authors analysed the results and reviewed the manuscript. Acknowledgements
Th
k h
b
ll g
This work has been partially funded by the European Union’s Horizon 2020 research and innovation programme
under the grant agreement No 686585 - LIAR, Living Architecture. This work has been partially funded by the European Union’s Horizon 2020 research and innovation programme
under the grant agreement No 686585 - LIAR, Living Architecture. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 13 www.nature.com/scientificreports/ Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-38223-w. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
i
tit ti
l ffili ti Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 14
|
https://openalex.org/W2913895432
|
https://www.itm-conferences.org/10.1051/itmconf/20192503007/pdf
|
English
| null |
How to Construct Effective Consultation System for S&T Decision-making
|
ITM web of conferences
| 2,019
|
cc-by
| 2,225
|
1 Introduction The consultation system of S&T decision-making is the
inevitable result of scientific and technological progress
and social development, and also the inherent requirement
for government decision-making. As a result of the new
round of scientific and technological revolution and
industrial transformation, the complexity of science and
technology strategy and policy making is increasing, and
the
comprehensive
participation
of
scientific,
technological, economic, educational and other social
factors and various think tanks is needed. China also
attaches great importance to the construction of the
consultation system of S&T decision-making. In January
2015, The Opinions on Strengthening the Construction of
a New Think Tank with Chinese Characteristics, issued by
the Office of the CPC Central Committee and General
Office of State Council, emphasized that "the consultation
system of the decision-making is an important part of the
construction of socialist democratic politics in China, and
the new think tank with Chinese characteristics is an
important content of the modernization of the national
governance system and also an important part of the
country's soft power "[1]. In September 2015, the Office of
the CPC Central Committee and General Office of State
Council issued The Implementation Plan for Deepening
the Reform of the Scientific and Technological System,
which called for "the establishment of a national
consultation mechanism of innovation and S&T decision-
making, the development of a good scientific and
technological community and think tank to support
innovation and S&T decision-making, the establishment of
a national advisory committee of S&T decision-making”
[2].On the May 30, 2016, General Secretary Xi Jinping How to Construct Effective Consultation System for S&T Decision-making
Zijun Tang1,a
1Office of Scientific Research Management Jishou University Jishou Hunan China Abstract. With the development of a new round of science and technology (S&T) revolution and industrial
transformation in the world, the complexity of S&T decision-making is increasing. It not only needs the comprehensive
participation of scientific, technological, economic, educational and other social factors but also an effective
consultation system to guarantee. To build an effective consultation system for S&T decision-making, a lot of measures
can be taken, such as establishing the system of the policy and regulation, perfecting the multi-mode operation system,
constructing the support system of talent team, developing of multi-channel financing system, optimizing the system
of social public participation and strengthening the system of interactive international cooperation. stressed in his important speech Striving to Build a World
Science and Technology Power, "We should speed up the
establishment of mechanism of S&T decision-making to
support administrative decision-making, strengthen the
consultation system of S&T decision-making, and build a
high level science and technology think tank to accelerate
the
institutionalization
of
major
scientific
and
technological decisions "[3] February 2017, the CPC
Central Committee passed the Plan of the Construction
Program of National Consultation System of S&T
Decision-making, pointed out to build a national
consultation system and a think tank at the highest level of
S&T decision-making. The Plan is a re-promotion of
China's current consultation mechanism of S&T decision-
making, marking the China’s consultation of S&T
decision-making is on the process of institutionalization
and systematization. There are a lot of measures can be
taken to construct the effective consultation system for
S&T decision-making in China. a Corresponding author: zijuntang@163.com © 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/). ITM Web of Conferences 25, 03007 (2019)
ICICCI 2018 ITM Web of Conferences 25, 03007 (2019)
ICICCI 2018 https://doi.org/10.1051/itmconf/20192503007 3 Perfecting the multi-mode operation
system It is a common direction for the development of
consultation system of S&T decision-making in various
countries to construct different and complementary
consultation systems at different levels and of various
types. It is a common practice in the international
community to set up a committee with members spanning
all fields, controlling the number of members and
highlighting their representativeness. China has already
established the construction plan of the national science
and technology advisory committee, and it is necessary to
further improve the operation mode of the institution. At
the horizontal level, to promote the coordinated
development of official advisory bodies and parastatal and
civil advisory bodies, encourage the Government to
purchase consultation services of S&T decision-making,
support the development of market-oriented decision-
making advisory bodies through corresponding policy
measures, strengthen their financial support, and enhance
the status and influence of civil decision-making advisory
bodies, finally form a useful supplement to the official
advisory body, make China's consultation of S&T
decision-making to the direction of specialization. At the
vertical level, there is a need for a multi-tiered and
complementary advisory body layout, based on the
hierarchical distribution of existing decision-making
advisory bodies, from the national level, the sectoral level
to the grass-roots level and social institutions. For example,
in the case of high-level consultation on major political and
diplomatic decisions, the national advisory committee on
science
and
technology
may
make
advisory
recommendations on such decisions. When it comes to
decision-making in the social and public fields, appropriate
decision-making consultation bodies may be set up
according to the local characteristics and actual conditions 4 Constructing the support system of
talent team The high-quality S&T decision-making consultation needs
the novelty, reliability and policy relevance of knowledge,
which will inevitably put forward special requirements for
the construction of talent team in decision-making
consultation bodies. According to the present situation of
decision-making consultation in China, it should be firstly
focused on cultivating interdisciplinary knowledge talents
and strengthening the ability of strategic research. In order
to better cope with increasingly complex decision-making
problems and enhance the effectiveness of decision-
making suggestions, it is necessary to cultivate research
talents with multi-disciplinary complementarity and cross-
disciplinary advantages, and pay attention to the selection
and training of talents in terms of professional depth,
disciplinary
breadth,
scientific
height
and
social
perspective. Secondly, the evaluation and incentive
mechanism of S&T decision consultation talents should be
constructed through targeted measures. It will be
formulated that unified and standardized evaluation
standards
for
the
employment,
professional
title
assessment, moral character and ability and social
contribution of talents, and take the social impact of the
consultation recommendations as the core assessment
indicators. At the same time, by encouraging original ideas,
respect for intellectual property rights, the establishment of
talent program projects and other methods, to stimulate the
healthy development of consulting talent team, to provide
sustainable
talent
support
for
decision-making
consultation, enhance the capacity and level of S&T
decision-making advisory bodies. Thirdly, it should be pay
more attention to cultivating and promoting the
professional ethics of decision-making consultation talents. In the selection of advisory members and experts, social
responsibility and academic ethics should be the basic
standards, and the professionalism of responsible
consultation should be actively promoted so that S&T
decision-making consultation can truly win the affirmation
and trust of the society. 2 Improving the policy and regulation
guarantee system The smooth development of consultation of S&T decision-
making is guaranteed by sound policies and regulations. Developed countries all regard the construction of perfect
legal system as the key to promote the consultation of
decision-making. The supporting policies and regulations
of consultation of S&T decision-making in China are in the
initial stage and need to be strengthened and improved in
the following four aspects. The first is to incorporate
consultation into the legal procedures for major decision-
making of government departments, protect the legal status ITM Web of Conferences 25, 03007 (2019) ITM Web of Conferences 25, 03007 (2019)
ICICCI 2018 https://doi.org/10.1051/itmconf/20192503007 of consulting bodies through relevant laws and regulations,
and specify their power and responsibility relationship,
expert selection, organizational structure and operation
mode; Secondly, the status and role of different types of
consultation bodies in decision-making should be clarified
through legislation. In particular, the consultation
procedures should be standardized and designed through
specific
legal
provisions
to
ensure
the
relative
independence
of
consultation
bodies. Meanwhile,
corresponding incentive measures should be formulated
according to different consultation situations; Thirdly,
establish the application and feedback system of important
S&T decision-making consultation achievements, and
strengthen the legal regulation of the formation, release
and use of consultation reports. Fourthly, establish a
supervision system and restriction mechanism for S&T
decision-making consultation, and a public disclosure and
hearing system for major decision-making matters, ensure
the openness of all links and procedures of decision-
making consultation, protect the right of supervision and
information of the public, and promote the openness,
transparency and fairness of decision-making consultation
activities. of various departments, institutions and regions. The
perfect operation mechanism is the premise to ensure the
decision-making level to grasp the relevant policy
information accurately, timely and systematically, provide
objective and reasonable scientific support for decision-
making, and is also the key to test its effectiveness. References 1. The Executive Office of the CPC Central Committee
and General Office. On strengthening Chinese
characteristics Views on the construction of a new
think
tank. [2016-09-30]. http://www.gov.cn/Xinwen/201501/20/content_2807
126.htm 2. The Executive Office of the CPC Central Committee
and General Office. Deepening the implementation of
scientific and technological system reform case. [2016-09-30]. http://www.gov.cn/guowuyuan/2015/09/24/Content_
2938314.htm 5 Developing multi-channel financing
system Of
course, whether it is from the public finances or corporate
sponsorship and social donations, there should be
transparent and open management of financial intervention
in order to prevent the possible existence of "undercover
operation" through financial support, affecting or shaping
the advisory opinions. Then the advisory bodies are not
disturbed by some kind of "external" factors and can
objectively provide independent advice on a fair attitude. 5 Developing multi-channel financing
system A certain amount of financial support is a necessary
condition for the normal operation of decision-making
advisory bodies. The advisory bodies of developed
countries are mainly funded by government grants,
corporate grants, private donations and dues. At present,
China mainly supports the operation of decision-making
advisory bodies through the way of government financial
allocation, while the proportion of enterprises and private 2 2 ITM Web of Conferences 25, 03007 (2019) ITM Web of Conferences 25, 03007 (2019)
ICICCI 2018 https://doi.org/10.1051/itmconf/20192503007 international research, operation and exchange in order to
obtain a voice on international issues. In order to meet the
requirements of the new era, China should further promote
the international development of S&T decision-making
consultation, and promote the competitive platform of
intellectual products to the international market. Firstly,
carry out cooperative research and exchange dialogue with
foreign S&T decision-making advisory bodies, draw on
their organizational methods, operating models and
discourse paradigms, and selectively integrate them into
the construction of our own advisory bodies in accordance
with the actual situation in China. Secondly, expand the
voice of domestic decision-making advisory bodies in the
international arena, strive to "lead" in some global issues
and China issues that arouse world concern, and build a
group
of
decision-making
advisory
bodies
with
international influence. Thirdly, cultivate a team of
consulting talents with critical spirit and innovative
consciousness,
international
discourse
ability
and
competitiveness, launch original research achievements
with the value of the times and global consciousness, and
improve the academic reputation in the international arena. The last is to actively promote the establishment of S&T
decision-making advisory branches abroad, as a means of
establishing effective international cooperative relations
for the exchange platform, and to expand the international
recognition and influence of advisory bodies through
advisory activities on China's foreign policy. financial support needs to be further improved. Therefore,
the management system of decision-making consultation
funds should be reformed, change the homogenization
mode of financing, encourage enterprises to invest in the
establishment of advisory bodies and promote their
market-oriented operation. At the same time, civil and
individual donations, etc., should also be given some
recognition and support, in order to further enrich the
funding sources of decision-making advisory bodies. Acknowledgement This paper was financially supported by the project of
Science and Technology Plan of Xiangxi Autonomous
Prefecture, "The Research on the Mechanism of University
-affiliated think tanks participating in Local Science and
Technology Innovation Decision-making consultation"
(No.2018SF5015) 6 Optimizing the system of public
participation in society. Many decision-making problems in social development
are often closely related to the public interest, and actively
promoting public participation is of great significance to
improve the sound consultation system of S&T decision-
making. Firstly, through the systematic formulation of
system rules, introducing orderly and effectively public
participation in the progress of decision-making advisory
activities can help to jointly promote the formation of
decision-making solutions, and also can make the
decision-making advisory bodies widely accept public
supervision, better promote the openness and transparency
of the consultation process; Secondly, the Government
should strengthen the guiding role of public participation
in decision-making, speed up the institutionalization of
public
participation,
create
a
good
institutional
environment, make public participation an integral part of
the decision-making consultation process, so that the
public really enjoy the right to participate in decision-
making consultation, and have a practical role and
influence in the process of decision-making consultation;
Thirdly, a multi-subject decision-making consultation
platform including government, S&T decision-making
advisory bodies and the public should be further built, so
as to smooth out the information communication channels
between the government and the society. 7 Strengthening the system of interactive
international cooperation In the context of globalization, international influence is
one of the important goals pursued by S&T decision-
making advisory bodies, for which most national decision-
making advisory bodies adopt open policies and conduct 3. Xi Jinping. Strive to build a world science and
technology power. People's Daily, 6(2016). 3
|
https://openalex.org/W4393149662
|
https://link.springer.com/content/pdf/10.1007/s00383-024-05683-3.pdf
|
English
| null |
A systematic review of Sandifer syndrome in children with severe gastroesophageal reflux
|
Pediatric surgery international
| 2,024
|
cc-by
| 4,266
|
Abstract Abstract
Purpose Sandifer syndrome (SS), which combines gastroesophageal reflux (GER) and a neurological or psychiatric disorder,
is an uncommon condition that often takes a long time to diagnosis. We aimed to systematically review available papers
regarding SS. Methods After presenting our two cases of SS, we systematically reviewed articles published in MEDILINE/PubMed,
Cochrane Library, and Web of Science. Results The meta-analysis included 54 reported cases and 2 of our own cases. Our results showed that all cases achieved
symptom improvement with appropriate treatment for GER. Notably, 19 of the 56 cases exhibited anatomical anomalies,
such as hiatal hernia and malrotation. Significantly more patients with than without anatomical anomalies required surgery
(p < 0.001). However, 23 of the 29 patients without anatomical anomalies (79%) achieved symptom improvement without
surgery. Patients who did not undergo surgery had a median (interquartile range) duration to symptom resolution of 1 (1–1)
month. Conclusion The primary care providers should keep SS in the differential diagnosis of patients presenting with abnormal
posturing and no apparent neuromuscular disorders. Fundoplication may be effective especially for patients with anatomical
anomalies or those whose symptoms do not improve after more than 1 month with nonsurgical treatment. Keywords Sandifer syndrome · Gastroesophageal reflux · Fundoplication · Laparoscopic surgery Pediatric Surgery International (2024) 40:91
https://doi.org/10.1007/s00383-024-05683-3 Pediatric Surgery International (2024) 40:91
https://doi.org/10.1007/s00383-024-05683-3 ORIGINAL ARTICLE ORIGINAL ARTICLE A systematic review of Sandifer syndrome in children with severe
gastroesophageal reflux Daiki Kato1 · Hiroo Uchida1 · Hizuru Amano1 · Kazuki Yokota2 · Chiyoe Shirota1 · Takahisa Tainaka1 · Wataru Sumida1 ·
Satoshi Makita1 · Akihiro Yasui1 · Yousuke Gohda1 · Takuya Maeda1 · Akinari Hinoki3 Daiki Kato1 · Hiroo Uchida1 · Hizuru Amano1 · Kazuki Yokota2 · Chiyoe Shirota1 · Takahisa Tainaka1 · Wataru Sumida1 ·
Satoshi Makita1 · Akihiro Yasui1 · Yousuke Gohda1 · Takuya Maeda1 · Akinari Hinoki3 Accepted: 19 March 2024
© The Author(s) 2024 * Hiroo Uchida
hiro2013@med.nagoya-u.ac.jp 3
Department of Rare/Intractable Cancer Analysis Research,
Nagoya University Graduate School of Medicine, 65
Tsurumai‑cho, Showa‑ku, Nagoya 466‑8550, Japan 1
Department of Pediatric Surgery, Nagoya University
Graduate School of Medicine, 65 Tsurumai‑cho, Showa‑ku,
Nagoya 466‑8550, Japan Fig. 1 a Upper gastrointestinal
series showing gastroesophageal
reflux. b Esophagogastroduo-
denoscopy showing reflux
esophagitis (Los Angeles clas-
sification: Grade D) and a hiatal
hernia Introduction abnormal posture and involuntary movements that disturb
clinicians or parents given that they can mimic seizures [2]. SS is often misdiagnosed as a neurological or musculoskel-
etal condition. The difficulty in accurately diagnosing this
clinical manifestation is that there are often no obvious gas-
trointestinal symptoms such as abdominal pain or vomit-
ing. This can lead to unnecessary and expensive neurologic
examinations such as MRI, EEG, and electromyography. These exams may lead to a missed and delayed diagnosis,
and mismanagement. When a patient has abnormal posture
or movement without neuromuscular disease, SS should be
one of the differential diagnoses. Fortunately, symptoms of
SS improve with GER treatments, such as medication and
surgery. Sandifer syndrome (SS) is an uncommon condition charac-
terized by a combination of gastroesophageal reflux (GER)
or hiatal hernia and a neurological or psychiatric disorder
[1]. SS is associated with abnormal posture and move-
ments of the neck and trunk. Patients with SS present with 1
Department of Pediatric Surgery, Nagoya University
Graduate School of Medicine, 65 Tsurumai‑cho, Showa‑ku,
Nagoya 466‑8550, Japan 1
Department of Pediatric Surgery, Nagoya University
Graduate School of Medicine, 65 Tsurumai‑cho, Showa‑ku,
Nagoya 466‑8550, Japan Recognizing SS and treating GER will quickly resolve
this disease. However, reports on SS have been infrequent,
with the diagnosis of SS often taking a long time. Moreo-
ver, to the best of our knowledge, only a few systematic
review and meta-analysis have been published on this 2
Department of Pediatric Surgery, Aichi Developmental
Disability Center Central Hospital, 713‑8 Kagiya‑cho,
Kasugai 480‑0392, Japan 2
Department of Pediatric Surgery, Aichi Developmental
Disability Center Central Hospital, 713‑8 Kagiya‑cho,
Kasugai 480‑0392, Japan (0123 3456789) Pediatric Surgery International (2024) 40:91 Page 2 of 7 Pediatric Surgery International 91 A 9‑year‑old boy This case involved a patient who was suffering from vomit-
ing and an abnormal left-leaning posture that started 4 years
prior to presentation. He initially sought consultation from
a pediatric neurologists. However, when no abnormalities
were found on blood examination and head magnetic reso-
nance imaging, he was placed under observation. Unfortu-
nately, his symptoms did not improve, prompting referral to
a pediatric psychiatrists 2 years prior to presentation. Elec-
troencephalography and development examinations showed
no abnormalities. As such, SS was suspected based on his-
tory, for which upper gastrointestinal examinations were per-
formed. Upper gastrointestinal series (UGI) showed GER
(Fig. 1a), whereas esophagogastroduodenoscopy (EGD)
showed reflux esophagitis (Los Angeles classification: Grade
D) and a hiatal hernia (Fig. 1b). Low esophageal pH (< 4)
accounted for 19% of the 24-h recording cycle. These find-
ings confirmed that SS was the correct diagnosis. Postural abnormalities temporarily disappeared after initi-
ating PPI treatment 1 month prior to presentation. However,
we opted to perform laparoscopic Toupet fundoplication
given the recurrence of symptoms despite PPI treatment. At
2 weeks after the surgery, his symptoms improved as shown
by the disappearance of postural abnormalities and cessation
of voluble speech. No symptom recurrence had been noted
for 9 months. Systematic review and meta‑analysis We subsequently reviewed articles published in MEDIL-
INE/PubMed, Cochrane Library, and Web of Science using
the following combinations of search terms: “Sandifer syn-
drome” and “gastroesophageal reflux.” A 7‑year‑old boy subject. Therefore, the current study first aimed to present
our experience with two cases of SS. Second, a systematic
review was conducted to evaluate all published studies on
patients with SS. Our article on the diagnosis and treatment
of patients with SS aimed to help consider SS as an early
differential diagnosis in children with these disorders. This case involved a patient who was suffering from abnor-
mal posture with backward bending of the neck, violent
speech, chronic cough, and vomiting 2 months prior to
presentation (Fig. 2a), which prompted him to initially seek
consultation from pediatricians. Computed tomography of
the head performed in the emergency department of the pre-
vious hospital showed no abnormality. The patient was then
transferred to our hospital. UGI showed GER (Fig. 2b), and
EGD showed reflux esophagitis (Los Angeles classification:
Grade D) and hiatal hernia (Fig. 2c). We could not perform
low esophageal pH 24-h recording due to his symptom of
abnormal posture. Results In total, 201 articles were identified via a comprehensive
review. After removing 116 duplicates and 61 studies that
satisfied the exclusion criteria, 24 articles and 54 cases were
ultimately identified (Table 1) [1–24]. Figure 3 shows the
flow diagram for study selection. Selection criteria i
Given the lack of improvement with proton pump inhibi-
tor (PPI) treatment for 1 month, laparoscopic Toupet fun-
doplication was performed, which improved the patients
symptoms 1 month after surgery. The left-leaning posture
disappeared, and the patient did not develop any symptom
recurrence for 2 years. Original articles and case reports reporting details regard-
ing patients with SS were included in the analysis. Two
reviewers independently scanned the titles and abstracts of
the identified articles. The exclusion criteria were as fol-
lows: (1) articles not written in English, (2) non-original Fig. 1 a Upper gastrointestinal
series showing gastroesophageal
reflux. b Esophagogastroduo-
denoscopy showing reflux
esophagitis (Los Angeles clas-
sification: Grade D) and a hiatal
hernia Page 3 of 7
91 Pediatric Surgery International (2024) 40:91 Page 3 of 7
91 Pediatric Surgery International ) Fig. 2 a Abnormal posture
with backward bending of the
neck was observed. b Upper
gastrointestinal series show-
ing gastroesophageal reflux. c
Esophagogastroduodenoscopy
showing reflux esophagitis (Los
Angeles classification: Grade D)
and a hiatal hernia articles or case reports (meeting abstracts, reviews), (3)
studies that did not focus on patients with SS, and (4)
studies with insufficient data on patients with SS. This
systematic review was conducted based on the PRISMA
principles. Data extraction l
g
y
All 54 published cases and both of our own were evalu-
ated via meta-analysis (Table 1). In all cases, symptoms
improved with appropriate GER treatment. In terms of ana-
tomical anomaly, there were 18 cases of hiatal hernia, 1 case
of malrotation, and 8 cases of no description. Notably, 17/19
(89%) cases with anatomical anomalies required surgery. Patients with anatomical anomalies required significantly
more surgeries than did those without anatomical anomalies
(p < 0.001). However, 23 of the 29 patients without anatomi-
cal anomalies (79%) showed symptom improvement with-
out surgery. In both of our cases, surgery promoted early
improvement in symptoms of hiatal hernia. Many patients
were diagnosed with GER by UGI, EGD, and low esopha-
geal pH 24-h recording. The pH measurement has increased
frequency over time and the diagnostic criteria for GER in
all cases. The median (interquartile range) age at diagnosis Two reviewers separately collected the following data from
the selected articles: the first author, year of publication,
study design, sample size, patient characteristics, treatment
course, and outcomes. Both reviewers reached a consensus
at each stage of the data extraction process. Fig. 2 a Abnormal posture
with backward bending of the
neck was observed. b Upper
gastrointestinal series show-
ing gastroesophageal reflux. c
Esophagogastroduodenoscopy
showing reflux esophagitis (Los
Angeles classification: Grade D)
and a hiatal hernia Statistical analysis Continuous variables were presented as medians and inter-
quartile ranges, whereas categorical variables were pre-
sented as frequencies and percentages. Fisher’s exact test
was used to evaluate categorical variables. p values < 0.05
were considered statistically significant. Statistical analysis 3 The flow diagram for
study inclusion based on the
PRISMA 2020 statement Fig. 3 The flow diagram for
study inclusion based on the
PRISMA 2020 statement was 2 (0–5) years. The median (interquartile range) dura-
tion to diagnosis was 6 (2–28) months. The duration from
nonsurgical treatment to surgery was more than 1 month,
although only a few articles described this. In patients who
did not undergo surgery, the median (interquartile range)
duration to resolution of symptom was 1 (1–1) month. various symptoms such as torticollis, dystonia, and seizures
[25, 26]. While the actual incidence of SS remains unknown,
estimates place it at probably < 1% of children with GER
[27, 28]. Given its lack of recognition, SS is often mistaken
for neuromuscular or neuropsychiatric disorders due to lim-
ited regarding the same. Available articles have shown that
SS takes several months or more to diagnose (Table 1), often
resulting in the repetition of unnecessary tests that further
delay diagnosis [29]. Statistical analysis Pediatric Surgery International (2024) 40:91 Pediatric Surgery International Page 4 of 7 91 91 Table 1 Data collected from reported and our cases
Author
Year
Age
Anatomical anomaly
Diagnostic examination
Definitive treatment
Duration to
diagnosis
(M)
Duration to
surgery (M)
Duration to
resolution
(M)
Shrestha
2021
4y
None
UGI
Drug
N/A
–
1
Sharif
2020
3y
None
UGI, EGD, pH
Drug
N/A
–
N/A
Bamji
2015
3m
None
Clinical history
Drug
2
–
1
2m
None
UGI
Drug
1
–
1
Nalbantoglu
2008
9m
None
EGD, pH
Diet
2
–
1
Tokuhara
2008
8y
None
UGI, EGD, pH
Surgery
36
3
3
Lehwald
2007
9y
Hiatal hernia
UGI, EGD
Surgery
60
N/A
3
Firat
2007
2y
Malrotation
UGI, pH
Surgery
21
N/A
1
Kabakus
2006
2m
None
Scintigraphy
Drug
5
–
N/A
4m
None
Scintigraphy
Drug
2
–
N/A
6m
None
Scintigraphy
Drug
3
–
N/A
1y
None
Scintigraphy
Drug
8
–
N/A
Corrado
2006
1y
None
UGI, pH
Drug
2
–
1
Frankel
2006
4y
None
UGI, EGD, pH
Surgery
2
30
1
Corrado
2000
15d
None
UGI, pH
Diet
14
–
N/A
Ybarrondo
2000
5y
None
UGI
Surgery
0
12
1
Olguner
1999
5y
None
UGI, pH
Surgery
12
N/A
2
Deskin
1995
2y
None
UGI
Surgery
12
6
3
Gorrotxategi
1995
N/A
N/A
UGI, EGD, pH
Drug
N/A
–
N/A
N/A
N/A
UGI, EGD, pH
Drug
N/A
–
N/A
N/A
N/A
UGI, EGD, pH
Drug
N/A
–
N/A
N/A
N/A
UGI, EGD, pH
Surgery
N/A
N/A
N/A
N/A
N/A
UGI, EGD, pH
Surgery
N/A
N/A
N/A
N/A
N/A
UGI, EGD, pH
Surgery
N/A
N/A
N/A
N/A
N/A
UGI, EGD, pH
Surgery
N/A
N/A
N/A
N/A
N/A
UGI, EGD, pH
Surgery
N/A
N/A
N/A
Senocak
1993
N/A
Hiatal hernia
N/A
Surgery
N/A
N/A
N/A
Puntis
1989
5y
Hiatal hernia
UGI, pH
Surgery
3
2
3
Nanayakarra
1985
2y
None
UGI, pH, scintigraphy,
Drug
19
–
N/A
2y
None
UGI, pH
Drug
18
–
1
2y
None
UGI, pH
Drug
19
–
1
Hadari
1984
13y
Hiatal hernia
UGI
Surgery
N/A
N/A
N/A
Werlin
1980
2w
None
UGI, pH
Drug
0
–
N/A
2w
None
UGI, pH
Drug
0
–
N/A
8m
None
UGI, pH
Drug
2
–
1
3m
None
UGI, pH
Drug
N/A
–
1
2w
None
UGI, pH
Drug
0
–
N/A
Murphy
1977
8m
Hiatal hernia
UGI
Drug
6
–
1
Bray
1977
3m
None
UGI
Drug
2
–
4
3m
None
UGI
Drug
2
–
2
2m
Hiatal hernia
UGI
Drug
3
–
2
4y
Hiatal hernia
UGI
Surgery
3
N/A
N/A
5y
Hiatal hernia
UGI
Surgery
30
N/A
1
5y
None
UGI
Surgery
54
N/A
N/A
7m
None
UGI
Drug
6
–
N/A
2m
None
UGI
Drug
1
–
N/A
Sutcliffe
1969
10y
Hiatal hernia
UGI
Surgery
N/A
N/A
N/A
6y
Hiatal hernia
UGI
Surgery
N/A
N/A
N/A Table 1 Data collected from reported and our cases Pediatric Surgery International (2024) 40:91 Page 5 of 7
91 Pediatric Surgery International 91 UGI upper gastrointestinal series, EGD esophagogastroduodenoscopy, pH low esophageal pH 24-h recording
Table 1 (continued)
Author
Year
Age
Anatomical anomaly
Diagnostic examination
Definitive treatment
Duration to
diagnosis
(M)
Duration to
surgery (M)
Duration to
resolution
(M)
4y
Hiatal hernia
UGI
Surgery
N/A
N/A
N/A
Kinsbourne
1964
1y
Hiatal hernia
UGI
Surgery
52
N/A
N/A
7y
Hiatal hernia
UGI
Surgery
36
N/A
N/A
4y
Hiatal hernia
UGI
Surgery
47
N/A
N/A
9y
Hiatal hernia
UGI
Surgery
72
N/A
N/A
14y
Hiatal hernia
UGI
Surgery
108
N/A
N/A
Kato
2023
9y
Hiatal hernia
UGI, EGD, pH
Surgery
54
1
1
7
Hiatal hernia
UGI, EGD
Surgery
3
1
1 UGI upper gastrointestinal series, EGD esophagogastroduodenoscopy, pH low esophageal pH 24-h recording
Table 1 (continued)
Author
Year
Age
Anatomical anomaly
Diagnostic examination
Definitive treatment
Duration to
diagnosis
(M)
Duration to
surgery (M)
Duration to
resolution
(M)
4y
Hiatal hernia
UGI
Surgery
N/A
N/A
N/A
Kinsbourne
1964
1y
Hiatal hernia
UGI
Surgery
52
N/A
N/A
7y
Hiatal hernia
UGI
Surgery
36
N/A
N/A
4y
Hiatal hernia
UGI
Surgery
47
N/A
N/A
9y
Hiatal hernia
UGI
Surgery
72
N/A
N/A
14y
Hiatal hernia
UGI
Surgery
108
N/A
N/A
Kato
2023
9y
Hiatal hernia
UGI, EGD, pH
Surgery
54
1
1
7
Hiatal hernia
UGI, EGD
Surgery
3
1
1
Fig. Discussion To date, the etiology of the muscle dystonia in SS remains
unclear. It may be related with the diaphragm and neck shar-
ing common innervation. Some authors have postulated the
movements and abnormal postures were a learned behav-
ior to relieve abdominal discomfort and improve esopha-
geal motility. It was proved the direct relationship between SS, which is named after neurologist Paul Sandifer, had first
been reported by Kinsbourne in 1964 after recognizing a
dysfunction in the upper gastrointestinal tract with neuro-
logical manifestations occurring in children and adolescents
[24]. SS consists of an unusual combination of GER and Pediatric Surgery International (2024) 40:91 Page 6 of 7 91 dystonic movements and low pH, and it revealed the pH in
a patient with SS. During 83 episodes of posturing, none of
these episodes occurred during a period of pH > 5 for ≥ 30
s[10]. SS could be caused by gastroesophageal reflux. Suc-
cessful treatment of the underlying GERD led to a com-
plete resolution of the symptoms. Although GER episodes in
patients with SS had initially been considered to be induced
by postural abnormalities, radiography during the torsion
episode suggested that GER was actually worsening [24]. Previous study reported that neurological manifestations
were the result of the vagal reflex [10]. However, it does not
explain why these postures cannot adopt this position dur-
ing sleep. The possible pathophysiologic relationship is that
GER episodes cause postural abnormalities. Several articles
have reported that esophageal motility improved during head
tilting as evidence by the increase in esophageal contraction
pressure from 47 to 74 mmHg and propagation velocity from
2.5 to 4 cm/s [13, 17]. The symptoms observed in SS are
thought to clear gastric acid, suggesting that GER causes
postural abnormalities, as supported by the disappearance
of symptoms after GER treatment in our cases and in the
reported literature. gastrointestinal examination. Fundoplication may be effec-
tive especially for patients with anatomical anomalies or
those whose symptoms fail to improve for over 1 month
with nonsurgical treatment. Quick detection of subtle
symptoms can facilitate early diagnosis and treatment and
omission of unnecessary examinations in patients with SS. Our study has several limitations. First, all eligible stud-
ies on patients with SS were case reports and non-rand-
omized in nature. A prospective study may provide addi-
tional insights into the diagnosis and outcomes of patients
with SS. Conclusion It is important that GER be resolved when treating SS. Should appropriate nonsurgical treatment fail to improve
symptoms, surgery may be necessary. Fundoplication, the
primary surgical procedure considered safe for the treat-
ment of GER [30], should always be considered as a pos-
sible treatment option for patients with SS. The current
study found that significantly more patients with anatomi-
cal anomalies required surgery than did those without the
same (Table 1). Fundoplication may be more effective than
drug or diet therapy in controlling SS should gross anatomi-
cal anomalies, such as hiatal hernia and malrotation, be the
cause of GER. Moreover, most patients treated with drug or
diet improved within 1 month (Table 1). Fundoplication may
need to be considered if symptoms do not improve for more
than 1 month with nonsurgical treatment. The primary care providers should keep SS in the dif-
ferential diagnosis of patients presenting with abnormal
posturing and no apparent neuromuscular disorders. Prompt treatment of GER may lead to early symptomatic
improvement and omission of unnecessary examinations. Fundoplication leads to early symptomatic improvement
and may be effective especially for patients with anatomi-
cal anomalies or those whose symptoms fail to improve for
over 1 month with nonsurgical treatment. Author contributions D.K. had the idea for the article, and wrote the
manuscript. D.K. and H.A. performed the literature search and data
analysis. H.U. drafted and critically revised the work. All authors
approved the final draft of this manuscript. The primary care providers should keep SS in the dif-
ferential diagnosis of patients presenting with abnormal
posturing and no apparent neuromuscular disorders. Rec-
ognition and treatment of GER in patients with SS is key
to medical management. The important first step is to sus-
pect SS and obtain a detailed medical history. If symptoms
appear immediately after feeding, SS may be present. As
the diagnosis of GER is most reliably made by assess-
ing the presence or absence of reflux, pH measurement
is recommended in principle. In reported articles, the pH
measurement has increased frequency over time (Table 1). The pH measurement is useful as it provides an objective
assessment of reflux and clinicians should always con-
sider performing this examination. Treatment should be
initiated immediately after confirming GER through upper Funding Open Access funding provided by Nagoya University. Funding Open Access funding provided by Nagoya University. Discussion Second, the sample size was small, suggesting the
need for large, high-quality randomized controlled trials
in the future. Third, the duration of conservative therapy
was unknown although we reviewed articles. If conserva-
tive treatment is effective, it may be acceptable to continue
treatment as is for long time. How long the effect of con-
servative treatment lasts is a major issue to be addressed
in the future. Conclusion Data availability No datasets were generated or analysed during the
current study. References 19. Hadari A, Azizi E, Lernau O, Nissan S (1984) Sandifer’s syn-
drome–a rare complication of hiatal hernia. A case report. Z
Kinderchir 39:202–203. https://doi.org/10.1055/s-2008-1044208 1. Shrestha AB, Rijal P, Sapkota UH et al (2021) Sandifer syndrome:
a case report. JNMA J Nepal Med Assoc 59:1066–1068. https://
doi.org/10.31729/jnma.6472 20. Werlin SL, D’Souza BJ, Hogan WJ et al (1980) Sandifer syn-
drome: an unappreciated clinical entity. Dev Med Child Neurol
22:374–378. https://doi.org/10.1111/j.1469-8749.1980.tb03719.x g
j
2. Sharif A, Carr L, Saliakellis E, Chakraborty H (2020) Paroxys-
mal head drops with ataxia-like symptoms presenting as Sandifer
syndrome in a 3-year old girl. BMJ Case Rep 13:e230989. https://
doi.org/10.1136/bcr-2019-230989 21. Murphy WJ, Gellis SS (1977) Torticollis with hiatus hernia in
infancy. Sandifer syndrome. Am J Dis Child 131:564–565. https://
doi.org/10.1001/archpedi.1977.02120180078015 22. Bray PF, Herbst JJ, Johnson DG et al (1977) Childhood gastroe-
sophageal reflux: neurologic and psychiatric syndromes mim-
icked. JAMA 237:1342–1345. https://doi.org/10.1001/jama.1977.
03270400046017 g
3. Bamji N, Berezin S, Bostwick H, Medow MS (2015) Treatment of
Sandifer syndrome with an amino-acid–based formula. AJP Rep
5:e51–e52. https://doi.org/10.1055/s-0035-1545672 p
g
4. Nalbantoglu B, Metin DM, Nalbantoglu A (2013) Sandifer’s syn-
drome: a misdiagnosed and mysterious disorder. Iran J Pediatr
23:715–716 23. Sutcliffe J (1969) Torsion spasms and abnormal postures in
childen with hiatus hernia, Sandifer’s syndrome. Prog Pediatr
Radiol 2:190–197 5. Tokuhara D, Yamano T, Okano Y (2008) Letters to the editor. J
Paediatr Child Health 44:311–312. https://doi.org/10.1111/j.1440-
1754.2008.01304-01305.x 24. Kinsbourne M (1964) Hiatus hernia with contortions of the neck. Lancet 283:1058–1061. https://doi.org/10.1016/S0140-6736(64)
91264-4 6. Lehwald N, Krausch M, Franke C et al (2007) Sandifer syndrome–
a multidisciplinary diagnostic and therapeutic challenge. Eur J
Pediatr Surg 17:203–206. https://doi.org/10.1055/s-2007-965145 25. Gellis SS, Feingold M (1971) Syndrome of hiatus hernia with
torsion spasms and abnormal posturing (Sandifer’s syndrome). Am J Dis Child 121:53–54 g
g
7. Firat AK, Karakas HM, Firat Y, Yakinci C (2007) Unusual symp-
tom of intestinal malrotation: episodic cervical dystonia due to
Sandifer syndrome. Pediatr Int 49:519–521. https://doi.org/10.
1111/j.1442-200x.2007.02409.x 26. Torticollis with hiatus hernia in infancy. Sandifer syndrome - Pub-
Med. https://pubmed.ncbi.nlm.nih.gov/855842/. Accessed 15 Sep
2023 27. Narchi H (2004) A Rare Under-Recognized Cause of Pseudo-
Seizures. Int Pediatr 19:39–41 8. Kabakuş N, Kurt A (2006) Sandifer Syndrome: a continuing prob-
lem of misdiagnosis. Pediatr Int 48:622–625. https://doi.org/10.
1111/j.1442-200X.2006.02280.x 28. Sommer A (1993) Occurrence of the Sandifer complex in the
Brachmann-de Lange syndrome. Am J Med Genet 47:1026–1028. https://doi.org/10.1002/ajmg.1320470719 9. Declarations Conflict of interest The authors declare no competing interests that are
relevant to the content of this article. Ethical approval All procedures performed in studies involving human
participants were in accordance with the ethical standards of the insti-
tutional or national research committee and with the 1964 Declaration
of Helsinki and its later amendments or comparable ethical standards. Informed consent Informed consent to participate in the study was
obtained from the parents of all individual participants by disclosing Page 7 of 7 91 Pediatric Surgery International (2024) 40:9 Pediatric Surgery International the study information and giving those who did not wish to participate
an opportunity to request it (opt-out). the study information and giving those who did not wish to participate
an opportunity to request it (opt-out). syndrome. Pediatr Int 41:321–322. https://doi.org/10.1046/j.1442-
200x.1999.01051.x 14. Deskin RW (1995) Sandifer syndrome: a cause of torticollis in
infancy. Int J Pediatr Otorhinolaryngol 32:183–185. https://doi.
org/10.1016/0165-5876(95)01130-4 Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, 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/. g
15. Gorrotxategi P, Reguilon MJ, Arana J et al (1995) Gastroesopha-
geal reflux in association with the Sandifer syndrome. Eur J Pedi-
atr Surg 5:203–205. https://doi.org/10.1055/s-2008-1066205 16. Senocak ME, Arda IS, Büyükpamukçu N (1993) Torticollis
with hiatus hernia in children. Sandifer syndrome. Turk J Pediatr
35:209–213f 17. Puntis JW, Smith HL, Buick RG, Booth IW (1989) Effect of dys-
tonic movements on oesophageal peristalsis in Sandifer’s syn-
drome. Arch Dis Child 64:1311–1313. https://doi.org/10.1136/
adc.64.9.1311 18. Nanayakkara CS, Paton JY (1985) Sandifer syndrome: an over-
looked diagnosis? Dev Med Child Neurol 27:816–819. https://doi.
org/10.1111/j.1469-8749.1985.tb03808.x References Corrado G, Fossati C, Turchetti A et al (2006) Irritable oesopha-
gus: a new cause of Sandifer’s syndrome. Acta Paediatr 95:1509–
1510. https://doi.org/10.1080/08035250600643251 29. Mandel H, Tirosh E, Berant M (1989) Sandifer syndrome recon-
sidered. Acta Paediatr Scand 78:797–799. https://doi.org/10.
1111/j.1651-2227.1989.tb11150.x p
g
10. Frankel EA, Shalaby TM, Orenstein SR (2006) Sandifer syndrome
posturing: relation to abdominal wall contractions, gastroesopha-
geal reflux, and fundoplication. Dig Dis Sci 51:635–640. https://
doi.org/10.1007/s10620-006-3184-1 30. Gordon N (2015) Sandifer’s syndrome: investigations and treat-
ment. J Pediatr Neurol 05:275–278. https://doi.org/10.1055/s-
0035-1557413 11. Corrado G, Cavaliere M, D’Eufemia P et al (2000) Sandifer’s syn-
drome in a breast-fed infant. Am J Perinatol 17:147–150. https://
doi.org/10.1055/s-2000-9285 Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. 12. de Ybarrondo L, Mazur JL (2000) Sandifer’s syndrome in a child
with asthma and cerebral palsy. South Med J 93:1019–1021 13. Olguner M, Akgür FM, Hakgüder G, Aktuğ T (1999) Gastroe-
sophageal reflux associated with dystonic movements: Sandifer’s
|
https://openalex.org/W2091457510
|
https://periodicos.ufsc.br/index.php/ref/article/download/S0104-026X2004000100003/7942
|
Portuguese
| null |
Engendrando desenvolvimento e etnicidade nas terras baixas do Pacífico colombiano
|
Revista Estudos Feministas
| 2,004
|
cc-by
| 7,516
|
Copyright 2004 by Revista
Estudos Feministas
* Excepcionalmente neste artigo,
as notas estão editadas ao final
do texto. Kiran Asher
Engendrando desenvolvimento e
Engendrando desenvolvimento e
Engendrando desenvolvimento e
Engendrando desenvolvimento e
Engendrando desenvolvimento e
etnicidade nas terras baixas do
etnicidade nas terras baixas do
etnicidade nas terras baixas do
etnicidade nas terras baixas do
etnicidade nas terras baixas do
PPPPPacífico colombiano
acífico colombiano
acífico colombiano
acífico colombiano
acífico colombiano
Clark University Kiran Asher
Clark University Estudos Feministas, Florianópolis, 12(1): 360, janeiro-abril/2004 15 Engendrando desenvolvimento e
Engendrando desenvolvimento e
Engendrando desenvolvimento e
Engendrando desenvolvimento e
Engendrando desenvolvimento e
etnicidade nas terras baixas do
etnicidade nas terras baixas do
etnicidade nas terras baixas do
etnicidade nas terras baixas do
etnicidade nas terras baixas do
PPPPPacífico colombiano
acífico colombiano
acífico colombiano
acífico colombiano
acífico colombiano RRRRResumo
esumo
esumo
esumo
esumo: Neste ensaio, exploro como as organizações e redes de mulheres afro-colombianas
moldam e são moldadas por iniciativas do Estado para desenvolver e modernizar a região do
Pacífico. Argumento que, agindo assim, elas mobilizam e vão além da retórica
desenvolvimentista do Estado e do discurso de uma etnia negra ‘gendrada’ e da tradição das
organizações políticas negras da região. PPPPPalavras-chave
alavras-chave
alavras-chave
alavras-chave
alavras-chave: mulheres afro-colombianas, estratégias de desenvolvimento, políticas étnicas
e de gênero. O Litoral Pacífico da Colômbia, ou a região Choco,
é uma área rica em recursos naturais, que se estende do
extremo sul do Panamá ao extremo norte do Equador, ao
longo da costa do Pacífico. Em 1991, a Colômbia adotou
uma nova Constituição que inclui leis e políticas com
implicações contraditórias para a região do Pacífico e seus/
suas habitantes. Por um lado, a Lei 70, de 1993, baseada
no Artigo Provisório 55 da Constituição de 1991, reconhece
os/as afro-colombianos/as (que compõem 90% da
população da região) como um grupo étnico separado,
com direitos a possuir terras coletivamente e utilizá-las dentro
dos moldes tradicionais.1 Por outro lado, as reformas
econômicas neoliberais esboçadas no ‘Charter’ de 1991
identificam áreas ‘marginais’, como as da região do Pacífico,
para vários projetos de macrodesenvolvimento, com vistas
a extrair seus recursos naturais e modernizá-las. Além disso,
em consonância com os apelos da Constituição pela
proteção do meio ambiente colombiano, as Terras Baixas 15 Estudos Feministas, Florianópolis, 12(1): 360, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... elas se mobilizam e vão além da retórica desenvolvimentista
do Estado e do discurso de uma etnia gendrada e da
tradição dos movimentos sociais negros na região. Concluo
discutindo como o ativismo das afro-colombianas complica
a nossa compreensão de como operam as intervenções
para o desenvolvimento. O ensaio mostra também como
as subjetividades e o agenciamento das mulheres negras
são moldados diferenciadamente, desigualmente e
discursivamente pelas, e contra as, intervenções políticas e
culturais na região. Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 17 Plan
P
Plan
P
Plan
P
Plan
P
Plan
Pacífico
acífico
acífico
acífico
acífico:
da
“inércia
do
:
da
“inércia
do
:
da
“inércia
do
:
da
“inércia
do
:
da
“inércia
do
subdesenvolvimento”
para
o
subdesenvolvimento”
para
o
subdesenvolvimento”
para
o
subdesenvolvimento”
para
o
subdesenvolvimento”
para
o
“desenvolvimento sem sujeitos”?
“desenvolvimento sem sujeitos”?
“desenvolvimento sem sujeitos”?
“desenvolvimento sem sujeitos”?
“desenvolvimento sem sujeitos”? Um tráfico incessante de mercadorias e pessoas fluía ao
longo do rio Atrato, em Quibdó (a capital do estado de
Choco). Os prédios de gesso e cimento, delineando o
malécon, rachavam e mofavam sob o intenso calor, com
sua pintura, outrora brilhante, empolando e descascando
na umidade do ar. Ao passar pelos vendedores, que
compravam peças artesanais de ouro de mulheres e
homens chocoanos, lembrei-me da vista aérea do rio Atrato
enquanto voava para Quibdó. Contaminado com o
mercúrio usado na mineração comercial, o rio Atrato
parecia uma corda grossa e verde insinuando-se
lentamente pela paisagem. Enquanto a mineração do ouro
e de outros metais preciosos (juntamente com a extração
da madeira e o comércio do marfim vegetal) tem sido a
base da economia extrativa da região desde os tempos
coloniais, a pesca, caça e a agropecuária são as principais
atividades de subsistência das comunidades negras dos
quatro estados do Litoral do Pacífico. Refletindo sobre a situação da região durante uma
visita a Quibdó nos anos 1980, Thomas Sanders, um ex-
professor de Religião e membro do Universities Field Staff
International, observou que “... o Choco continua a
simbolizar um tipo de pobreza e de subdesenvolvimento em
que cultura e raça têm influência”.4 De acordo com Sanders,
a região do Choco continuou a sofrer da mesma “inércia
do subdesenvolvimento” que ele havia testemunhado
durante sua primeira visita em 1970, apesar do aumento
do preço do ouro no mundo. Para Sanders, o futuro da região
residia no exercício do poder político e da responsabilidade
econômica para desenvolver os ricos recursos naturais e
humanos e melhorar o padrão de vida.5 Mais de uma década depois, um relatório feito pelo
Departamento Nacional de Planejamento (DNP) reflete o
diagnóstico de Sanders, observando que “a pobreza é a Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 17 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 19 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... coligados a uma política partidária formal, opõem-se a tais
medidas modernizadoras. Janeth Rojas, uma feminista
mestiça de Cali, com experiência em projetos de
desenvolvimento a longo prazo na região do Pacífico,
resume sucintamente as reações de muitos/as afro-
colombianos/as: Essa visão de desenvolvimento com respeito às
comunidades negras baseia-se fundamentalmente em [a
noção de] “ter” (TENER) mais do que na possibilidade de
“ser” (SER). Com isso subentende-se que a melhoria de
qualidade de vida é medida mais através da satisfação
das necessidades básicas, melhores salários e aumento
do consumo, do que do fortalecimento do direito de ser
diferente.11 Talvez os mais notáveis críticos das iniciativas do
Estado colombiano para o desenvolvimento sejam os
membros de um grupo de intelectuais negros/as e ativistas
que uniram forças coletivamente sob o nome de Processo
de Comunidades Negras (PCN).12 Carlos Rosero, um antropólogo de Buenaventura e
membro da liderança do PCN, rejeita a representação que
o Estado faz da região e do seu povo como “marginal e
pobre”.13 Ele argumenta que o Plan Pacífico foi concebido
nos escritórios de planejamento do governo e é uma forma
de “desenvolvimento sem sujeitos”, porque ignora as
percepções, necessidades e agenciamento dos/as afro-
colombianos/as.14 Rosero e outros/as citam como evidência
o fato de que as primeiras versões do Plan Pacífico
enfocavam
primordialmente
planos
de
macrodesenvolvimento e investimento a fim de promover a
atividade econômica na região.15 Eles/as corretamente
mencionam que as questões sócio-culturais e ecológicas
foram introduzidas na agenda desse plano inicial após muito
lobby de grupos étnicos e de ONGs ambientalistas. y
g
p
Refletindo a respeito da onda anterior de
desenvolvimento no Pacífico, os/as críticos/as observam
como tais projetos – planejados na capital andina e
incompatíveis com as realidades sócio-culturais da região
– foram malsucedidos e fizeram surgir ondas de migração
em direção a cidades do Pacífico e além.16 A população
rural deslocada, inclusive um grande número de mulheres,
foi incorporada à economia monetária como mão-de-obra
barata na agroindústria e na cultura aquática, o que levou
a um colapso dos sistemas de subsistência locais e,
geralmente, deixou as comunidades mais empobrecidas
que antes.17 Betty Ruth Lozano18 observa que a intensificação
dos sistemas de acumulação de capital levou a uma
duplicação ou triplicação da carga de trabalho das
mulheres e reforçou a subordinação de gênero das mulheres 19 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 21 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... articula essa preocupação em termos coloquiais: Pra que serve água potável, eletricidade, escolas, clínicas,
aeroportos, reservas florestais, grandes empresas e dinheiro
se temos vergonha de sermos negros, se denunciamos
nossas fazendas e vendemos nossas terras, se nos
acanhamos de comer o que tiramos da floresta, ou as
nossas comidas tradicionais, e se nos esquecemos de como
fazer uma catanga [uma armadilha para lagostas], se
abandonamos o território que tem sido a nossa vida, se
todo esse desenvolvimento traz violência e pobreza? Nossa
busca principal agora é pelo direito a “ser” [SER] sem negar
a importância de “ter” [TENER] condições materiais para
gozar a vida como camponeses dignos, aqui nos nossos
rios.25 Em reuniões públicas, em negociações com o Estado,
assim como em entrevistas e conversas, os/as ativistas do
PCN chamam a atenção para as várias práticas de
subsistência das comunidades negras – agricultura, pesca
artesanal e caça, catação de mariscos, extração de vários
produtos renováveis, como também extração de madeira
e mineração em pequena escala. Observam que os/as afro-
colombianos/as vivem na região desde o período colonial
e desenvolveram esses sistemas de produção econômica
ecologicamente sustentáveis baseados/as nas tradições
africanas e ameríndias. Para os/as ativistas do PCN, são essas
práticas ‘ancestrais’ e as relações sociais associadas a elas,
baseadas no parentesco e nos laços de família, que
representam o locus da etnia e da diferença dos negros,
que devem ser o fulcro do “desenvolvimento culturalmente
apropriado” na região. Leyla Arroyo, um outro membro ativo
no PCN de Buenaventura, observa o seguinte: Acredito que a identidade étnica está no centro do
processo organizativo do povo negro. O reconhecimento
das comunidades negras como um grupo étnico é uma
de nossas maiores vitórias. Trabalhamos para que o nosso
povo sinta orgulho de ser negro; reafirmamos quem nós
somos, conquistamos auto-estima. [...] Acredito que
podemos desenvolver uma visão de vida alternativa, um
modelo econômico diferente, baseado nas nossas
tradições, que seja justo e que inclua todos os aspectos da
sociedade.26 Contudo, os/as afro-colombianos são marcados/as
por muitas distinções regionais, ideológicas, de classe e de
gênero. Dada essa heterogeneidade, os debates em torno
do que significa ser ‘negro/a’ ou ‘afro-colombiano/a’ e o
estágio incipiente das organizações negras em nível
nacional, não existe uma visão comum ou politicamente
viável de um “desenvolvimento culturalmente apropriado”. Dentro deste contexto, os movimentos sociais de negros na 21 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 23 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... observa que os primeiros planos de desenvolvimento para
a região surgiram nas décadas de 1950 e 1960, durante o
período de desenvolvimento do Terceiro Mundo pós-
Segunda Guerra Mundial. Uma série de desastres naturais
na região (inclusive um incêndio em Quibdó em 1966 e um
maremoto em Tumaco em 1979) também abriram espaço
para grandes projetos de reconstrução apoiados pelo
Estado. Nas décadas seguintes, vários eventos, instituições
e práticas – evangelização, concessões para florestamento,
estudos para avaliar o potencial agroindustrial,
desenvolvimento de culturas aquáticas, projetos para a
construção de portos e canais – levaram o desenvolvimento
e integração da região ao interior do país.28 No final da década de 1960, projetos de
desenvolvimento como o PLADEICOP (Projeto de
Desenvolvimento Integrado para o Pacífico Colombiano),
do presidente Belisario Betancur, tinham as mulheres como
alvos de projetos de bem-estar social, contra a pobreza e
de saúde rural. Essas iniciativas continuaram na forma de
programas de assistência como o El Plan de Hogares de
Bienestar (Plano para Casas de Bem-Estar), que era parte
do plano sócio-econômico do presidente Virgilio Barcos
para a região do Pacífico.29 Programas do convênio entre
a Corporación Autônoma Regional Del Valle Del Cauca e
a Holanda introduziram novas tecnologias e técnicas para
a intensificação da produção agrícola em pequena escala
na região. Embora esses programas produtivos não visassem
às mulheres, como Lozano e Rojas demonstram,30 eles
incorporaram com sucesso as mulheres negras ao processo
de desenvolvimento. Por exemplo, na região de Tumaco,
no Pacífico Sul, mulheres, em grande número, foram
empregadas em pequenas tarefas em fazendas de
camarão e em plantações de palmeiras para a extração
de óleo, e integradas ao setor agroindustrial. Em 1988, um
PLADEICOP mais abrangente lançou um projeto direcionado
às mulheres para facilitar a contribuição delas ao setor
produtivo.31 Esses projetos de desenvolvimento
correspondiam à tendência internacional das abordagens
Mulheres-no-Desenvolvimento (Women-in-Development/
WID), que defendia a necessidade de integrar as mulheres
ao processo de desenvolvimento e demonstrava que, além
da contribuição ao bem-estar da família, as mulheres são
uma parte importante do setor econômico produtivo e
devem ter acesso igual aos seus benefícios.32 Como a pequena descrição no início desta seção
indica, as mulheres negras são uma parte ativa e visível da
economia da região. Na década de 1980, mulheres
padeiras, costureiras, peixeiras, vendedoras de frutas em
Guapi e Tumaco formavam pequenos grupos em torno de 23 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 2 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 25 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... princípio fundamental da rede era “criar organizações de
mulheres autônomas que manifestassem e refletissem nosso
desenvolvimento, interesses e identidade étnica e cultural”.40
CoopMujeres, Ser Mujer e Fundemujer estavam entre as
muitas organizações ligadas à Red. princípio fundamental da rede era “criar organizações de
mulheres autônomas que manifestassem e refletissem nosso
desenvolvimento, interesses e identidade étnica e cultural”.40
CoopMujeres, Ser Mujer e Fundemujer estavam entre as
muitas organizações ligadas à Red. Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 27 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Desarollo de la Mujer de Buenaventura (FUNDEMUJER), era
a maior das três cooperativas, consistindo de 25 grupos de
mulheres, com um total de 800 membros. Com uma história
organizacional e institucional estabelecida, as mulheres
negras começaram a questionar, nas cooperativas, sobre
suas identidades étnicas e de gênero, e procuraram ir além
das atividades produtivas.36 Em abril de 1995, houve um encontro entre o
CoopMujeres de Guapi com um novo programa para
mulheres negras fundado pelo governo do Canadá e
administrado pela Fundación FES, uma fundação privada
colombiana. Os executores desse fundo colombiano-
canadense encontraram-se com membros das
cooperativas para discutir suas propostas e planos para os
anos seguintes. No encontro, Sylveria Rodríguez, a diretora
do CoopMujeres, disse que os membros queriam direcionar
novos fundos para o treinamento técnico das mulheres. Além
disso, ela e membros da cooperativa queriam realizar
oficinas sobre mecanismos de participação política,
cidadania, relações interpessoais e familiares. Quando
perguntada a respeito de oficinas sobre os direitos das
mulheres, a resposta de Sylveria Rodríguez foi: Já conhecemos os nossos direitos; agora queremos
aprender como obter nossos direitos; ensinar a outras
pessoas sobre os nossos direitos. Precisamos educar os
nossos homens sobre os direitos das mulheres. No ano
passado, comemoramos o Dia dos Pais no Coop Mujeres. Este ano, estamos tentando fazer com que cada membro
“conquiste” o seu parceiro e o traga para a oficina. No ano anterior, uma oficina sobre cozinha regional
foi realizada paralelamente à comemoração do Dia dos
Pais. Tal evento combinou atividades produtivas (cozinhar)
com o resgate de tradições culinárias locais e exibiu as
atividades dos membros das cooperativas para os pais e
esposos presentes. No dia seguinte ao encontro, Cipriana Diuza, uma
professora e membro recente do CoopMujeres, contou-me
que se engajou na cooperativa porque era “organizada,
fundamentada, e ajudava ativamente mulheres solteiras,
mães solteiras, mulheres pobres e chefes de famílias”. Mas
confessou ter curiosidade a respeito de “toda essa confusão
em torno de ser mulher negra”. Ela sentia que “nós somos
mulheres negras, alegres, mas ainda escravizadas, ainda
temerosas. Ainda precisamos aprender a valorizar o nosso
dialeto, a nossa religião, as nossas danças”. Mulheres nas
cooperativas de Buenaventura, Guapi e Tumaco
expressaram sentimentos semelhantes. Elas queriam manter
o enfoque nas atividades produtivas geradoras de renda, 25 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... número um, o amigo-homem” tratava-se de uma alusão
não apenas aos homens em casa, como também aos
homens inseridos nas lutas mais amplas dos/as negro/as na
região. Os membros do Fundemujer afirmam: Queremos ver as lutas étnico-políticas com os olhos de
mulheres negras. Mas o PCN não quer as duas lutas juntas. A opinião deles é que a luta de gênero enfraquece a luta
étnica.46 Apontando exemplos das muitas mulheres negras
que estavam envolvidas no processo de mobilização para
a ratificação da AT 55, membros do Fundemujer ecoaram
a observação de Teofila de que as mulheres negras e suas
organizações desempenharam um papel crucial na luta
pela Lei 70. Mercedes, Patrícia, Dora e Myrna observaram
também que as mulheres negras ofereceram um apoio
logístico importante (cozinhando para o encontro,
administrando o escritório, controlando as finanças) dentro
do PCN. No entanto, como Teofila, elas sentiram que os
membros do PCN não reconheceram nem valorizaram o
trabalho e a importância das mulheres negras enquanto
mulheres.47 De acordo com elas, até as mulheres que têm
um papel importante no PCN não possuem uma
“consciência de gênero”. Patricia Moreno continua: Armando [do PCN] disse que eles tinham medo que nós
falássemos sobre gênero e esquecêssemos a luta étnica. Eles acham que questões de gênero vindas do “interior”
[referindo-se às capitais andinas da região e do país] irão
diluir a luta étnica. Isso os apavora. Eles acreditam que nós
não sentimos a nossa própria opressão. São as forças
externas que nos “pesquisam”. Como se não sentíssemos
nossa subordinação sexual e física! Os ativistas do PCN contestam tal caracterização de
seu posicionamento em relação às mulheres e às questões
de gênero. Eles enfatizam que não apenas reconhecem o
papel central desempenhado pelas mulheres negras na
vida social, cultural e política do Pacífico, como também
admitem a necessidade de se abordar questões de gênero
dentro do PCN.48 Entretanto, reiteram seu posicionamento
firme em relação às intervenções do Estado na região,
inclusive “a nova política de gênero”. Os membros do PCN
afirmam que as relações de gênero são relações sociais
fundamentais, e que a dinâmica entre homens e mulheres
negras são mais bem abordadas através de lutas para se
conquistar direitos étnicos e não através de programas
estatais direcionados às mulheres. VVVVVendo através dos olhos das mulheres
endo através dos olhos das mulheres
endo através dos olhos das mulheres
endo através dos olhos das mulheres
endo através dos olhos das mulheres
negras: as redes afro
negras: as redes afro
negras: as redes afro
negras: as redes afro
negras: as redes afro-colombianas
-colombianas
-colombianas
-colombianas
-colombianas Teofila Betancur é uma guapireña e membro ativo da Red
de Mujeres Negras. Durante uma conversa com ela em
Cali, em 1995, ela me disse: “As mulheres negras ajudaram
a Lei 70 a partir da sua condição de mulher (ser mujer). Contudo, elas têm pouca ou nenhuma participação
pública ou política enquanto mulheres negras. Isso também
é verdade nas cooperativas de mulheres negras. As
mulheres negras são discriminadas triplamente – enquanto
pobres, enquanto mulheres e enquanto negras. Isso as leva
a se subvalorizar. As mulheres negras na costa assim como
nas cidades estão alisando, pintando ou fazendo
permanentes em seus cabelos para ficarem ‘brancas’ e
adotam hábitos da alta sociedade colombiana em uma
tentativa de conseguir aceitação social. Isso representa
uma perda de valores e de beleza. Então, membros da
Rede organizaram oficinas de cabelos e concursos de
penteados entre as mulheres negras de Guapi. Agora, mais
de metade das guapireñas usam trancinhas, tranças e
outros penteados afro-colombianos tradicionais. Precisamos trabalhar a partir da ‘cabeça’ para recuperar
nossa identidade externa como mulheres negras, para
refletirmos a respeito e afirmarmo-nos como negras”.41 Teofila Betancur e suas companheiras acreditam que
o enfoque nas identidades e na auto-estima das mulheres
negras representa um verdadeiro primeiro passo em direção
aos objetivos mais amplos da Red de trabalhar para a
melhoria das mulheres negras e suas comunidades.42
Quando encontrei Teofila novamente em 1999, ela me disse
que a seção da Rede em Guapi agora incluía 74 grupos
locais, ribeirinhos e regionais de mulheres, no estado de
Cauca. Cada grupo está envolvido em vários tipos de
atividades, inclusive projetos ‘produtivos’ como o cultivo e a
recuperação de grãos alimentícios e plantas medicinais
nativas, assim como projetos de ‘conservação’ com foco
nas práticas agrícolas e silvícolas para conservar o meio
ambiente e a biodiversidade.43 De acordo com Teofila, as
diversas atividades e projetos empreendidos por grupos
dentro da Rede surgem de “suas necessidades, percepções
e experiências como mulheres negras, e representam a sua
força coletiva”. Para simbolizar essa força, a Rede de Guapi
adotou o nome Red de Organizaciones Femeninas del 27 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 29 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... étnicas e de gênero, para contestar as forças hegemônicas
do desenvolvimento apoiado pelo Estado. Abaixo, ofereço
uma leitura diferente do ativismo das mulheres negras. Argumento que as subjetividades e atividades das mulheres
negras são moldadas através e contra as intervenções
políticas e culturais na região. Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 31 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... A posição do PCN é a
de que as organizações e redes de mulheres negras
funcionando de maneira independente dos movimentos
étnicos mais amplos da região terminariam sendo 29 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Através de
Através de
Através de
Através de
Através de ser y tener
ser y tener
ser y tener
ser y tener
ser y tener: constituindo etnia,
: constituindo etnia,
: constituindo etnia,
: constituindo etnia,
: constituindo etnia,
gênero e desenvolvimento no P
gênero e desenvolvimento no P
gênero e desenvolvimento no P
gênero e desenvolvimento no P
gênero e desenvolvimento no Pacífico
acífico
acífico
acífico
acífico Ponderando sobre uma política de desenvolvimento
para e por mulheres no Pacífico colombiano, Maria Del
Rosario Minaescreve: Temos um interesse básico: trabalhar conjuntamente entre
nós mesmas e com outras para propor uma política de
desenvolvimento para as mulheres afrodescendentes da
região do Pacífico que corresponda à nossa existência
básica (SER), a partir da qual seja possível identificar as
condições e necessidades materiais e sociais (TENER) que
garantam essa existência.49 Para Mina, um enfoque nas necessidades materiais
não se choca com a recuperação e o orgulho da
identidade. Pelo contrário, na sua formulação da “política
de desenvolvimento para mulheres negras do Pacífico”, ‘ser’
(existir) e ‘tener’ (ter as necessidades que garantam essa
existência) estão dialeticamente ligados. Para as mulheres
negras do Pacífico, participar dos projetos de modernização
do Estado não significa apenas abandonar suas identidades
e tradições para conquistar aquilo que o desenvolvimento
promete. Pelo contrário, para as mulheres negras, engajar-
se no desenvolvimento significa engajar-se na luta pelo
poder de definir o que se entende por ‘necessidades’ e de
criar meios de atendê-las. Isso requer que as mulheres se
engajem estrategicamente com o poder estatal e com suas
intervenções desenvolvimentistas, assim como com as lutas
étnicas. Também requer que recorram ao seu conhecimento
das práticas tradicionais e culturais de sobrevivência no
Pacífico. Minha discussão sobre o ativismo das mulheres afro-
colombianas refere-se a pelo menos duas questões sobre o
debate em torno do desenvolvimento: primeiro, as lutas dos
grupos locais contra o Estado e a modernização; e, segundo,
os problemas do ativismo político e ‘agenciamento’ das
mulheres do Terceiro Mundo.50 A década de 1980 presenciou muitos países latino-
americanos enfrentando graves crises financeiras, tumultos
políticos e levantes sociais. Estudiosos/as dos novos
movimentos sociais (NMSs) na América Latina afirmam que
coalizões pouco organizadas de trabalhadores/as de 31 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 3 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... estados do sudoeste do Pacífico), assim como de representantes de
organizações da Costa Atlântica e da capital Bogotá. Grupos do estado
de Choco, que fica ao noroeste, preferiram se organizar
separadamente. estados do sudoeste do Pacífico), assim como de representantes de
organizações da Costa Atlântica e da capital Bogotá. Grupos do estado
de Choco, que fica ao noroeste, preferiram se organizar
separadamente. p
Minhas discussões sobre o posicionamento e estratégias do PCN
baseiam-se em vários encontros e conversas com membros de
organizações do PCN durante trabalho de campo na região. Líderes
do PCN, como Carlos Rosero, Libia Grueso, Hernan Cortés e Leyla Andréa
Arroyo também expressam seus pontos de vista em seminários,
conferências e fóruns com vistas a discutir os direitos dos/as negros/as,
que são amplamente publicados em minutas ou anais das conferências. Baseio-me nesses pareceres coletados durante o meu trabalho de
campo e forneço citações paralelas de fontes publicadas sempre que
possível. 13 Tais críticas ao desenvolvimento são ecoadas largamente por
acadêmicos/as e ativistas, no norte e no sul. Por exemplo, os/as vários/
as colaboradores/as do Dicionário do desenvolvimento: um guia para
o conhecimento como poder (Wolfgang SACHS, 1992) analisam certos
conceitos-chave do desenvolvimento – tais como Planejamento,
Pobreza, Produção, Progresso, Padrão de Vida, etc. – em um esforço
de expor os limites do pensamento acerca do desenvolvimento e de
imaginar uma “era pós-desenvolvimento”. Esses/as críticos/as
argumentam que, através de alegações enganosas de superioridade
dos sistemas econômico e político do Ocidente, os discursos sobre o
desenvolvimento constroem um Terceiro Mundo ‘subdesenvolvido’ e
impõem uma ‘racionalidade’ e conhecimento ocidental a esses
espaços. p ç
14 ROSERO, 1996, p. 182. 15 Alguns projetos propostos no Plan Pacífico pelo Conselho Nacional
de Política Econômica e Social (CONPES) e pelo Departamento Nacional
de Planejamento (DNP) da Colômbia incluem: a construção de estradas
ligando centros regionais-chave com centros de troca e comércio
andinos, construindo oleodutos e usinas hidroelétricas, e construindo e
modernizando vários portos. Ver em Diego PIEDRAHÍTA e María Estella
PINEDA, 1993, uma lista detalhada e uma descrição das propostas desses
projetos. Ver também www.dnp.gov.co para uma visão geral das
agendas de desenvolvimento da Colômbia. g
16 Gustavo DE ROUX, 1990-1991; Alvaro PEDROSA, 1996; ROJAS, 1994. ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... alternativas para o desenvolvimento e as literaturas dos NMSs
histórica e analiticamente, a fim de argumentar que o
desenvolvimento e os movimentos contra ele estão
implicados uns nos outros. Afirma que as buscas que vemos
hoje por alternativas às forças destrutivas do capitalismo
precisam começar a examinar como o moderno e o não-
moderno produzem um ao outro em inúmeros locais. p
Minha discussão do ativismo das mulheres negras no
Pacífico mostra que gênero desempenha um papel crucial
nessa produção e contestação do desenvolvimento. Enquanto os primeiros estudos acerca de gênero e
desenvolvimento retratavam as mulheres como vítimas e
recipientes passivas das políticas de bem-estar social,
estudos feministas recentes revelam a multiplicidade de
localidades e de questões em torno das quais uma
diversidade de mulheres age.59 Feministas pós-coloniais
como Chandra Mohanty, Aihwa Ong e Gayatri Spivak60
elucidam como estruturas do capitalismo, discursos de
desenvolvimento e de gênero, patriarcados locais e noções
de cultura constituem as subjetividades das mulheres de
maneiras específicas e são utilizados para reforçar certos
significados e práticas, e para evitar outros. Essas feministas
chamam a atenção para a natureza dinâmica e oposicional
da dominação e da resistência das mulheres dentro de
relações desiguais de poder. Por exemplo, em Women
Workers and Capitalist Scripts, Mohanty61 enfoca não
apenas a exploração de mulheres trabalhadoras do Terceiro
Mundo pela atual expansão capitalista, com também “seu
agenciamento enquanto trabalhadoras, em nome dos
interesses comuns das mulheres trabalhadoras, baseado na
compreensão de localidades e necessidades
compartilhadas, e nas estratégias/práticas de organização
que estão ancoradas na, e levam à, transformação das
vidas cotidianas das mulheres trabalhadoras”.62
Similarmente, no seu estudo sobre as operárias de fábricas
na Malásia, Ong63 discute como essas mulheres camponesas
que se tornaram assalariadas são moldadas pelas, e resistem
às, relações industriais capitalistas. O exemplo das cooperativas de mulheres negras e
da Red de Mujeres Negras destaca como as subjetividades
e as organizações afro-colombianas são moldadas pela
linguagem e pelas práticas de instituições para o
desenvolvimento e suas políticas de gênero atuais. Por sua
vez, as mulheres negras utilizam-se dos termos e recursos
das iniciativas desenvolvimentistas e dos discursos culturais
predominantes na região para abordar as suas necessidades
e subverter as agendas tanto do Estado como das lutas
étnicas. Através dessa constituição mútua, elas ajudam a
institucionalizar as intervenções do Estado e, simultaneamente, 33 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 35 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... manuscritos publicados e não-publicados sobre e pela rede de
mulheres, e de um videoteipe do segundo encontro da rede de mulheres
da região de Cauca e Nariño, realizado em Timbiqui, Cauca, no período
de 1 a 4 de maio de 1997. manuscritos publicados e não-publicados sobre e pela rede de
mulheres, e de um videoteipe do segundo encontro da rede de mulheres
da região de Cauca e Nariño, realizado em Timbiqui, Cauca, no período
de 1 a 4 de maio de 1997. 43 Ver ASHER, 2002, para uma análise mais detalhada sobre o
envolvimento de mulheres negras (inclusive do grupo de Teofila) em
projetos de desenvolvimento sustentável e de preservação da
biodiversidade, no estado de Cauca, no sudoeste colombiano. Ver
Juana CAMACHO, 1999 e 2001, para uma discussão sobre o papel
central das mulheres na administração de recursos agrícolas e da
biodiversidade, e na manutenção de redes sociais nas áreas costeiras
do estado de Choco, no noroeste. 44 Observação participativa no seminário e na marcha organizada pela
Red de Mujeres Chocoanas, no Dia Internacional da Mulher, 8 de março
de 1995, e em entrevistas com as organizadoras do evento, Adriana
Eliza Fox Porro e Nimia Teresa Cuesta. 45 Talvez a explicação por que as mulheres da OBAPO resolveram se
organizar sob os auspícios de uma organização negra resida no fato
de que a maior parte das posições de liderança na OBAPO é de
mulheres. 46 Isso me traz à mente as reflexões de Audre Lorde sobre o movimento
negro nos Estados Unidos: “Dentro das comunidades negras onde o racismo é uma realidade
viva, as diferenças entre nós geralmente parecem suspeitas e perigosas. A necessidade de unidade é normalmente mal interpretada como uma
necessidade de homogeneidade, e o olhar de uma feminista negra,
confundido com traição dos nossos interesses comuns enquanto um
povo” (LORDE, 1992, p. 51). p
(
p
)
47 Ver ASHER, 1997, para uma discussão sobre a questão contraditória
da presença de mulheres negras como líderes em movimentos sociais
negros e a sua invisibilidade enquanto mulheres negras nessas
organizações. 48 OCN, 1996, p. 256-260. 49 MINA, 1995. ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... 17 Por exemplo, a pesquisa de Carmen Diana DEERE e Magdalena LEÓN
(1981), em três regiões andinas que se desenvolvem com maior rapidez
na Colômbia e no Peru, mostrou que o maior envolvimento de mulheres
no trabalho agrícola caminha lado a lado com o empobrecimento
geral de grandes setores rurais. Ver também seu volume editado Rural
Women and State Policy: Feminist Perspectives on Latin American
Agricultural Development (DEERE e LEÓN, 1987) para discussões sobre
como as políticas agrícolas do Estado afetem as mulheres em várias
partes da América Latina e da bacia caribenha. 19 GRUESO, 1998; GRUESO et al., 1998. 20 OCN, 1996; ROSERO, 1995 e 1996. 21 Julio CARRIZOSA UMANÃ, 1993; Arturo ESCOBAR, 1996; ROJAS, 1994;
Juan Pablo RUIZ, 1993. 21 Julio CARRIZOSA UMANÃ, 1993; Arturo ESCOBAR, 1996; ROJAS, 1994;
Juan Pablo RUIZ, 1993. 23 Como era de se esperar, há debates intensos na Colômbia, inclusive
entre negros/as, sobre o que constitui a etnia e a diferença ‘afro-
colombiana’. Muitas pesquisas têm enfocado a forma como as
diferenças culturais e de identidade dos/as negros/as ou afro-
colombianos/as são entendidas e construídas pelos movimentos sociais
negros depois da Lei 70. Ver ASHER, 1998; Eduardo RESTREPO, 1998 e
2001; e Peter WADE, 1996, para algumas discussões importantes. Ver 35 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 37 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Ourselves as Women, Rescuing our Black Identity: Ethnicity
and Gender in the Pacific Lowlands.” Current World
Leaders, v. 40, n. 6, 1997. p. 106-127. Leaders, v. 40, n. 6, 1997. p. 106-127. ____. “Culture/Politics and Black Ethnicities in the Pacific
Coast of Colombia.” Paper presented at XXI International
Conference of the Latin American Studies Association,
Chicago, September 24-26, 1998. ____. “Mobilizing the Discourses of Sustainable Economic
Development and Biodiversity Conservation in the Pacific
Lowlands of Colombia.” Strategies: A Journal of Theory,
Culture and Politics, v. 13, n. 1, 2000. p. 111-125. ____. “Texts in Context: Afro-Colombian Women’s Activism
in the Pacific Lowlands of Colombia.” Feminist Review,
2002. Under review. Í ___. “Culture/Politics and Black Ethnicities in the Pacific
Coast of Colombia.” Paper presented at XXI International
C
f
f th
L ti
A
i
St di
A
i ti Culture and Politics, v. 13, n. 1, 2000. p. 111 125. ____. “Texts in Context: Afro-Colombian Women’s Activism
in the Pacific Lowlands of Colombia.” Feminist Review,
2002. Under review. BALANTA, Olivia; RODRÍGUEZ, Betty; SINISTERRA, Sonia;
Quiñonez, Piedad; ARROYO, Leila; EQUIPO DINAMIZADOR. “Red de Mujeres Negras del Pacífico: tejiendo procesos
organizativos autonomos”. Esteros, n. 9, p. 37-42, feb. 1997. BRAIDOTTI, Rosi, CHARKIEWICZ, Ewa, HÄUSLER, Sabine, and
WIERINGA, Saskia (eds.). Women, the Environment and
Sustainable Development: Towards a Theoretical
Synthesis. London: Zed Books, 1994. CAMACHO, Juana. “‘Todos tenemos derecho a su parte’:
derechos de herencia, acceso y control de bienes en
comundiades negras de la costa Pacífica chocoana”. In: CAMACHO, Juana; RESTREPO, Eduardo (Coords.). De
montes, ríos, y ciudades: territorios e identidades de la
gente negra en Colombia. Santa Fe de Bogotá:
Fundación Natura, Ecofondo, Instituto Colombiano de
Antropología, 1999. p. 107-130. _____. “Mujeres, zoteas y hormigas arrieras: prácticas de
manejo de flora en la costa Pacífica chocoana”. In:
ARROYO, Jesús; CAMACHO, Juana; LEYTON, Mireya;
GONZÁLEZ, Maribel (Coords.). Zoteas: biodiversidad y
relaciones culturales en el Chocó biogeográfico
colombiano. Bogotá: Instituto de Investigaciones
Ambientales del Pacífico-IIAP, Fundación Natura,
Fundación Swissaid-Colombia, 2001. p. 35-58. CAMACHO, Juana. “‘Todos tenemos derecho a su parte’:
derechos de herencia, acceso y control de bienes en
comundiades negras de la costa Pacífica chocoana”. In: CAMACHO, Juana; RESTREPO, Eduardo (Coords.). De
montes, ríos, y ciudades: territorios e identidades de la
gente negra en Colombia. Santa Fe de Bogotá:
Fundación Natura, Ecofondo, Instituto Colombiano de
Antropología, 1999. p. 107-130. p
g
,
p
_____. ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... 50 O ativismo das mulheres afro-colombianas baseado na ligação entre
‘ser’ e ‘tener’ também problematiza a caracterização da luta das
mulheres do Terceiro Mundo como sendo ou lutas ‘femininas’ para
atender às ‘necessidades práticas’ ou lutas ‘feministas’ pelo poder de
redefinir as relações de gênero e contestar as posturas políticas e
econômicas do Estado. Ver Marianne MARCHAND, 1995, para uma
discussão sobre como essas visões sobre as lutas das mulheres são
aplicadas para uma melhor compreensão do ativismo das mulheres. 51 Susan ECKSTEIN, 1989; David SLATER, 1985; e Arturo ESCOBAR e Sonia
ALVAREZ, 1992. 52 A literatura dos NMSs é influenciada em vários níveis pelos debates
pós-estruturalistas nas ciências sociais e humanas e textos ‘anti-
desenvolvimento’ que surgiram nas décadas de 1980 e 1990. Muitos/as
estudiosos/as e ativistas do Terceiro Mundo (ESCOBAR, 1992; SACHS, 1992;
e Vandana SHIVA, 1988) denunciam os projetos desenvolvimentistas como
um subgrupo da modernidade hegemônica, eurocêntrica. Baseando-
se em trabalhos recentes em historiografia pós-colonial, eles chamam a
atenção para a necessidade de se reconhecer racionalidades ‘não-
ocidentais’. De acordo com esses/as críticos/as, alternativas às tendências
homogeneizantes e excludentes do capitalismo global são encontradas
nas práticas tradicionais dos grupos locais. p
g
p
53 ESCOBAR e PEDROSA, 1993 e 1996. 54 Ou para usar a frase de Verónica Schild, questiono a “separação tão
definida entre Estado e sociedade civil” (SCHILD, 1998, p. 111), que
está implícita em muito da literatura dos NMSs sobre as lutas locais contra
o desenvolvimento. Escrevendo sobre o projeto de democratização e 37 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... _____.“Viejas y nuevas formas de capital y los dilemas de la
biodiversidad”. In: ESCOBAR, Arturo and PEDROSA, Alvaro
(eds.). Pacífico ¿Desarrollo o Diversidad?: Estado, capital
y movimientos sociales en el Pacífico colombiano. Bogotá: CEREC y Ecofondo 1996 p 109 131 _____.“Viejas y nuevas formas de capital y los dilemas de la
biodiversidad”. In: ESCOBAR, Arturo and PEDROSA, Alvaro
(eds.). Pacífico ¿Desarrollo o Diversidad?: Estado, capital
y movimientos sociales en el Pacífico colombiano. Bogotá: CEREC y Ecofondo 1996 p 109 131 _____.“Viejas y nuevas formas de capital y los dilemas de la
biodiversidad”. In: ESCOBAR, Arturo and PEDROSA, Alvaro
(eds.). Pacífico ¿Desarrollo o Diversidad?: Estado, capital
y movimientos sociales en el Pacífico colombiano. B
tá CEREC
E
f
d
1996
109 131 _____.“Cultural Politics and Biological Diversity: State, Capital,
and Social Movements in the Pacific Coast of Colombia.”
In: LOWE, Lisa, and LLOYD, David (eds.). The Politics of
Culture in the Shadow of Capital. Durham, NC: Duke
University Press, 1997. p. 201-225. ESCOBAR, Arturo, and ALVAREZ, Sonia. The Making of Social
Movements in Latin America: Identity, Strategy, and
Democracy. Boulder, CO: Westview Press, 1992. ESCOBAR, Arturo; PEDROSA, Alvaro. “¿Laboratorio para el
postdesarrollo?” Revista Universidad del Valle, n. 5, p. 34-45, 1993. ESCOBAR, Arturo; PEDROSA, Alvaro. “Camaroneras
comunitarias: ¿Alternativas al capital? Entrevista con
Harold Moreno y Relato de Doña Ester Caicedo”. In:
ESCOBAR, Arturo, and PEDROSA, Alvaro (Coords.). Pacífico
¿Desarrollo o Diversidad?: Estado, capital y movimientos
sociales en el Pacífico colombiano. Bogotá: CEREC y
Ecofondo, 1996. p. 144-175. ESTEROS. Mujeres negras e indigenas definiendo futuro, v. 5, n. 9, feb. 1997. Número especial. FEMPRESS: Red de comunicación alternativa de la mujer. Mujeres negras, Latinoamerica, 1995. Número especial. FEMPRESS: Red de comunicación alternativa de la mujer. Mujeres negras, Latinoamerica, 1995. Número especial. FERGUSON, James. The Anti-Politics Machine: “Development”,
Depoliticization, and Bureaucratic Power in Lesotho. Cambridge, UK: Cambridge University Press, 1990. GROS, Christian. Colombia indigena: identidad cultural y
cambio social. Bogotá, Colombia: CEREC, 1991. Cambridge, UK: Cambridge University Press, 1990. GROS, Christian. Colombia indigena: identidad cultural y
cambio social. Bogotá, Colombia: CEREC, 1991. GRUESO, Jesus Alberto; ESCOBAR, Arturo. “Las cooperativas
agrarias y la modernización de los agricultores”. In:
ESCOBAR, Arturo, and PEDROSA, Alvaro (eds.). Pacífico
¿Desarrollo o Diversidad?: Estado, capital y movimientos
sociales en el Pacífico colombiano. Bogotá: CEREC y
Ecofondo. 1996. p. 90-108. GRUESO, Libia Rosario. ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... “Mujeres, zoteas y hormigas arrieras: prácticas de
manejo de flora en la costa Pacífica chocoana”. In:
ARROYO, Jesús; CAMACHO, Juana; LEYTON, Mireya;
GONZÁLEZ, Maribel (Coords.). Zoteas: biodiversidad y
relaciones culturales en el Chocó biogeográfico
colombiano. Bogotá: Instituto de Investigaciones
Ambientales del Pacífico-IIAP, Fundación Natura,
Fundación Swissaid-Colombia 2001 p 35-58 CAMACHO, Juana; RESTREPO, Eduardo (Coords.). De montes,
ríos, y ciudades: territorios e identidades de la gente
negra en Colombia. Santa Fe de Bogotá: Fundación
Natura, ECOFONDO, Instituto Colombiano de
Antropología, 1999. CARRIZOSA UMAÑA, Julio. “El Chocó y el resto del mundo.”
EcoLogica: Politica, Medio Ambiente, Cultura, v. 4, n. 15-
16, p. 12-17, 1993. 39 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... “El ejericio del derecho al territorio
en la comunidad negra del Pacífico Sur: un reto en doble
vía”. Buenaventura, Colombia, 1998. Manuscrito. GRUESO, Libia, ROSERO, Carlos, and ESCOBAR, Arturo. “The
Process of Black Community Organizing in the Southern
Pacific Coast Region of Colombia.” In: ALVAREZ, Sonia,
DAGNINO, Evelina and ESCOBAR, Arturo (eds.). Cultures
of Politics/Politics of Cultures: Re-visioning Latin American
Social Movements. Boulder, CO: Westview Press, 1998. p. 196-219. 41 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... TINKER, Irene (ed.). Persistent Inequalities: Women and World
Development. Oxford: Oxford University Press, 1990. TINKER, Irene (ed.). Persistent Inequalities: Women and World
Development. Oxford: Oxford University Press, 1990. VILLA, William. “El Pacífico colombiano: políticas de
desarrollo”. Esteros, n. 1, p. 15-19, enero/marzo, 1993. _____.“El plan de desarrollo de comunidades negras en la
ley 70 de 1993”. Esteros, n. 7, p. 36-40, agosto 1995. VILLA, William. “El Pacífico colombiano: políticas de
desarrollo”. Esteros, n. 1, p. 15-19, enero/marzo, 1993. _____.“El plan de desarrollo de comunidades negras en la
ley 70 de 1993”. Esteros, n. 7, p. 36-40, agosto 1995. ,
, p
,
/
,
_____.“El plan de desarrollo de comunidades negras en la
ley 70 de 1993”. Esteros, n. 7, p. 36-40, agosto 1995. WADE, Peter. “The Language of Race, Place and Nation in
Colombia.” America Negra, n. 2, 1991. p. 41-68. _____. Blackness and Race Mixture: The Dynamics of Racial
Identity in Colombia. Baltimore and London: The Johns
Hopkins Press, 1993. _____. “The Cultural Politics of Blackness in Colombia.”
American Ethnologist, n. 22, v. 2, 1995. p. :341-357. _____. “Identidad y etnicidad”. In: ESCOBAR, Arturo; PEDROSA,
Al
(C
d ) P
ífi
¿D
ll
Di
id d? WADE, Peter. “The Language of Race, Place and Nation in
Colombia.” America Negra, n. 2, 1991. p. 41-68. WADE, Peter. “The Language of Race, Place and Nation in
Colombia.” America Negra, n. 2, 1991. p. 41-68. _____. Blackness and Race Mixture: The Dynamics of Racial
Identity in Colombia. Baltimore and London: The Johns
Hopkins Press, 1993. _____. “The Cultural Politics of Blackness in Colombia.”
American Ethnologist, n. 22, v. 2, 1995. p. :341-357. _____. “Identidad y etnicidad”. In: ESCOBAR, Arturo; PEDROSA,
Alvaro (Coords.). Pacífico ¿Desarrollo o Diversidad?:
Estado, capital y movimientos sociales en el Pacífico
colombiano. Bogotá: CEREC y Ecofondo, 1996. p. 283-
298. WADE, Peter. “The Language of Race, Place and Nation in
Colombia.” America Negra, n. 2, 1991. p. 41-68. _____. Blackness and Race Mixture: The Dynamics of Racial
Identity in Colombia. Baltimore and London: The Johns
Hopkins Press, 1993. _____. Blackness and Race Mixture: The Dynamics of Racial
Identity in Colombia. Baltimore and London: The Johns
Hopkins Press, 1993. Hopkins Press, 1993. _____. “The Cultural Politics of Blackness in Colombia.”
American Ethnologist, n. 22, v. 2, 1995. p. :341-357. American Ethnologist, n. 22, v. 2, 1995. p. :341 357. _____. “Identidad y etnicidad”. In: ESCOBAR, Arturo; PEDROSA,
Alvaro (Coords.). ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... OCN (Organización de Comunidades Negras). “Movimiento
negro, identidad y territorio: entrevista con la
Organización
de
Comunidades
Negras
de
Buenaventura”. In: ESCOBAR, Arturo; PEDROSA, Alvaro
(Coords.). Pacífico ¿Desarrollo o Diversidad?: Estado,
capital y movimientos sociales en el Pacífico
colombiano. Bogotá: CEREC y Ecofondo, 1996. p. 245-
265. ONG, Aihwa. Spirits of Resistance and Capitalist Discipline:
Factory Women in Malaysia. Albany, NY: State University
of New York Press, 1987. _____.“Colonialism and Modernity: Feminist Re-presentations
of Women in Non-Western Societies.” Inscriptions, v. 3-4,
n. 2, 1988. p. 79-93. PARDO, Mauricio (Coord.). Acción colectiva, Estado y
etnicidad en el Pacífico colombiano. Bogotá: ICANH,
Colciencias, 2001. PARDO, Mauricio; ALVAREZ, Manuela. “Estado y movimiento
negro en el Pacífico colombiano”. In: PARDO, Mauricio
(Coord.). Acción colectiva, Estado y etnicidad en el
Pacífico colombiano. Bogotá: ICANH, Colciencias, 2001. p. 229-258. PCN (Proceso de Comunidades Negras). “El Proceso de
Comunidades Negras en el centro-sur del Pacífico y el
problema del ‘desarrollo’”. Buenaventura, Colombia, n.d. Manuscrito. PEDROSA, Alvaro. “La institucionalizacion del desarrollo”. In:
ESCOBAR, Arturo; PEDROSA, Alvaro (Coords.). Pacífico
¿Desarrollo o Diversidad?: Estado, capital y movimientos
sociales en el Pacífico colombiano. Bogotá: CEREC y
Ecofondo, 1996. p. 66-89. PEET, Richard, and WATTS, Michael (eds.). Liberation
Ecologies: Environment, Development, Social
Movements. London and New York: Routledge, 1996. Í PIEDRAHÍTA, Diego; PINEDA, María Estella. “En vías de
desarrollo: lista de proyectos de desarrollo para el
Pacífico”. EcoLogica: Politica, Medio Ambiente, Cultura,
v. 4, n. 15-16, 1993. p. 83-86. p
RESTREPO, Eduardo. “La construcción de la etnicidad:
comunidades negras en Colombia”. In: SOTOMAYOR,
Maria Lucia (Coord.). Modernidad, identidad y
desarrollo: construcción de sociedad y re-creación
cultural en contextos de modernización. Bogotá: ICAN,
1998. p. 341-358. _____. “Imaginando comunidad negra: etnografía de la
etnización de poblaciones negras en el Pacífico sur
colombiano”. In: PARDO, Mauricio (Coord.). Acción
colectiva, Estado y etnicidad en el Pacífico colombiano. Bogotá: ICANH, Colciencias, 2001. p. 41-70. RESTREPO, Eduardo. “La construcción de la etnicidad:
comunidades negras en Colombia”. In: SOTOMAYOR,
Maria Lucia (Coord.). Modernidad, identidad y
desarrollo: construcción de sociedad y re-creación
cultural en contextos de modernización. Bogotá: ICAN,
1998. p. 341-358. _____. “Imaginando comunidad negra: etnografía de la
etnización de poblaciones negras en el Pacífico sur
colombiano”. In: PARDO, Mauricio (Coord.). Acción
colectiva, Estado y etnicidad en el Pacífico colombiano. Bogotá: ICANH, Colciencias, 2001. p. 41-70. 43 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 45 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Pacífico ¿Desarrollo o Diversidad?:
Estado, capital y movimientos sociales en el Pacífico
colombiano. Bogotá: CEREC y Ecofondo, 1996. p. 283-
298. WATTS, Michael. “‘A New Deal in Emotions’: Theory and
Practice and the Crisis of Development.” In: CRUSH,
Jonathan (ed.). Power of Development. London and NY:
Routledge, 1995. p. 44-62. WESTWOOD, Sallie, and RADCLIFFE, Sarah A. “Gender, Racism
and the Politics of Identities in Latin America.” In:
RADCLIFFE, Sallie A., and WESTWOOD, Sarah (eds.). ‘Viva’:
Women and Popular Protest in Latin America. London
and New York: Routledge, 1993. p. 1-29. Ser y T
Ser y T
Ser y T
Ser y T
Ser y Tener
ener
ener
ener
ener: Engendering Development and Ethnicity in the P
: Engendering Development and Ethnicity in the P
: Engendering Development and Ethnicity in the P
: Engendering Development and Ethnicity in the P
: Engendering Development and Ethnicity in the Pacific L
acific L
acific L
acific L
acific Lowlands of Colômbia
owlands of Colômbia
owlands of Colômbia
owlands of Colômbia
owlands of Colômbia
Abstract
Abstract
Abstract
Abstract
Abstract: In this paper I explore how Afro-Colombian women’s organizations and networks shape
and are shaped by state initiatives to develop and modernize the Pacific region. I argue that in
doing so these mobilize and go beyond the developmentalist rhetoric of the state and the
discourse of gendered black ethnicity and tradition of black political organizations in the region. Key words
Key words
Key words
Key words
Key words: Afro-Colombian women, development strategies, gendered ethnicity in politics. Tradução de Ana Cecília Acioli
Revisão da tradução: Susana Bornéo Funck Tradução de Ana Cecília Acioli
Revisão da tradução: Susana Bornéo Funck Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 45 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 45
|
https://openalex.org/W3032797196
|
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0233640&type=printable
|
English
| null |
Computational model of tranexamic acid on urokinase mediated fibrinolysis
|
PloS one
| 2,020
|
cc-by
| 5,254
|
PLOS ONE RESEARCH ARTICLE Tie Bo WuID1*, Thomas Orfeo2, Hunter B. Moore3, Joshua J. Sumislawski3, Mitchell
J. Cohen3, Linda R. PetzoldID1 1 Department of Mechanical Engineering, University of California Santa Barbara, Santa Barbara, California,
United States of America, 2 Department of Biochemistry, University of Vermont, Burlington, Vermont, United
States of America, 3 Department of Surgery, Denver Health and Hospital Authority, Denver, Colorado, United
States of America * tiebo@ucsb.edu * tiebo@ucsb.edu * tiebo@ucsb.edu a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 OPEN ACCESS Citation: Wu TB, Orfeo T, Moore HB, Sumislawski
JJ, Cohen MJ, Petzold LR (2020) Computational
model of tranexamic acid on urokinase mediated
fibrinolysis. PLoS ONE 15(5): e0233640. https://
doi.org/10.1371/journal.pone.0233640 Citation: Wu TB, Orfeo T, Moore HB, Sumislawski
JJ, Cohen MJ, Petzold LR (2020) Computational
model of tranexamic acid on urokinase mediated
fibrinolysis. PLoS ONE 15(5): e0233640. https://
doi.org/10.1371/journal.pone.0233640 Editor: Raghavan Raju, Medical College of Georgia,
Augusta, UNITED STATES Received: February 6, 2020
Accepted: May 9, 2020
Published: May 26, 2020 Received: February 6, 2020
Accepted: May 9, 2020
Published: May 26, 2020 Abstract Understanding the coagulation process is critical to developing treatments for trauma and
coagulopathies. Clinical studies on tranexamic acid (TXA) have resulted in mixed reports on
its efficacy in improving outcomes in trauma patients. The largest study, CRASH-2, reported
that TXA improved outcomes in patients who received treatment prior to 3 hours after the
injury, but worsened outcomes in patients who received treatment after 3 hours. No consen-
sus has been reached about the mechanism behind the duality of these results. In this
paper we use a computational model for coagulation and fibrinolysis to propose that defi-
ciencies or depletions of key anti-fibrinolytic proteins, specifically antiplasmin, a1-antitrypsin
and a2-macroglobulin, can lead to worsened outcomes through urokinase-mediated
hyperfibrinolysis. Computational model of tranexamic acid on
urokinase mediated fibrinolysis Tie Bo WuID1*, Thomas Orfeo2, Hunter B. Moore3, Joshua J. Sumislawski3, Mitchell
J. Cohen3, Linda R. PetzoldID1 PLOS ONE PLOS ONE Introduction Copyright: © 2020 Wu 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. Severe trauma often induces a coagulopathic state known as Acute Traumatic Coagulopathy
(ATC) that manifests in increased bleeding and resultant mortality [1]. The mechanisms
underlying ATC are not yet fully understood, making treatment difficult [2]. One treatment in
particular, tranexamic acid (TXA), has been found to be often effective [3, 4]. However, there
are unknown circumstances in which the treatment has been reported to worsen the condi-
tion, with increased bleeding and mortality. A meta-analysis of the CRASH-2 study found that
the relative risk of death due to bleeding in patients who received TXA compared to placebo
was 0.68 for patients who arrived within 1 hour after injury, 0.79 for patients who arrived
between 1 and 3 hours after injury, but 1.44 for patients who arrived more than 3 hours after
injury [5, 6]. The risk of worsening the situation is one of the main reasons why many health
care organizations are hesitant to incorporate TXA into their trauma protocol. Developing a
mechanistic understanding of how TXA can produce such contrasting results is necessary for
widespread adoption of the treatment. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: This material is based upon work
supported by the US Army Contracting Command,
Aberdeen Proving Ground, Natick Contracting
Division under Contract No. W911QY-15-C-0026. Funding was granted to Linda Petzold (LP). The
funders had no role in study design, analysis,
decision to publish, or preparation of the
manuscript. Tranexamic acid is an antifibrinolytic amino acid derivative that prevents the binding of
plasmin(ogen) to fibrin. This binding inhibits fibrinolysis in two ways (Fig 1). First, it slows 1 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis Competing interests: The authors have declared
that no competing interests exist. down tissue-plasminogen-activator(tPA) mediated conversion of plasminogen to plasmin, as
fibrin acts as a substrate that increases the catalytic efficiency of the reaction 500-fold. Even
after activation, TXA-bound plasmin cannot bind to fibrin, directly preventing the digestion
of fibrin [7]. Competing interests: The authors have declared
that no competing interests exist. Introduction The mechanisms behind the anti-fibrinolytic properties of TXA are well under-
stood, but the mechanism responsible for the possible increased bleeding and mortality have
yet to be determined. Competing interests: The authors have declared
that no competing interests exist. A possible explanation for the varying effectiveness of TXA treatment is the interaction
between TXA and urokinase plasminogen activator (uPA or urokinase) [8]. Studies have
shown that uPA levels are elevated in many instances in traumatic injury [9, 10], and since
uPA-mediated plasmin activation occurs in solution, the inhibition of fibrin binding by TXA
is inconsequential to this process. Furthermore, there is another binding site for TXA on plas-
minogen that induces a conformation change that speeds up plasmin activation approximately
3-fold [11]. However this explanation is incomplete, as multiple studies have shown that
despite the increase of plasmin generation, TXA still inhibits fibrinolysis in uPA-mediated sys-
tems [12, 13]. This suggests that other conditions must be met to change the behavior of TXA
from anti-fibrinolytic to pro-fibrinolytic. To explore the possible conditions under which TXA
can increase fibrinolysis through uPA-mediated plasmin generation, we constructed a differ-
ential equation based computational model. This model builds on previous ODE models [14–
18] of the coagulation and fibrinolysis system, but focuses specifically on investigating the
interaction between uPA and TXA, which are not present in these models. Fig 1. TXA interaction with plasminogen. Tranexamic Acid(TXA) inhibits fibrinolysis through its binding to plasminogen (Pg). This binding
prevents plasminogen from binding to fibrin, which inhibits activation through tPA. https://doi.org/10.1371/journal.pone.0233640.g001 Fig 1. TXA interaction with plasminogen. Tranexamic Acid(TXA) inhibits fibrinolysis through its binding to plasminogen (Pg). This binding
prevents plasminogen from binding to fibrin, which inhibits activation through tPA. Fig 1. TXA interaction with plasminogen. Tranexamic Acid(TXA) inhibits fibrinolysis through its binding to plasminogen (Pg). This binding
prevents plasminogen from binding to fibrin, which inhibits activation through tPA. https://doi.org/10.1371/journal.pone.0233640.g001 https://doi.org/10.1371/journal.pone.0233640.g001 https://doi.org/10.1371/journal.pone.0233640.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 2 / 11 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis Fig 2. Simplified diagram of reaction network. This network diagram shows the important reactions in our model. Tissue factor (TF) initiates a chain
of reactions in the coagulation cascade resulting in thrombin (IIa). Thrombin converts fibrinogen into fibrin, forming the clot. Introduction The clot is broken down
by plasmin (Pn) which is activated from plasminogen (Pg) in one of two ways, by tissue-plasminogen activator (tPA) or urokinase-plasminogen
activator (uPA). This diagram illustrates how tranexamic acid (TXA) has properties that both inhibit and promote fibrinolysis. The purpose of this
paper is to explore which conditions cause the net effect to change from anti-fibrinolytic to pro-fibrinolytic. Additional abbreviation: Fibrin
Degradation Productions (FDP). Fig 2. Simplified diagram of reaction network. This network diagram shows the important reactions in our model. Tissue factor (TF) initiates a chain
of reactions in the coagulation cascade resulting in thrombin (IIa). Thrombin converts fibrinogen into fibrin, forming the clot. The clot is broken down
by plasmin (Pn) which is activated from plasminogen (Pg) in one of two ways, by tissue-plasminogen activator (tPA) or urokinase-plasminogen
activator (uPA). This diagram illustrates how tranexamic acid (TXA) has properties that both inhibit and promote fibrinolysis. The purpose of this
paper is to explore which conditions cause the net effect to change from anti-fibrinolytic to pro-fibrinolytic. Additional abbreviation: Fibrin
Degradation Productions (FDP). https://doi.org/10.1371/journal.pone.0233640.g002 https://doi.org/10.1371/journal.pone.0233640.g002 We found that additional plasmin inhibitors such as α1-antitrypsin(A1AT) and α2-macro-
globulin(A2M), two proteins not often associated with fibrinolysis, play a critical role in pre-
venting TXA-induced hyperfibrinolysis (Fig 2. If the other plasmin inhibitors are depleted,
α2-antiplasmin (AP or antiplasmin) is not sufficient in preventing fibrinolysis, and the pres-
ence of TXA acts to increase the rate of fibrinolysis. Computational model In this paper we constructed an ordinary differential equation (ODE) model of the coagulation
process. This includes reactions for thrombin generation, fibrin formation and fibrin degrada-
tion (Fig 2). Most of these reactions and their associated rate constants are taken from models
based on in vitro experimental results, and most of them can be found in previously published
ODE models [14–18]. In our model we also make the assumption that A2M and A1AT are not
being consumed outside of the coagulation and fibrinolysis systems. Because of the difficulty 3 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis Fig 3. Diagram of different variants of TXA-bound plasminogen. Plasminogen has 2 binding sites that interact with TXA (labeled x and y in the
model). One of the binding sites with Kd = 600 μM changes the conformation that speeds up activation by uPA. The other binding site with Kd = 1.1 μM
prevents binding to fibrin, which prevents activation via tPA as well as fibrin degradation. UPA can activate all 4 of these variants, whereas tPA can
activate only the variants with the second binding site free. Fig 3. Diagram of different variants of TXA-bound plasminogen. Plasminogen has 2 binding sites that interact with TXA (labeled x and y in the
model). One of the binding sites with Kd = 600 μM changes the conformation that speeds up activation by uPA. The other binding site with Kd = 1.1 μM
prevents binding to fibrin, which prevents activation via tPA as well as fibrin degradation. UPA can activate all 4 of these variants, whereas tPA can
activate only the variants with the second binding site free. https://doi.org/10.1371/journal.pone.0233640.g003 of accurately modeling these additional systems and their low impact on the overall system, we
took the conservative approach of excluding them. If these reactions were included, the rate of
A2M and A1AT consumption would increase and the results would be amplified in the favor
of our conclusions. Our model focuses on the interactions between TXA and fibrinolysis, so it includes a more
detailed model of TXA-plasminogen binding (Fig 3). We model plasminogen with 2 binding
sites for TXA (labeled x and y), resulting in 4 species of Pg, depending on whether TXA is
bound to one or both of those sites (Pg, Pgx, Pgy, Pgxy). The x site controls the pro-fibrinolytic
activity of TXA. PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 Computational model If TXA is bound to this site (Kd = 600 μM), then its activation rate via uPA is
increased 3-fold. The y site controls the anti-fibrinolytic activity of TXA and has a much higher
binding affinity (Kd = 1.1 μM). This is the binding site that fibrin and plasmin inhibitors such
as α2-antiplasmin bind to. If the y site is occupied by TXA, it cannot bind to fibrin, which pre-
vents it from being activated by tPA and prevents it from degrading fibrin even after being
converted to plasmin. While tPA cannot activate the plasminogen species with its y site bound
(Pgy, Pgxy), uPA can activate all of these species of plasminogen. Therefore, in uPA-mediated
systems, TXA will increase the amount of plasmin generated [11, 12]. 4 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis The reversibility of the TXA binding with plasmin(ogen) has an important consequence. It
means that eventually TXA bound to the y site will be replaced by a irreversible plasmin inhibi-
tor as long as there is some left in the system. This means that TXA is effective at inhibiting
fibrinolysis only if there is a sufficient supply of plasmin inhibitors [18]. A complete descrip-
tion of the model, a complete list of reactions, rate constants and initial conditions for our
model can be found in S1 Appendix in S1 File. The full model can be found online at https://
github.com/taicheeze/coag_ode_julia. We initiated coagulation with 5 pM of tissue factor (TF), which is frequently used in other
computational models [15]. We examined the rate of fibrinolysis initiated with 2.5 nM tPA or
5 nM uPA, and varied the amount of TXA in the system (0, 1, 3, 14, 54 and 3470 μM) to com-
pare to experimental results to those of Longstaff 2019 [13]. PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 UPA mediated fibrinolysis The interactions between TXA and uPA are more complex. Overall, fibrinolysis with uPA is
slower than with tPA, which can be for a variety of reasons. UPA activates plasmin in solution
rather than on the fibrin surface, making it easier to inhibit by plasmin inhibitors. In addition,
uPA is inhibited by protein C inhibitor (PCI) [20, 21] which is present in the body at a higher
concentration than the inhibitors of tPA. Since we are assuming that A2M and A1AT are not
being consumed by reactions outside of the coagulation and fibrinolysis systems, this affects
uPA-activated plasmin more than tPA activated plasmin. Focusing on the plasmin inhibition
role of these proteins, we simulated the effects of consumption of these proteins by varying
their initial concentrations. Under conditions simulated with high concentrations of plasmin
inhibitors (Fig 5A), TXA behaves in an anti-fibrinolytic manner. Despite increasing the
amount of plasmin in the system, the activated plasmin is quickly bound to TXA, preventing it
from degrading fibrin. The TXA bound to the active plasmin is later replaced by one of the
non-reversible plasmin inhibitors, which permanently inhibits it. Higher concentrations of
TXA shift the equilibrium between TXA-bound plasmin and free plasmin, lowering the
amount of free plasmin at any given time, resulting in quick inhibition by other plasmin
inhibitors. When α1-antitrypsin is removed from the system (Fig 5B), the behavior of the system
remains mostly unchanged. The effects of A1AT are not obvious until both A1AT and A2M
are removed. In this system, A1AT appears to play a role redundant to A2M except much
weaker, due to its much slower binding to plasmin [22]. When A2M is removed from the sys-
tem (Fig 5C), uPA mediated fibrinolysis becomes much more apparent. However, TXA retains
its anti-fibrinolytic behavior, with a small change in behavior in the 10-100 μM range, which
has also been shown experimentally [12]. However, once both A2M and A1AT are removed from the system(Fig 5D), the behavior of
TXA flips. In this system, the only plasmin inhibitor left is antiplasmin. Since the ratio of plas-
minogen to antiplasmin is nominally 2:1, a large amount of plasmin activation can deplete
antiplasmin. The presence of TXA increases the rate of plasmin activation, and since the bind-
ing between TXA and plasmin is reversible, antiplasmin will eventually deplete, and any
remaining activated plasmin will rapidly degrade fibrin, resulting in hyperfibrinolysis. UPA mediated fibrinolysis Addi-
tional results of uPA mediated fibrinolysis with varying levels of plasmin inhibitors can be
found in S1 Fig in the S1 File. TPA mediated fibrinolysis The effects of TXA on tPA mediated fibrinolysis in our computational model behave as
expected and are confirmed by results of previous empirical experiments(Fig 4) [12, 13, 19]. As more TXA was added to the system, fibrinolysis slowed down in a dose-dependent manner. This behavior is consistent through different initial concentrations of plasmin inhibitors (not Fig 4. Effects of TXA on tPA mediated fibrinolysis. Modeling results show that Tranexamic Acid (TXA) affects tPA
mediated fibrinolysis in a dose dependent fashion. As the concentration of TXA increases, the rate of fibrinolysis
decreases. At the highest dose of TXA, fibrinolysis appears to be completely inhibited. These simulations are initiated
with 5 pM tissue factor, and an initial tPA concentration of 2.5 nM. https://doi.org/10.1371/journal.pone.0233640.g004 Fig 4. Effects of TXA on tPA mediated fibrinolysis. Modeling results show that Tranexamic Acid (TXA) affects tPA
mediated fibrinolysis in a dose dependent fashion. As the concentration of TXA increases, the rate of fibrinolysis
decreases. At the highest dose of TXA, fibrinolysis appears to be completely inhibited. These simulations are initiated
with 5 pM tissue factor, and an initial tPA concentration of 2.5 nM. https://doi.org/10.1371/journal.pone.0233640.g004 5 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis shown) as expected since the interactions between TXA and tPA are straightforward and well
established. These results show that the bulk of the model, with the exception of the uPA path-
way of fibrinolysis, is in agreement with empirical data. TPA and uPA mediated fibrinolysis https://doi org/10 1371/journal pone 0233640 g005 https://doi.org/10.1371/journal.pone.0233640.g005 https://doi.org/10.1371/journal.pone.0233640.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 TPA and uPA mediated fibrinolysis The behavior of TXA also depends on the balance between tPA and uPA in the system. In sys-
tems depleted of A1AT and A2M, TXA remains anti-fibrinolytic in the tPA pathway, but
becomes pro-fibrinolytic in the uPA pathway. By modulating the ratio of tPA to uPA, the over-
all effect of TXA can go from pro-fibrinolytic (Fig 6A and 6B), to no effect(Fig 6C) to anti-
fibrinolytic(Fig 6D). We can see that across these concentrations of tPA, the curves for the
highest TXA concentrations are more-or-less identical. This shows that at high concentrations
of TXA, the tPA pathway is completely inhibited. 6 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis Fig 5. Effects of TXA on uPA mediated fibrinolysis. Modeling results reveal that the effect of TXA on uPA mediated fibrinolysis greatly depends on
the presence or absence of plasmin inhibitors in the system. When A2M is in the system, with (A) or without (B) A1AT, fibrinolysis is inhibited and
TXA behaves in an anti-fibrinolytic fashion. When A2M is removed from the system, but A1AT is still present (C), fibrinolysis can be observed. Under
these circumstances, TXA exhibits anti-fibrinolytic behavior and acts to slow down fibrinolysis in a dose dependent manner. When both A2M and
A1AT are removed from the system (D), TXA becomes pro-fibrinolytic, and speeds up the rate of fibrinolysis through increased plasmin generation
and antiplasmin depletion. These simulations are initiated with 5 pM tissue factor, and an initial uPA concentration of 5 nM. https://doi.org/10.1371/journal.pone.0233640.g005 Fig 5. Effects of TXA on uPA mediated fibrinolysis. Modeling results reveal that the effect of TXA on uPA mediated fibrinolysis greatly depends on
the presence or absence of plasmin inhibitors in the system. When A2M is in the system, with (A) or without (B) A1AT, fibrinolysis is inhibited and
TXA behaves in an anti-fibrinolytic fashion. When A2M is removed from the system, but A1AT is still present (C), fibrinolysis can be observed. Under
these circumstances, TXA exhibits anti-fibrinolytic behavior and acts to slow down fibrinolysis in a dose dependent manner. When both A2M and
A1AT are removed from the system (D), TXA becomes pro-fibrinolytic, and speeds up the rate of fibrinolysis through increased plasmin generation
and antiplasmin depletion. These simulations are initiated with 5 pM tissue factor, and an initial uPA concentration of 5 nM. Discussion In this paper we have proposed a mechanistic explanation for the variance of outcomes in
patients treated with TXA. In situations with elevated uPA and depletion of plasmin inhibitors
such as A1AT and A2M, TXA can speed up fibrinolysis, leading to increased bleeding. With-
out A1AT and A2M, antiplasmin is quickly depleted and the generated plasmin rapidly breaks
down any fibrin clots uninhibited. One possible mechanism for depletion of plasmin inhibi-
tors during trauma may arise from the interactions between A1AT and A2M with activated
Protein C (APC) [23]. Many studies have shown that high levels of protein C are correlated
with trauma patients with worse outcomes [24, 25]. This is more likely to occur in patients that
arrive late, as there is more time for the plasmin inhibitors to deplete. Furthermore, the likely
concurrence of high tPA in the system during trauma can work in tandem to push a system 7 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis Fig 6. Effects of TXA on systems with both tPA and uPA. Modeling results show that the effect of TXA on systems with both tPA and uPA depend on
the balance between the two pathways. The concentration of uPA is held constant at 5nM, as tPA is increased from 0.1nM to 2.5nM. When tPA is low
compared tp uPA (A and B), TXA exhibits the pro-fibrinolytic behavior similar to Fig 5D. This behavior shifts toward anti-fibrinolytic as tPA increases. At 1nM tPA (C), the hyperfibrinolysis occurs at similar times regardless of the concentration of TXA. At 2.5nM tPA (D), TXA is anti-fibrinolytic and
acts to slow down hyperfibrinolysis. These simulations are initiated with 5 pM tissue factor. https://doi.org/10.1371/journal.pone.0233640.g006 Fig 6. Effects of TXA on systems with both tPA and uPA. Modeling results show that the effect of TXA on systems with both tPA and uPA depend on
the balance between the two pathways. The concentration of uPA is held constant at 5nM, as tPA is increased from 0.1nM to 2.5nM. When tPA is low
compared tp uPA (A and B), TXA exhibits the pro-fibrinolytic behavior similar to Fig 5D. This behavior shifts toward anti-fibrinolytic as tPA increases. At 1nM tPA (C), the hyperfibrinolysis occurs at similar times regardless of the concentration of TXA. At 2.5nM tPA (D), TXA is anti-fibrinolytic and
acts to slow down hyperfibrinolysis. PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 Discussion These simulations are initiated with 5 pM tissue factor. https://doi.org/10.1371/journal.pone.0233640.g006 https://doi.org/10.1371/journal.pone.0233640.g006 toward hyperfibrinolysis [26]. Under these conditions, TXA increases the amount of plasmin
produced by uPA in solution that rapidly consumes antiplasmin. Once antiplasmin has been
reduced significantly, TXA loses its ability to prevent tPA-mediated fibrinolysis and any plas-
min generated on fibrin surfaces will go uninhibited as A2M and A1AT cannot bind to fibrin-
bound plasmin. This model also provides an explanation for empirical studies that show how TXA can
increase plasmin generation in uPA systems, but at the same time slow down fibrinolysis. This
model shows that TXA binding to plasmin alone cannot prevent antiplasmin from depleting,
because the binding between TXA and plasmin is reversible. Regardless of the amount of TXA
in the system, free plasmin will always be present, but higher concentrations of TXA decreases
the amount of free plasmin available. This free plasmin will be consumed by plasmin inhibitors
and shifts the equilibrium to generate more free plasmin. In healthy plasma, there is an ample 8 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis supply of plasmin inhibitors, and TXA serves to lower the amount of free plasmin at any given
time, leading to quick inhibition. However, if A2M and A1AT are depleted, TXA can act to
speed up the rate of antiplasmin depletion because the concentration of plasminogen is
approximately twice the concentration of antiplasmin. This leaves the remaining free plasmin
free to break down fibrin unimpeded. Because of this, the presence of additional irreversible
inhibitors is necessary in a functional fibrinolytic system. Even in the absence of TXA, these additional inhibitors are necessary in healthy fibrinolysis. During an injury, tPA deposits in endothelial cells are quickly released in large amounts into
the site of injury. During the time immediately after injury, plasmin activators greatly outnum-
ber its inhibitors such as PAI-1 and can quickly activate large amounts of plasmin [18]. With-
out additional inhibitors, antiplasmin would quickly deplete, resulting in hyperfibrinolysis. As TXA continues to be explored as treatment for different situations [27–29], a mechanis-
tic understanding of this phenomena becomes increasingly important in preventing unfore-
seen side effects of TXA treatment. Currently, the decision on whether to use TXA in
treatment depends on whether the patient arrived before or after 3 hours post injury. Discussion Although
this heuristic protocol can be helpful, it will undoubtedly lead to instances in which patients
who can benefit from TXA treatment are not receiving it and patients who would suffer
adversely from it will be given it. With an understanding of the conditions under which TXA
helps or worsens a patient’s condition, we can identify patients that should receive TXA and
ones that shouldn’t. In addition, TXA treatment can potentially be improved by using plasmin
inhibitors in conjection with TXA to mitigate the risk of hyperfibrinolysis through the deple-
tion of plasmin inhibitors. Conclusion This paper uses computational modeling to present a mechanistic explanation for the different
behaviors of tranexamic acid. TXA usually has anti-fibrinolytic properties, but in systems with
high levels of uPA and low levels of plasmin inhibitors, TXA can become pro-fibrinolytic,
resulting in worse outcomes. This understanding can potentially be used to adjust treatment
protocols and stratify patients according to TXA risk to ensure that treatment is beneficial to
patients receiving it. As we have done with previous models [18], this model can also be used
to test potential treatments such as the replenishment of key proteins such as α2-antiplasmin. Author Contributions Conceptualization: Thomas Orfeo, Hunter B. Moore, Joshua J. Sumislawski, Mitchell J. Cohen. References 1. Cohen MJ. Acute traumatic coagulopathy: Clinical characterization and mechanistic investigation. Thrombosis Research. 2014; 133(SUPPL. 1):S25–S27. https://doi.org/10.1016/j.thromres.2014.03.013
PMID: 24759135 2. Godier A, Susen S. Trauma-induced coagulopathy. Annales Francaises d’Anesthesie et de Reanima-
tion. 2013; 32:527–530. https://doi.org/10.1016/j.annfar.2013.07.013 PMID: 23916515 3. Cap AP, Baer DG, Orman JA, Aden J, Ryan K, Blackbourne LH. Tranexamic Acid for Trauma Patients:
A Critical Review of the Literature. The Journal of Trauma: Injury, Infection, and Critical Care. 2011; 71
(supplement):S9–S14. https://doi.org/10.1097/TA.0b013e31822114af 4. Scho¨chl H, Schlimp CJ, Maegele M. Tranexamic Acid, Fibrinogen Concentrate, and Prothrombin Com-
plex Concentrate. Shock. 2014; 41(SUPPL. 1):44–46. https://doi.org/10.1097/SHK. 0000000000000093 PMID: 24296431 5. Olldashi F, Kerc¸i M, Zhurda T, Ruc¸i K, Banushi A, et al. The importance of early treatment with tranexa-
mic acid in bleeding trauma patients: an exploratory analysis of the CRASH-2 randomised controlled
trial. The Lancet. 2011; 377(9771):1096–1101.e2. 6. Williams-Johnson JA, McDonald AH, Strachan GG, Williams EW. Effects of tranexamic acid on death,
vascular occlusive events, and blood transfusion in trauma patients with significant haemorrhage
(CRASH-2): a randomised, placebo-controlled trial. The Lancet. 2010; 376(9734):23–32. https://doi. org/10.1016/S0140-6736(10)60835-5 7. P L M. Tranexamic Acid: A review of its use in the treatment of hyperfibrinolysis. Drugs. 2012; 72
(5):585–617. https://doi.org/10.2165/11209070-000000000-00000 8. Takada A, Takada Y. Conversion of glu-plasminogen to plasmin by urokinase in the presence of tra-
nexamic acid. Thrombosis Research. 1981; 22(4):437–443. https://doi.org/10.1016/0049-3848(81)
90105-5 PMID: 6457415 9. Hijazi N, Abu Fanne R, Abramovitch R, Yarovoi S, Higazi M, Abdeen S, et al. Endogenous plasminogen
activators mediate progressive intracerebral hemorrhage after traumatic brain injury in mice. Blood. 2015; 125(16):2558–2567. https://doi.org/10.1182/blood-2014-08-588442 PMID: 25673638 10. Saes JL, Schols SEM, van Heerde WL, Nijziel MR. Hemorrhagic disorders of fibrinolysis: a clinical
review. Journal of Thrombosis and Haemostasis. 2018; 16(8):1498–1509. https://doi.org/10.1111/jth. 14160 11. Ellis V, Behrendt N, Dano K. Plasminogen activation by receptor-bound urokinase: A kinetic study with
both cell-associated and isolated receptor. Journal of Biological Chemistry. 1991; 266(19):12752–
12758. PMID: 1829461 12. Hoylaerts M, Lijnen HR, Collen D. Studies on the mechanism of the antifibrinolytic action of tranexamic
acid. BBA—General Subjects. 1981; 673(C):75–85. https://doi.org/10.1016/0304-4165(81)90312-3
PMID: 7193484 13. Longstaff C, Locke M. Increased urokinase and consumption of α2-antiplasmin as an explanation for
the loss of benefit of tranexamic acid after treatment delay. Journal of Thrombosis and Haemostasis. 2018; p. jth.14338. 14. Hockin MF, Jones KC, Everse SJ, Mann KG. A model for the stoichiometric regulation of blood coagula-
tion. J Biol Chem. 2002; 277(21):18322–18333. Formal analysis: Tie Bo Wu. Formal analysis: Tie Bo Wu. Investigation: Hunter B. Moore, Joshua J. Sumislawski. Methodology: Tie Bo Wu. Methodology: Tie Bo Wu. Project administration: Mitchell J. Cohen, Linda R. Petzold. Project administration: Mitchell J. Cohen, Linda R. Petzold. Resources: Thomas Orfeo, Hunter B. Moore. Resources: Thomas Orfeo, Hunter B. Moore. Supervision: Mitchell J. Cohen, Linda R. Petzold. 9 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis Visualization: Tie Bo Wu. Writing – original draft: Tie Bo Wu. Writing – review & editing: Tie Bo Wu, Thomas Orfeo, Linda R. Petzold. Writing – review & editing: Tie Bo Wu, Thomas Orfeo, Linda R. Petzold. References https://doi.org/10.1074/jbc.M201173200 PMID:
11893748 15. Brummel-Ziedins KE, Orfeo T, Callas PW, Gissel M, Mann KG, Bovill EG. The Prothrombotic Pheno-
types in Familial Protein C Deficiency Are Differentiated by Computational Modeling of Thrombin Gen-
eration. PLoS ONE. 2012; 7(9):e44378. https://doi.org/10.1371/journal.pone.0044378 PMID:
22984498 16. Mitrophanov AY, Wolberg AS, Reifman J. Kinetic model facilitates analysis of fibrin generation and its
modulation by clotting factors: implications for hemostasis-enhancing therapies. Mol BioSyst. 2014; 10
(9):2347–2357. https://doi.org/10.1039/c4mb00263f PMID: 24958246 10 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis 17. Wootton DM, Popel AS, Rita Alevriadou B. An experimental and theoretical study on the dissolution of
mural fibrin clots by tissue-type plasminogen activator. Biotechnology and Bioengineering. 2002; 77
(4):405–419. https://doi.org/10.1002/bit.10127 PMID: 11787013 18. Wu TB, Wu S, Buoni M, Orfeo T, Brummel-Ziedins K, Cohen M, et al. Computational Model for Hyperfi-
brinolytic Onset of Acute Traumatic Coagulopathy. Annals of Biomedical Engineering. 2018; 46
(8):1173–1182. https://doi.org/10.1007/s10439-018-2031-9 PMID: 29675813 19. SILVA MMCG, THELWELL C, WILLIAMS SC, LONGSTAFF C. Regulation of fibrinolysis by C-terminal
lysines operates through plasminogen and plasmin but not tissue-type plasminogen activator. Journal
of Thrombosis and Haemostasis. 2012; 10(11):2354–2360. https://doi.org/10.1111/j.1538-7836.2012. 04925.x PMID: 22974122 20. Stump DC, Thienpont M, Collen D. Purification and characterization of a novel inhibitor of urokinase
from human urine. Quantitation and preliminary characterization in plasma. The Journal of biological
chemistry. 1986; 261(27):12759–66. PMID: 3091604 21. Heeb MJ, España F, Geiger M, Collen D, Stump DC, Griffin JH. Immunological identity of heparin-
dependent plasma and urinary protein C inhibitor and plasminogen activator inhibitor-3. The Journal of
biological chemistry. 1987; 262(33):15813–6. PMID: 2824468 22. Kolev K, Le´ra´nt I, Tenekejiev K, Machovich R. Regulation of fibrinolytic activity of neutrophil leukocyte
elastase, plasmin, and miniplasmin by plasma protease inhibitors. The Journal of biological chemistry. 1994; 269(25):17030–4. PMID: 7516329 23. Martos L, Ramo´n LA, Oto J, Ferna´ndez-Pardo A´ , Bonanad S, Cid AR, et al. α 2 -Macroglobulin Is a Sig-
nificant in Vivo Inhibitor of Activated Protein C and Low APC:α 2 M Levels Are Associated with Venous
Thromboembolism. Thrombosis and Haemostasis. 2018; 118(4):630–638. https://doi.org/10.1055/s-
0038-1629902 PMID: 29448296 24. Brohi K, Cohen MJ, Ganter MT, Schultz MJ, Levi M, Mackersie RC, et al. Acute coagulopathy of trauma:
hypoperfusion induces systemic anticoagulation and hyperfibrinolysis. The Journal of trauma. 2008; 64
(5):1211–7; discussion 1217. https://doi.org/10.1097/TA.0b013e318169cd3c PMID: 18469643 25. PLOS ONE | https://doi.org/10.1371/journal.pone.0233640
May 26, 2020 References Howard BM, Kornblith LZ, Cheung CK, Kutcher ME, Miyazawa BY, Vilardi RF, et al. Inducing Acute
Traumatic Coagulopathy In Vitro: The Effects of Activated Protein C on Healthy Human Whole Blood. PLOS ONE. 2016; 11(3):e0150930. https://doi.org/10.1371/journal.pone.0150930 PMID: 27008408 26. Chapman MP, Moore EE, Moore HB, Gonzalez E, Gamboni F, Chandler JG, et al. Overwhelming tPA
release, not PAI-1 degradation, is responsible for hyperfibrinolysis in severely injured trauma patients. The journal of trauma and acute care surgery. 2016; 80(1):15–16. 27. Shakur H, Elbourne D, Gu¨lmezoglu M, Alfirevic Z, Ronsmans C, Allen E, et al. The WOMAN Trial
(World Maternal Antifibrinolytic Trial): tranexamic acid for the treatment of postpartum haemorrhage: an
international randomised, double blind placebo controlled trial. Trials. 2010; 11(1):40. https://doi.org/10. 1186/1745-6215-11-40 PMID: 20398351 28. The Crash-3 Trial CollaboratorsEffects of tranexamic acid on death, disability, vascular occlusive
events and other morbidities in patients with acute traumatic brain injury (CRASH-3): a randomised, pla-
cebo-controlled trial. The Lancet. 2019; 394(10210):1713–1723. 29. Ahmadzia HK, Phillips JM, Katler QS, James AH. Tranexamic Acid for Prevention and Treatment of
Postpartum Hemorrhage. Obstetrical & Gynecological Survey. 2018; 73(10):587–594. 11 / 11
|
https://openalex.org/W4288441213
|
https://www.frontiersin.org/articles/10.3389/fpubh.2022.969523/pdf
|
English
| null |
The impact and challenges of digital marketing in the health care industry during the digital era and the COVID-19 pandemic
|
Frontiers in public health
| 2,022
|
cc-by
| 2,342
|
Introduction Pasaribu SB, Novitasari D,
Goestjahjanti FS and Hendratono T
(2022) The impact and challenges of
digital marketing in the health care
industry during the digital era and the
COVID-19 pandemic. According to Arni and Laddha (1), the healthcare industry was under great pressure
due to the emergence of COVID-19. The COVID-19 pandemic has had devastating
effects on the global economy, industries, and organizations, affecting marketing and
spending strategies. Due to the total lockdown in various regions, digital marketing is
crucial because traditional marketing strategies are no longer working. Due to a large
number of quarantine patients at home, as well as concerns about the possibility of
contracting COVID-19, people have chosen not to visit health care facilities, both clinics
and hospitals, resulting in a decrease in the volume and income from prescribing drugs
from 2021 until now. Khan and Nawaz (2) stated that digital marketing strategies have
increased over the years, and spending on such plans has also increased. The COVID-19
pandemic accelerates the growth rate of digital marketing in the health industry and is
expected to increase the return of both direct visits and patient telemedicine to hospitals
so that hospital revenues also increase. Front. Public Health 10:969523. doi: 10.3389/fpubh.2022.969523 COPYRIGHT
© 2022 Pasaribu, Novitasari,
Goestjahjanti and Hendratono. This is
an open-access article distributed
under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution or reproduction
in other forums is permitted, provided
the original author(s) and the copyright
owner(s) are credited and that the
original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution
or reproduction is permitted which
does not comply with these terms. The development of the Internet, the World Wide Web, and digital technologies
such as technology platforms from desktops, laptops, smartphones, to tablet devices
used by consumers has changed marketing. Patients, who are convinced that they are
consumers of health services and products, are increasingly using the internet or other
digital technologies to find the right information, as well as choose and buy those goods
or services. For organizations like hospitals, especially during the COVID-19 pandemic
conditions, digital media and new technology platforms provide opportunities to expand
into new markets, offer new services, apply new online communication techniques, and
compete on a more equal footing with larger businesses. According to Arni and Laddha
(1) and Al-Weshah et al. TYPE Opinion
PUBLISHED 28 July 2022
DOI 10.3389/fpubh.2022.969523 TYPE Opinion
PUBLISHED 28 July 2022
DOI 10.3389/fpubh.2022.969523 The impact and challenges of
digital marketing in the health
care industry during the digital
era and the COVID-19 pandemic OPEN ACCESS
EDITED BY
Mochammad Fahlevi,
Binus University, Indonesia
REVIEWED BY
Simon Grima,
University of Malta, Malta
Magdalena Syrkiewicz-Switala,
Medical University of Silesia, Poland
*CORRESPONDENCE
Sahala Benny Pasaribu
bennypasaribu@trilogi.ac.id
SPECIALTY SECTION
This article was submitted to
Health Economics,
a section of the journal
Frontiers in Public Health
RECEIVED 15 June 2022
ACCEPTED 07 July 2022
PUBLISHED 28 July 2022
CITATION
Pasaribu SB, Novitasari D,
Goestjahjanti FS and Hendratono T
(2022) The impact and challenges of
digital marketing in the health care
industry during the digital era and the
COVID-19 pandemic. Front Public Health 10:969523 OPEN ACCESS
EDITED BY
Mochammad Fahlevi,
Binus University, Indonesia
REVIEWED BY
Simon Grima,
University of Malta, Malta
Magdalena Syrkiewicz-Switala,
Medical University of Silesia, Poland
*CORRESPONDENCE
Sahala Benny Pasaribu
bennypasaribu@trilogi.ac.id
SPECIALTY SECTION
This article was submitted to
Health Economics,
a section of the journal
Frontiers in Public Health
RECEIVED 15 June 2022 Sahala Benny Pasaribu1*, Dewiana Novitasari2,
Francisca Sestri Goestjahjanti2 and Tonny Hendratono3 1Faculty of Economics and Business, Trilogi University, South Jakarta, Indonesia, 2Economics and
Management Department, Sekolah Tinggi Ilmu Ekonomi Insan Pembangunan, Tangerang,
Indonesia, 3Tourism Department, Sekolah Tinggi Pariwisata Ambarrukmo, Yogjakarta, Indonesia digital marketing, health care industries, digital era, COVID-19, pandemic, hospital frontiersin.org Introduction (3), most consumers searched for information online about
treatment options or more generally, to learn about health problems or health care
providers. Digital marketing can simply be defined as achieving marketing goals through
the application of technology and digital media. Hospital use of digital technology
increased by 50% to reach healthcare consumers. According to de Ruyter et al. (4)
and Khan and Nawaz (2), 48% of healthcare provider executives see revenue growth increased by 50% to reach healthcare consumers. According to de Ruyter et al. (4)
and Khan and Nawaz (2), 48% of healthcare provider executives see revenue growth Frontiers in Public Health Frontiers in Public Health 01 frontiersin.org 10.3389/fpubh.2022.969523 10.3389/fpubh.2022.969523 Pasaribu et al. as a key benefit of digital investment. Today, consumers are
looking for a stress-free and trustworthy user experience. With
the
advancement
of
technology,
the
demand
for
innovative healthcare applications has increased. Therefore,
the implementation of health marketing strategies on digital
platforms will enable the health sector to grow. for other people in the future. Many people go to hospitals or
doctor websites for reviews, and use Google reviews to examine
and share medical experiences. In addition, a large number of
people use social media platforms to share their experiences. In previous research, we can see in Figure 1 that the opinion
framework shows a shift in digital marketing in the current
health industry. Content marketing has a positive impact on factors such
as customer engagement, trust, and loyalty. In comparison,
content marketing was found to be more effective at persuading
customer loyalty compared to trust and loyalty. Furthermore,
customer trust in a brand has a strong positive relationship with
loyalty. A positive effect of customer engagement on trust has
also been found. Digital marketing increases patient satisfaction,
loyalty, and engagement with hospital services. According to
Savitri et al. (7), Tancharoenwong (8), and Qian et al. (9), one
of the benefits of digital marketing is that it can be closer
to consumers. Hospitals should adopt a multi-channel content
marketing approach to take full advantage. Discussion and opinion Digital methods can promote medical services in order to
expand the business. The strategic way of thinking, in this
case, implies attracting new patients and offering them quality
health services, which ensures satisfaction and the possibility for
them to recommend further health facilities. This is consistent
with the data from Mishra that digital marketing increases
satisfaction, loyalty, and patient engagement with hospital
services. According to Wijaya et al. (5) and Wisetsri et al. (6), one of the benefits of digital marketing is to expand the
brand/business online. The results of a study in Bangladesh
stated that in order to collect information about doctors, people
rely on hospital websites because it is the most accurate and up-
to-date source of information, and collect information posted on
social media such as Facebook groups. Before deciding to visit
a health service, people research health care professionals and
share reviews about their experiences so that they can be useful According to Nunan and Di Domenico (10), Purcarea (11),
Purwanto (12), and Pourkarim et al. (13), digital marketing
effects and barriers are something to manage during COVID-19. A digital marketing strategy is adopted to manage work online
and persist with safety precautions. Technology brings both
negative and positive impacts to the society. However, during
the pandemic, digital marketing tools and management are still FIGURE 1
Opinion framework. 02 Frontiers in Public Health frontiersin.org Pasaribu et al. 10.3389/fpubh.2022.969523 effective and helpful in moving normal life into the new norm. Digital marketing has gained a lot of advantages in terms of
online businesses, small, or large entrepreneurs, excelling in the
advertising market well. and promoting the services to patients’ relatives and family. The selection of methods or channels for digital marketing
depends on the goals or targets to be achieved by the hospital. After determining the goals of digital marketing, the hospital
determines the choice of methods or digital media channels that
will be used, then determines the goals, targets, and objectives
to be achieved from each of the selected media, analyzes the
situation and audience/market share, and determines the budget
and marketing frequency. Furthermore, for its implementation,
it is necessary to determine who will carry out the marketing
strategy that has been chosen. Moreover, monitoring and
evaluation of each digital marketing strategy that has been
implemented must be carried out so that the hospital can
achieve its goals or targets effectively and efficiently. Author contributions All
authors
listed
have
made
a
substantial,
direct,
and intellectual contribution to the work and approved it
for publication. 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. Discussion and opinion According to Savitri et al. (7), Tancharoenwong (8), Qian
et al. (9), Wijayaa et al. (5), and Wisetsri et al. (6), several
challenges and barriers to the use of social media in hospital
digital marketing, which make health services lag behind the
industry in digital marketing, include security issues, patient
privacy, regulatory issues, lack of guidance on how to use
digital platforms properly, lack of staffinterest to use social
media or the right infrastructure to respond to complaints, and
unclear responsibilities for various internet marketing activities. As a result, management commitment and consistency are
needed because the use of digital technology requires adequate
resources in terms of finance, infrastructure, and manpower
to be more effective. Electronic communications should be
conducted through an experimental approach rather than a
planned approach to avoid poor integration of online and offline
marketing communications. Conflict of interest Digital technology marketing has a tremendous impact,
including
increasing
engagement
on
social
media
and
marketing, serving as a key performance indicator for analyzing
organizational values, increasing demand for digital, growing
product searches among users, and increasing demand for
content platforms. All of them have been demonstrated in
many companies and organizations. During the COVID-19
pandemic, digital marketing has hit its growth charts and
has made technological advances all over the world. Digital
marketing in hospitals during this pandemic is a marketing
strategy that has many benefits, including attracting new
patients,
expanding
business,
increasing
customer/patient
trust, strengthening customer/patient loyalty, increasing brand
awareness, encouraging patients to use hospital services, 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. 6. Wisetsri W, Soni N, Singh RK, Chaurasia PK, Gupta SK. The healthcare
sector: a development of digital marketing methods. Linguist Antverpiensia.
(2021) 3:2602–21. 7. Savitri C, Hurriyati R, Wibowo L, Hendrayati H. The role of social
media
marketing
and
brand
image
on
smartphone
purchase
intention.
Int
J
Data
Network
Sci.
(2022)
6:185–92.
doi:
10.5267/j.ijdns.2021.
9.009 8. Tancharoenwong AS. Facebook as a digital marketing tool: Facebook’s
engagement strategy for maternal and child health page. UTCC Int J Bus Econ.
(2018) 10:3–17. interaction and word-of-mouth on purchase intention: an empirical study from
Indonesian smartphone consumers. Int J Data Network Sci. (2021) 5:231–
8. doi: 10.5267/j.ijdns.2021.6.011 interaction and word-of-mouth on purchase intention: an empirical study from
Indonesian smartphone consumers. Int J Data Network Sci. (2021) 5:231–
8. doi: 10.5267/j.ijdns.2021.6.011 2. Khan RZ, Nawaz H. Impact and challenges of digital marketing during
Covid-19 pandemic. Gorteria J. (2021) 34:31–9. 11. Purcarea EVL. The impact of marketing strategies in healthcare systems. J
Med Life. (2019) 12:93–9. 9. Qian ZW, Wan GS, Du XL, Shi YF, Huang G. Performance evaluation of
digital marketing in health care industry with the application of data envelopment
analysis. Revista De Cercetare Si Interventie Sociala. (2018) 60:39–50. 10. Nunan D, Di Domenico M. Older consumers, digital marketing, and public
policy: a review and research agenda. J Public Policy Mark. (2019) 38:469–
83. doi: 10.1177/0743915619858939 Pasaribu et al. 13. Pourkarim M, Nayebzadeh S, Alavian SM, Hataminasab SH. Digital
Marketing: a unique multidisciplinary approach towards the elimination
of
viral
hepatitis.
Pathogens.
(2022)
11:626.
doi:
10.3390/pathogens
11060626 9. Qian ZW, Wan GS, Du XL, Shi YF, Huang G. Performance evaluation of
digital marketing in health care industry with the application of data envelopment
analysis. Revista De Cercetare Si Interventie Sociala. (2018) 60:39–50.
10. Nunan D, Di Domenico M. Older consumers, digital marketing, and public
policy: a review and research agenda. J Public Policy Mark. (2019) 38:469–
83. doi: 10.1177/0743915619858939
11. Purcarea EVL. The impact of marketing strategies in healthcare systems. J
Med Life. (2019) 12:93–9. 10.3389/fpubh.2022.969523 References 1. Arni P, Laddha S. Adoption of digital marketing in health industry. SIES J
Manage. (2017) 13:3–10. 1. Arni P, Laddha S. Adoption of digital marketing in health industry. SIES J
Manage. (2017) 13:3–10. interaction and word-of-mouth on purchase intention: an empirical study from
Indonesian smartphone consumers. Int J Data Network Sci. (2021) 5:231–
8. doi: 10.5267/j.ijdns.2021.6.011 2. Khan RZ, Nawaz H. Impact and challenges of digital marketing during
Covid-19 pandemic. Gorteria J. (2021) 34:31–9. 3. Al-Weshah GA, Kakeesh DF, Al-Ma’aitah NA. Digital marketing strategies
and international patients’ satisfaction: an empirical study in Jordanian
health service industry. Stud Appl. Econ. (2021) 39:90–99 doi: 10.25115/eea. v39i7.4811 4. de
Ruyter
K,
Keeling
DI,
Ngo
LV. When
nothing
is
what
it
seems:
a
digital
marketing
research
agenda. Aust
Mark
J. (2018)
26:199–203. doi: 10.1016/j.ausmj.2018.07.003 4. de
Ruyter
K,
Keeling
DI,
Ngo
LV. When
nothing
is
what
it
seems:
a
digital
marketing
research
agenda. Aust
Mark
J. (2018)
26:199–203. doi: 10.1016/j.ausmj.2018.07.003 8. Tancharoenwong AS. Facebook as a digital marketing tool: Facebook’s
engagement strategy for maternal and child health page. UTCC Int J Bus Econ. (2018) 10:3–17. 5. Wijayaa O, Sulistiyanib S, Pudjowatic J, Kurniasih N, Purwanto A. The role of social media marketing, entertainment, customization, trendiness, Frontiers in Public Health frontiersin.org 03 Pasaribu et al. 10.3389/fpubh.2022.969523 12. Purwanto
A. How
the
role
of
digital
marketing
and
brand
image
on
food
product
purchase
decisions? An
empirical
study
on
Indonesian SMEs in the digital era. J Indus Eng Manage Res. (2022) 3:
34–41. 13. Pourkarim M, Nayebzadeh S, Alavian SM, Hataminasab SH. Digital
Marketing: a unique multidisciplinary approach towards the elimination
of
viral
hepatitis. Pathogens. (2022)
11:626. doi:
10.3390/pathogens
11060626 Frontiers in Public Health 04 Frontiers in Public Health frontiersin.org
|
https://openalex.org/W2135886685
|
http://www.ajnr.org/content/ajnr/33/10/1964.full.pdf
|
English
| null |
Assessment of Salivary Gland Dysfunction after Radioiodine Therapy for Thyroid Carcinoma Using Non-Contrast-Enhanced CT: The Significance of Changes in Volume and Attenuation of the Glands
|
American journal of neuroradiology
| 2,012
|
cc-by
| 5,932
|
ORIGINAL
RESEARCH B. Nabaa
K. Takahashi
T. Sasaki
A. Okizaki
T. Aburano B. Nabaa
K. Takahashi
T. Sasaki
A. Okizaki
T. Aburano BACKGROUND AND PURPOSE: Although radiation induced damage to the salivary gland is a known
complication of radioactive iodine (131I) therapy for thyroid carcinoma, prediction of the severity and
reversibility of sialoadenitis is difficult. Our aim was to correlate the extent of salivary dysfunction
assessed by salivary gland scintigraphy with changes in the volume and attenuation of salivary glands
on nonenhanced CT in postoperative patients with thyroid cancer treated with RIT. MATERIALS AND METHODS: Forty patients with thyroid carcinoma, 13 men (age range, 21–80 years)
and 27 women (age range, 28–75 years) who underwent a total thyroidectomy and were treated with
RIT were assessed retrospectively. On CT, the percentage of volume reduction and the difference in
attenuation of the parotid and submandibular glands after RIT were determined and correlated with the
extent of radiation-induced salivary dysfunction on scintigraphy. RESULTS: The salivary gland volume significantly decreased with an increase in the dysfunction grade
on scintigraphy for both the parotid and submandibular glands (P .001). The attenuation significantly
increased with an increase in the dysfunction grade on scintigraphy for the parotid gland (P .001), but
not for the submandibular gland. The cutoff value of volume reduction to diagnose severe gland
dysfunction was 19.5% (sensitivity, 86.0%; specificity, 100%) for the parotid gland and 31.0%
(sensitivity, 100%; specificity, 97.0%) for the submandibular gland, and that of the attenuation change
was 9.8 HU (sensitivity, 81.0%; specificity, 95%) for the parotid gland. CONCLUSIONS: The reduction in volume of the parotid and submandibular glands and the increase in
attenuation of the parotid gland on nonenhanced CT can be indicators of the grade of RIT-induced
salivary dysfunction. ABBREVIATIONS: HU hounsfield unit; RIT radioiodine therapy; ROC receiver operating
characteristic analysis ABBREVIATIONS: HU hounsfield unit; RIT radioiodine therapy; ROC receiver operating
characteristic analysis obtained during the follow-up period after RIT. These
changes are likely due to radiation-induced sialoadenitis and
could be a predictor of salivary gland dysfunction. A few
studies have reported dose-dependent volume loss of the
salivary gland in patients undergoing external radiation for
head and neck cancers,10-13 but no previous studies have as-
sessed the effect of RIT on gland volume or attenuation, to
our knowledge. Received October 26, 2011; accepted after revision January 24, 2012.
From the Department of Radiology, Asahikawa Medical University, Asahikawa, Japan.
Please address correspondence to Koji Takahashi, MD, PhD, Department of Radiology,
Asahikawa Medical University, 2-1-1-1, Midorigaoka-higashi, Asahikawa, Japan 078-8510;
e-mail: taka1019@asahikawa-med.ac.jp
Indicates article with supplemental on-line figures.
http://dx.doi.org/10.3174/ajnr.A3063 Aburano
B. Nabaa, K. Takahashi, T. Sasaki, A. Okizaki and T. Aburano
B. Nabaa, K. Takahashi, T. Sasaki, A. Okizaki and T. of October 23, 2024. This information is current as of October 23, 2024. This information is current as http://www.ajnr.org/content/33/10/1964
https://doi.org/10.3174/ajnr.A3063
doi:
2012, 33 (10) 1964-1970
AJNR Am J Neuroradiol http://www.ajnr.org/content/33/10/1964
https://doi.org/10.3174/ajnr.A3063
doi:
2012, 33 (10) 1964-1970
AJNR Am J Neuroradiol ORIGINAL
RESEARCH T
he use of RIT for ablation of residual thyroid tissue after
surgery has been established for the management of differ-
entiated thyroid cancer.1 Due to simultaneous accumulation
of 131I in the salivary glands, radiation damage to the salivary
gland is a known immediate and long-term complication of
RIT.2-4 Patients with RIT-induced salivary dysfunction have a
variety of symptoms, including swelling and pain in the pa-
rotid region, dryness of the mouth, altered taste, and difficulty
swallowing. With RIT, however, calculation of the actual ab-
sorbed dose in the salivary glands and prediction of the sever-
ity and reversibility of associated sialoadenitis are difficult. Generally, qualitative and quantitative salivary gland scintig-
raphy has been used for the assessment and follow-up of
salivary gland dysfunction after RIT.5-9 T In this study, we examine the relationship between the ex-
tent of salivary gland dysfunction evaluated on scintigraphy
and changes in volume and attenuation of the salivary glands
on CT in postoperative patients with thyroid carcinoma
treated with therapeutic doses of radioiodine. CT of the neck and chest is usually performed in patients
who have undergone RIT for evaluation of the recurrence
and metastasis of thyroid carcinoma. We have noticed that
the salivary glands, especially the parotid glands, show
some shrinkage and an increase in attenuation on CT scans ORIGINAL RESEARCH ORIGINAL RESEARCH All CT studies were assessed by 2 radiologists (T.S. and B.N.,
with 9 and 3 years’ experience in head and neck imaging, respec-
tively), who were blinded to the results of scintigraphy. The volume
and attenuation of the bilateral parotid and submandibular glands
were retrospectively measured in each patient on both CT images
obtained before the initial RIT and after the last RIT. The volume of
the salivary gland was measured by summation of products of an area
of the salivary gland on each section and section thickness. The area of
the salivary gland was obtained by a manually contoured region-of-
interest measurement. The percentage of volume reduction of each
gland after treatment was calculated with the following formula:
(Pretreatment Volume Posttreatment Volume)/Pretreatment Vol-
ume 100. The attenuation of each gland was determined by a re-
gion-of-interest measurement (region-of-interest size, 40–60 mm2) on
CT scans obtained before and after the RIT, and their difference
(Posttreatment Attenuation Pretreatment Attenuation) was de-
fined as an attenuation change of the salivary gland. The mean of
the above-mentioned percentage of volume reduction and attenua-
tion change of the bilateral parotid and submandibular glands was
used as a CT index for each salivary gland. Patients The protocol for this retrospective study was approved by our insti-
tutional review board without requirement of informed consent. The
study subjects were 40 patients: Thirteen were men (age range, 21–80
years; mean age, 61 years) and 27 were women (age range, 28–75
years; mean age, 55 years). All patients had undergone a total thyroid-
ectomy for well-differentiated thyroid carcinoma and subsequent
RIT from January 2006 to January 2011. We divided the patients into
4 groups according to the number of doses of RIT: 20, 10, 4, and 6
patients received 1, 2, 3, and 4 doses, respectively. The individual 1964
Nabaa AJNR 33 Nov 2012 www.ajnr.org 1964
Nabaa AJNR 33 Nov 2012 www.ajnr.org 1964 Table 2: The correlation of the grade of submandibular gland
dysfunction on scintigraphy with the number of radioiodine
treatments
No. of
Radioiodine
Treatments
Grade of Salivary Dysfunction on Scintigraphy
1
2
3
4
Total
Pretreatment
31
9
0
0
40
1
11
5
4
0
20
2
0
4
4
2
10
3
0
1
2
1
4
4
0
2
2
2
6
Table 1: The correlation of the grade of parotid gland dysfunction
on scintigraphy and the number of radioiodine treatments
No. of
Radioiodine
Treatments
Grade of Salivary Dysfunction on Scintigraphy
1
2
3
4
Total
Pretreatment
32
8
0
0
40
1
8
3
5
4
20
2
0
1
1
8
10
3
0
0
0
4
4
4
1
0
0
5
6 doses ranged from 5.1 to 5.6 GBq (139–150 mCi), with a mean of
5.3 GBq (144.5 mCi). None of these patients had known salivary
diseases, including Sjo¨gren disease, chronic sialolithiasis, sialadenitis,
or sialosis. Table 1: The correlation of the grade of parotid gland dysfunction
on scintigraphy and the number of radioiodine treatments No. of
Radioiodine
Treatments
Grade of Salivary Dysfunction on Scintigraphy
1
2
3
4
Total
Pretreatment
32
8
0
0
40
1
8
3
5
4
20
2
0
1
1
8
10
3
0
0
0
4
4
4
1
0
0
5
6 Imaging Examinations Salivary gland scintigraphy and non-contrast-enhanced neck CT
were performed before the initial RIT in all patients. The average
time interval from pretreatment salivary gland scintigraphy and CT to
the initial RIT was 8 days (range, 1–15 days). Changes in the salivary
glands after RIT were evaluated on scintigraphy and CT scans ob-
tained on the same day, with an average interval of 9 months (range,
6–12 months) since the last therapy and of 17 months (range, 8–38
months) since the first dose of RIT. Table 2: The correlation of the grade of submandibular gland
dysfunction on scintigraphy with the number of radioiodine
treatments Scintigraphy Studies. Salivary gland function was estimated by
salivary gland scintigraphy by using 185-MBq (5 mCi) technetium
Tc99m pertechnetate. All salivary gland scintigraphic data were as-
sessed by a radiologist (A.O., with 15 years’ experience in nuclear
medicine). Depending on the salivary gland uptake and excretion, its
function was classified into the following 4 grades: grade 1, normal
uptake and excretion; grade 2, mild dysfunction, decreased salivary
uptake, and delayed excretion, with oral activity equal to salivary up-
take at 30–40 minutes; grade 3, moderate dysfunction, markedly de-
creased salivary gland uptake, and delayed excretion, with higher sal-
ivary gland activity than oral activity at 30–40 minutes; and grade 4,
severe dysfunction, severely decreased salivary uptake and higher
background than salivary activity during the entire study.7,8 To judge
the grade of function in the parotid and submandibular glands, we
initially determined the grade of each side gland separately. For the
final grade, we used the worst grade in cases with different grades of
the bilateral glands. No. of
Radioiodine
Treatments
Grade of Salivary Dysfunction on Scintigraphy
1
2
3
4
Total
Pretreatment
31
9
0
0
40
1
11
5
4
0
20
2
0
4
4
2
10
3
0
1
2
1
4
4
0
2
2
2
6 AJNR Am J Neuroradiol 33:1964–70 Nov 2012 www.ajnr.org
1965 Results Grade of Dysfunction on Scintigraphy and Its Correlation
with the Treatment Dose Statistical Analyses All statistical analyses were performed by using a commercially avail-
able software program (Statistical Package for the Social Sciences,
Version 15.0 for Windows; SPSS, Chicago, Illinois). The correlation
between the grade of dysfunction of the gland on scintigraphy and the
number of RIT sessions was assessed by using the Spearman rank
correlation test. The correlations of the volume reduction and atten-
uation change of the salivary glands with the dysfunction grade of the
salivary gland on scintigraphy were assessed by using a 1-way analysis
of variance. Multiple comparisons of volume reduction and attenua-
tion changes of the group with each dysfunction grade were assessed
by using the Tukey Honestly Significant Difference test. The compar-
isons of the incidences of each grade of dysfunction after RIT and
those of the mean values of pretreatment attenuation and attenuation
change after RIT between the parotid and submandibular glands were
assessed by using the 2 test and a paired t test, respectively. We cal-
culated a correlation coefficient to assess the correlation between 2
datasets. A P value .05 was considered statistically significant. The
cutoff value of the volume-reduction percentage and attenuation
change of the salivary gland to diagnose grade 4 (severe) dysfunction
of the gland was determined by an ROC curve. NECK
ORIGINAL RESEARCH CT Studies. CT examinations were performed by using a 16-
detector row scanner (Aquilion 16; Toshiba Medical Systems, Tokyo,
Japan). CT images were obtained with the following parameters:
the tube voltage, 120 kV; tube current, 280–400 mA (automatically
adjusted for the patient body build); gantry rotation time, 0.5 sec-
onds; detector collimation, 1 mm; and a table feed, 12 mm per
gantry rotation. Contiguous 3-mm-thick-section axial images were
reconstructed. All CT studies were performed without contrast
enhancement. Grade of Dysfunction on Scintigraphy and Its Correlation
with the Treatment Dose A 71-year-old woman who underwent 3 radioiodine treatments showed severe dysfunction of the parotid gland on scintigraphy. Non-contrast-enhanced CT images (A) before and
(B) after the treatment. The bilateral parotid glands (arrows) showed prominent volume reduction of 53% and an increase in attenuation of 32 HU on CT after the treatment. Fig 2. A 71-year-old woman who underwent 3 radioiodine treatments showed severe dysfunction of the parotid gland on scintigraphy. Non-contrast-enhanced CT images (A) before and
(B) after the treatment. The bilateral parotid glands (arrows) showed prominent volume reduction of 53% and an increase in attenuation of 32 HU on CT after the treatment. 13.9% (28.7%–41.4%) for grade 4 (n 21) (Figs 1 and 2). The
parotid gland volume after the treatment significantly de-
creased with an increase in the grade of dysfunction on scin-
tigraphy (P .001, 1-way analysis of variance). A significant
difference in the reduction volume was present between
grade 1 and grade 4 (P .001) and grades 2 and 3 and grade 4
(P .001) groups, but not between grade 1 and grades 2 and 3
groups, as determined by the Tukey Honestly Significant Dif-
ference test (Fig 3). of dysfunction in the submandibular glands also increased
with an increase in the number of treatments (r 0.636,
P .001, Spearman rank correlation test) (Table 2). When we compared the incidence of each grade of dysfunc-
tion after RIT in all patients, the parotid gland showed a sig-
nificantly higher grade of dysfunction than the submandibular
gland (P .002, 2 test). Results on CT and Their Correlation with the Grade of
Dysfunction on Scintigraphy The reduction volume (mean SD, 95% confidence inter-
val) in the submandibular gland after RIT, depending on the
grade of scintigraphy, was 4.0 3.1% (2.0%–6.1%) for
grade 1 (n 11); 14.7 9.2% (10.8%–18.5%) for grades 2
and 3 (n 24); and 42.3 11.3% (28.3%–56.3%) for grade 4
(n 5) (Figs 4 and 5). The submandibular gland volume after
the treatment significantly decreased with an increase in the
grade of dysfunction on scintigraphy (P .001, 1-way analysis
of variance). There was a significant difference in the reduc-
tion volume between grade 1 and grades 2 and 3 (P .001),
grades 2 and 3 and grade 4 (P .001), and grade 1 and grade 4
(P .001) groups, as determined by the Tukey Honestly Sig-
nificant Difference test (Fig 3). Due to the small number of total patients and to simplify the
analysis, we combined grades 2 and 3 salivary dysfunction into
1 category and divided all of the patients into 3 groups, de-
pending on the results of scintigraphy; grade 1, grades 2 and 3,
and grade 4 in the subsequent analyses. Grade of Dysfunction on Scintigraphy and Its Correlation
with the Treatment Dose The grade of function in the parotid gland in all patients before
RIT was grade 1 in 32, grade 2 in 8, grade 3 in 0, and grade 4 in
0 patients, and that after RIT was grade 1 in 9, grade 2 in 4,
grade 3 in 6, and grade 4 in 21 patients. The deterioration in
the parotid gland function on scintigraphy increased with an
increase in the number of radiation treatments (r 0.593,
P .001, Spearman rank correlation test) (Table 1). The grade of function in the submandibular gland in all
patients before RIT was grade 1 in 31, grade 2 in 9, grade 3 in 0,
and grade 4 in 0 patients, and that after RIT was grade 1 in 11,
grade 2 in 12, grade 3 in 12, and grade 4 in 5 patients. The grade AJNR Am J Neuroradiol 33:1964–70 Nov 2012 www.ajnr.org
1965 AJNR Am J Neuroradiol 33:1964–70 Nov 2012 www.ajnr.org
1965 Fig 1. A 73-year-old woman who underwent 2 radioiodine treatments showed mild dysfunction of the parotid gland on scintigraphy. Non-contrast-enhanced CT images (A) before and
(B) after the treatment. Bilateral parotid glands (arrows) showed only mild shrinkage (volume reduction of 12%) and an increase in attenuation (6 HU) on the CT scan obtained after the
treatment. Fig 1. A 73-year-old woman who underwent 2 radioiodine treatments showed mild dysfunction of the parotid gland on scintigraphy. Non-contrast-enhanced CT images (A) before and
(B) after the treatment. Bilateral parotid glands (arrows) showed only mild shrinkage (volume reduction of 12%) and an increase in attenuation (6 HU) on the CT scan obtained after the
treatment. Fig 2. A 71-year-old woman who underwent 3 radioiodine treatments showed severe dysfunction of the parotid gland on scintigraphy. Non-contrast-enhanced CT images (A) before and
(B) after the treatment. The bilateral parotid glands (arrows) showed prominent volume reduction of 53% and an increase in attenuation of 32 HU on CT after the treatment. Fig 1. A 73-year-old woman who underwent 2 radioiodine treatments showed mild dysfunction of the parotid gland on scintigraphy. Non-contrast-enhanced CT images (A) before and
(B) after the treatment. Bilateral parotid glands (arrows) showed only mild shrinkage (volume reduction of 12%) and an increase in attenuation (6 HU) on the CT scan obtained after the
treatment. Fig 2. Attenuation Change in the Gland on CT versus the
Grade of Dysfunction on Scintigraphy The attenuation change (mean SD, 95% confidence inter-
val) in the parotid gland after RIT, depending on the grade of
scintigraphy, was 4.3 3.7 HU (1.5–7.2 HU) for grade 1
(n 9); 4.3 3.4 HU (1.8–6.8 HU) for grades 2 and 3
(n 10); and 23.9 17.7 HU (15.8–31.9 HU) for grade 4
(n 21) (Figs 1 and 2). The attenuation change in the parotid
gland after the treatment significantly increased with an in-
crease in the grade of dysfunction on scintigraphy (P .001,
1-way analysis of variance). A significant difference in the
attenuation change was present between the grade 1 and
grade 4 (P .002) and between the grades 2 and 3 and grade 4
(P .001) groups, but not between the grade 1 and grades 2
and 3 groups, as determined by the Tukey Honestly Significant
Difference test (Fig 6). Generally, salivary dysfunction after RIT occurs in a dose-
dependent manner.2,7 If the patient receives a single dose of
6 GBq, it typically results in a 30% loss of parenchymal func-
tion, while a cumulative administered dose of 35 GBq results
in complete loss of glandular function, as determined by sali-
vary gland scintigraphy.15 Radiation-induced salivary gland
dysfunction is also a common complication in patients under-
going external radiation therapy for head and neck can-
cers.10,11 A complete loss of salivary gland function from ex-
ternal radiation therapy occurs at a dose level of 60 Gy. Given
the absorbed functional effect of complete loss in the salivary
gland by RIT and external radiation, the absorbed dose per
administered 131I activity was 1.7 Gy/GBq. However, the pub-
lished absorbed dose per administered activity exhibits a large
variation, with a range of 0.03–14 Gy/GBq.15 These data sug-
gest that the biologic effectiveness of external radiation and
RIT is not equivalent. The attenuation change (mean SD, 95% confidence in-
terval) in the submandibular gland after RIT, depending on
the grade of scintigraphy, was 4.4 3.5 HU (2.0–6.7 HU) for
grade 1 (n 11); 5.3 5.9 HU (2.8–7.8 HU) for grades 2 and
3 (n 24); and 10.2 2.8 HU (6.8–13.7 HU) for grade 4
(n 5) (Figs 4 and 5). In the submandibular glands, there was
no significant correlation between the attenuation change and
the grade of dysfunction on scintigraphy. Discussion Radioactive iodine (131I) is a well-known adjunctive treatment
for thyroid carcinoma, especially in patients with a high risk of
recurrence.1 In addition to accumulation in the thyroid tissue,
radioactive iodine also concentrates in the salivary glands and
typically leads to transient or permanent damage to the glands. In the salivary glands, serous cells are more susceptible to the
deleterious effects of ionizing radiation than mucous acini. Therefore, the serous-dominant parotid gland demonstrates a
more intense radiation sialoadenitis than the mixed mucous
and serous cell–containing submandibular and sublingual
glands.14 In line with this, our results showed that the grades of
salivary dysfunction after RIT were significantly higher in the
parotid glands than in the submandibular glands. ROC Curves and Cutoff Values We determined the cutoff values of volume reduction and
attenuation changes to diagnose grade 4 dysfunction of the
salivary glands by using ROC curves in both the parotid and
submandibular glands (On-line Figs 1 and 2). The best cutoff
value for the volume reduction to diagnose severe dysfunction
was 19.5% in the parotid gland and 31.0% in the submandib-
ular gland, and that in the attenuation change was 9.8 HU in
the parotid gland. The sensitivity, specificity, positive predic-
tive value, negative predictive value, positive likelihood ratio,
and negative likelihood ratio for each cutoff value are shown in
Table 3. Fig 3. The volume reduction of the parotid and submandibular glands on CT after
radioiodine therapy in relation to the dysfunction grade on scintigraphy (Tukey Honestly
Significant Difference test). In the parotid gland, a significant difference in the volume
reduction is present between the following groups: grade 1 and grade 4 (P .001) and
grades 2 and 3 and grade 4 (P .001), but not between grade 1 and grades 2 and 3. In
the submandibular gland, a significant difference in the volume reduction is present
between the following groups: grade 1 and grades 2 and 3 (P .001), grades 2 and 3 and
grade 4 (P .001), and grade 1 and grade 4 (P .001). The horizontal line in the box
indicates median, E, outliers; asterisk, extreme outliers (3 times the box height). The means of attenuation change after RIT of the parotid
gland and submandibular gland were 14.6 16.3 HU and
5.6 5.2 HU, respectively and the attenuation change of
the parotid gland was significantly larger than that in the sub-
mandibular gland (P .001). The attenuation change after RIT in the parotid gland
showed a significant inverse correlation with its pretreatment
attenuation (r 0.47, P .002), while no significant corre-
lation was noted between the attenuation change after RIT in
the submandibular gland and its pretreatment attenuation
(r 0.12, P .449). AJNR Am J Neuroradiol 33:1964–70 Nov 2012 www.ajnr.org
1967 Volume Reduction of the Gland on CT versus the
Grade of Dysfunction on Scintigraphy The reduction volume (mean SD; 95% confidence interval)
in the parotid gland after RIT, depending on the grade of scin-
tigraphy, was 2.5 4.2% (0%–5.8%) for grade 1 (n 9); 7.9
5.2% (4.2%–11.7%) for grades 2 and 3 (n 10); and 35.0 1966
Nabaa AJNR 33 Nov 2012 www.ajnr.org Attenuation Change in the Gland on CT versus the
Grade of Dysfunction on Scintigraphy No significant dif-
ferences were noted among the attenuation changes for each
group (Fig 6). The means of pretreatment attenuation of the parotid
and submandibular gland were 5.5 25.6 HU and 40.5
10.8 HU, respectively, and the attenuation of the submandib-
ular gland was significantly higher than that of the parotid
gland (P .001). The dose-effect relationship in the salivary glands is well-
known with regard to external radiation therapy,10-13 whereas
for RIT, predicting the severity of radiation-induced salivary
dysfunction can be difficult for the following reasons: First, AJNR Am J Neuroradiol 33:1964–70 Nov 2012 www.ajnr.org 1967 Fig 4. A 46-year-old woman who underwent 2 radioiodine treatments shows mild dysfunction of the submandibular gland on scintigraphy. Non-contrast-enhanced CT images (A) before
and (B) after the treatment. The bilateral submandibular glands (arrows) show a mild volume reduction of 11% and an increase in attenuation of 12 HU on CT after the treatment. Fig 4. A 46-year-old woman who underwent 2 radioiodine treatments shows mild dysfunction of the submandibular gland on scintigraphy. Non-contrast-enhanced CT images (A) before
and (B) after the treatment. The bilateral submandibular glands (arrows) show a mild volume reduction of 11% and an increase in attenuation of 12 HU on CT after the treatment. Fig 5. A 68-year-old woman who underwent 4 radioiodine treatments shows severe dysfunction of the submandibular gland on scintigraphy. Non-contrast-enhanced CT images (A) before
and (B) after the treatment. The bilateral submandibular glands (arrows) show a prominent volume reduction of 56% but a minimal increase in attenuation of 14 HU on CT after the
treatment. Fig 4. A 46-year-old woman who underwent 2 radioiodine treatments shows mild dysfunction of the submandibular gland on scintigraphy. Non-contrast-enhanced CT images (A) before
and (B) after the treatment. The bilateral submandibular glands (arrows) show a mild volume reduction of 11% and an increase in attenuation of 12 HU on CT after the treatment. ( )
g
(
)
Fig 5. A 68-year-old woman who underwent 4 radioiodine treatments shows severe dysfunction of the submandibular gland on scintigraphy. Non-contrast-enhanced CT images (A) before
and (B) after the treatment. The bilateral submandibular glands (arrows) show a prominent volume reduction of 56% but a minimal increase in attenuation of 14 HU on CT after the
treatment. Fig 5. A 68-year-old woman who underwent 4 radioiodine treatments shows severe dysfunction of the submandibular gland on scintigraphy. 1968
Nabaa AJNR 33 Nov 2012 www.ajnr.org Our results suggest that we can assess the extent of radia-
tion-induced salivary gland dysfunction in patients after RIT
on a noncontrast-enhanced CT study, which is performed as a
routine examination to screen for recurrence and metastasis. We might thereby avoid the routine survey with salivary scin-
tigraphy after RIT because the CT prediction of salivary dys-
function allows clinicians to inform patients of the extent of
salivary gland dysfunction without scintigraphy. Scintigraphy
could be reserved for cases in which acute deterioration of the
salivary gland function is suggested from clinical signs and
symptoms. The advantages of CT over scintigraphy for the
assessment of salivary function are its widespread availability
in most hospitals; the simplicity and objectiveness of
the widely used method of measurement of volume and atten-
uation of the structures of interest, including salivary glands;
and less expensive cost. There are some limitations to this study. First, although the
aging process could significantly affect salivary gland function
and attenuation, we did not consider the patient age owing to
the small number of subjects examined in this study. Second,
we could not determine the appropriate interval for perform-
ing CT after RIT for the assessment of associated salivary dys-
function because we did not sequentially perform CT studies
after the treatment. Third, because scintigraphy and CT were
performed on the same day for correlation in this study, we
could not assess the potential sensitivity of scintigraphy versus
CT as a function of time. Scintigraphy as a measure of salivary
function might be able to detect salivary dysfunction sooner
than CT, which is presumably measuring chronic changes
such as fibrosis. Fig 6. The attenuation change of the parotid and submandibular glands on CT after
radioiodine therapy, depending on the dysfunction grade on scintigraphy (Tukey Honestly
Significant Difference test). In the parotid gland, a significant difference in the attenuation
increase is present between the following groups: grade 1 and grade 4 (P .002), grades 2
and 3 and grade 4 (P .001), but not between grade 1 and grades 2 and 3. In the
submandibular gland, no significant difference is noted among the attenuation changes for
each group. The horizontal line in the box indicates the median; E, outliers; asterisk,
extreme outliers (3 times the box height). Conclusions A reduction in volume of the parotid and submandibular
glands and an increase in attenuation in the parotid gland on
noncontrast-enhanced CT can be indicators of the grade of
salivary dysfunction in patients who have undergone postop-
erative RIT for thyroid carcinomas. Acknowledgments
h
k h
l We thank the nuclear medicine technologist Junichi Sato and
the CT technologist Masahiko Suzuki for their contribution
and expertise in scanning technique. increase of nearly 10 HU indicated severe dysfunction of the
gland, with a sensitivity of 81% and a specificity of 95%. Such
a correlation was not present in the submandibular glands,
possibly because of the high attenuation already present in the
glands before treatment.17 In the current study, the pretreat-
ment attenuation of the submandibular gland was signifi-
cantly higher than that in the parotid gland. Moreover, its
attenuation before treatment showed an inverse correlation
with the attenuation change in the parotid gland, while that in
the submandibular gland did not show such a correlation. Salivary dysfunction occasionally becomes more evident dur-
ing a period of several months to 1 year after the last RIT,
indicating that slowly progressing fibrosis is part of the patho-
logic process involved in radiation-induced sialoadenitis.2,8
We think that replacement of the fat tissue by fibrosis within increase of nearly 10 HU indicated severe dysfunction of the
gland, with a sensitivity of 81% and a specificity of 95%. Such
a correlation was not present in the submandibular glands,
possibly because of the high attenuation already present in the
glands before treatment.17 In the current study, the pretreat-
ment attenuation of the submandibular gland was signifi-
cantly higher than that in the parotid gland. Moreover, its
attenuation before treatment showed an inverse correlation
with the attenuation change in the parotid gland, while that in
the submandibular gland did not show such a correlation. Salivary dysfunction occasionally becomes more evident dur-
ing a period of several months to 1 year after the last RIT,
indicating that slowly progressing fibrosis is part of the patho-
logic process involved in radiation-induced sialoadenitis.2,8
We think that replacement of the fat tissue by fibrosis within Table 3: The performance of the determined cutoff values of the
percentage of volume reduction and attenuation increase in the
salivary gland for the diagnosis of severe salivary dysfunction
Parotid
Gland
Submandibular
Gland
Cutoff Value
of the
Attenuation
Increase (HU)
Cutoff value of volume reduction
19.5%
31.0%
9.8
Sensitivity
0.86
1.00
0.81
Specificity
1.00
0.97
0.95
PPV
1.00
0.83
0.94
NPV
0.86
1.00
0.82
Positive LR
–
34.5
15.3
Negative LR
0.14
0
0.20
Note:—NPV indicates negative predictive value; PPV, positive predictive value; LR
likelihood ratio; –, not appreciable. Table 3: The performance of the determined cutoff values of the
percentage of volume reduction and attenuation increase in the
salivary gland for the diagnosis of severe salivary dysfunction Table 3: The performance of the determined cutoff values of the
percentage of volume reduction and attenuation increase in the
salivary gland for the diagnosis of severe salivary dysfunction Attenuation Change in the Gland on CT versus the
Grade of Dysfunction on Scintigraphy Non-contrast-enhanced CT images (A) before
and (B) after the treatment. The bilateral submandibular glands (arrows) show a prominent volume reduction of 56% but a minimal increase in attenuation of 14 HU on CT after the
treatment. the activity distribution of 131I in the salivary gland is in-
homogeneous. Second, the uptake of 131I in the salivary gland
is individually variable and is also affected by the cumulative
activity of previous treatments.4,7 Third, the biologic half-life
of 131I differs in each patient. Therefore, a clinically available
indicator for the salivary dysfunction is required in patients
undergoing RIT. contraindicated for a few weeks before the RIT, we obtained
noncontrast CT images. Our current results suggested that
the volume change of the salivary gland measured on CT could
be an indicator for the assessment of salivary dysfunction
after RIT. A volume reduction of nearly 20% in the parotid
and 30% in the submandibular gland suggested severe dys-
function of the gland, with sensitivities of 86% and 100%
and specificities of 100% and 97%, respectively. A few authors
have reported radiation-induced volume loss in the salivary
glands in patients receiving external radiation,10-13 and
Teshima et al10 suggested a correlation between the loss of
gland volume and decreased saliva production. However, no
previous reports have investigated the relationship between
the changes in the volume of the salivary gland on CT and the
grade of salivary gland dysfunction in patients who undergo
RIT. Salivary gland scintigraphy has been widely accepted as
a sensitive and valid method for evaluation of the function
of the salivary glands.5-9,16 Several authors have used salivary
gland scintigraphy for the assessment of salivary gland dys-
function after RIT and reported a good correlation with
the clinical and histopathologic features of the salivary
glands.5-9,16 In our study, salivary gland scintigraphy showed a
dose-dependent salivary dysfunction after RIT, similar to the
findings described in previous reports.5-9 The increase in attenuation after RIT on non-contrast-
enhanced CT was also significantly correlated with gland dys-
function on scintigraphy in the parotid glands. An attenuation CT examinations of the head and neck are commonly
performed during the follow-up period after RIT to screen for
recurrence and metastasis. Because iodine contrast material is 1968
Nabaa AJNR 33 Nov 2012 www.ajnr.org the salivary gland contributes to its attenuation increase after
RIT. References 1. Samaan NA, Schults PN, Hickey RC, et al. The results of various modalities of
treatment of well differentiated thyroid carcinomas; a retrospective review of
1599 patients. J Clin Endocrinol Metab 1992;75:714–20 2. AlexanderC,BaderJB,SchaeferA,etal.Intermediateandlongtermsideeffects
of high-dose radioiodine therapy for thyroid carcinoma. J Nucl Med 1998;39:
1551–54 3. Newkirk KA, Ringel MD, Wartofsky L, et al. The role of radioactive iodine in
salivary gland dysfunction. Ear Nose Throat J 2000;79:460–68 y g
y
4. Almeida JP, Sanabria AE, Lima EN, et al. Late side effects of radioactive iodine
on salivary gland in patients with thyroid cancer. Head Neck 2011;33:686–90 5. Bohuslavizki KH, Brenner W, Lassmann S, et al. Quantitative salivary gland
scintigraphy in the diagnosis of parenchymal damage after treatment with
radioiodine. Nucl Med Commun 1996;17:681–86 6. Malpani BL, Samuel AM, Ray S. Quantification of salivary gland function in
thyroid cancer patients treated with radioiodine. Int J Radiat Oncol Biol Phys
1996;35:535–40 7. Solans R, Bosch JA, Gaofre P, et al. Salivary and lacrimal gland dysfunction
(Sicca syndrome) after radioiodine therapy. J Nucl Med 2001;42:738–43 AJNR Am J Neuroradiol 33:1964–70 Nov 2012 www.ajnr.org
1969 AJNR Am J Neuroradiol 33:1964–70 Nov 2012 www.ajnr.org
1969 8. Caglar M, Tuncel M, Alpar R. Scintigraphic evaluation of salivary gland dys-
function in patients with thyroid cancer after radioiodine treatment. Clin
Nucl Med 2002;27:767–71 cancer using integrated CT/linear acceleratory system. Int J Radiat Oncol Biol
Phys 2004;59:960–70 cancer using integrated CT/linear acceleratory system. Int J Radiat Oncol Biol
Phys 2004;59:960–70 13. Robar JL, Day A, Clancey J, et al. Spatial and dosimetric variability of organs at
risk in head-and-neck intensity-modulated radiotherapy. Int J Radiat Oncol
Biol Phys 2007;68:1121–30 9. Raza H, Khan AH, Hameed A, et al. Quantitative evaluation of salivary gland
dysfunction after radioiodine therapy using salivary gland scintigraphy. Nucl Med Commun 2006;27:495–99 14. Mandel SJ, Mandel L. Radioactive iodine and salivary glands. Thyroid
2003;13:265–71 10. Teshima K, Murakami R, Tomitaka E, et al. Radiation-induced parotid gland
changes in oral cancer patients: correlation between parotid volume and sa-
liva production. Jpn J Clin Oncol 2010;40:42–46 15. Jentzen W, Schneider E, Freudenberg L. Relationship between cumulative
radiation dose and salivary gland associated with radioiodine therapy of
thyroid cancer. Nucl Med Commun 2006;27:669–76 11. Wang ZH, Yan C, Zhang ZY, et al. Radiation induced changes in parotid
and submandibular gland in patients with head and neck cancer receiving
postoperative radiotherapy: a longitudinal study. 8. Caglar M, Tuncel M, Alpar R. Scintigraphic evaluation of salivary gland dys-
function in patients with thyroid cancer after radioiodine treatment. Clin
Nucl Med 2002;27:767–71 References Laryngoscope 2009;119:
1966–74 16. Loutfi I, Nair MK, Ebrahim AK. Salivary gland scintigraphy: the use of semi-
quantitative analysis for uptake and clearance. J Nucl Med Technol 2003;31:
81–85 12. Baker JL Jr, Garden AS, Ang KK, et al. Quantification of volumetric and geo-
metricchangesoccurringduringfractionatedradiotherapyforheadandneck 17. Bryan RN, Miller RH, Ferreyro RI, et al. Computed tomography of the major
salivary glands. AJR Am J Roentgenol 1982;139:547–54 1970
Nabaa AJNR 33 Nov 2012 www.ajnr.org
|
https://openalex.org/W4361266941
|
https://journal.binayfoundation.org/article/73764.pdf
|
English
| null |
Disease characteristics and clinical outcomes of endometrial cancer in Asian Indian and Pakistani American (AIPA) women
|
International journal of cancer care and delivery
| 2,023
|
cc-by-sa
| 567
|
International Journal of Cancer Care and Delivery International Journal of Cancer Care and Delivery Vol. 3, Issue Supplement 1, 2023 Vol. 3, Issue Supplement 1, 2023 619 (59%) were Asian Indians, 362 (35%) were Indian/Pak
istanis, not specified, and 62 (6%) were Pakistanis. AIPAs
were significantly younger at diagnosis (Median age: 60 and
63 years, p = 0.00). Endometroid carcinoma was the most
common cancer. When compared, 87% of AIPA and 91% of
NHW had endometroid cancer. Likewise, serous cell carci
noma was seen in 11% AIPAs and 8% of the NHW popula
tion. Endometroid cancer was more common compared to
serous cell cancer among NHW when compared to AIPAs
(p < 0.001). AIPAs were less likely to receive chemother
apy than NHW (81% and 86%, p = 0.000). AIPAs were found
to have better survival than NHW in univariate analysis
(p=0.0001) and multivariate analysis after adjusting other
co-variates (HR = 0.68, CI: 0.72 - 0.98, p = 0.04). METHODS We utilized the SEER database to derive disease charac
teristics, therapy, and survival data for AIPA and Non-His
panic White women aged 18 years and above from 2000 to
2018. Baseline characteristics were compared among these
groups. Univariate survival analysis was done using a Ka
plan Meier plot with a log-rank test, and the Cox pro
portional-hazards model was used for multivariate survival
analysis. BACKGROUND Endometrial cancer is the fourth most common cancer in
the United States females. While the disparity in the out
comes of this cancer has been studied extensively among
other minority populations, it has not yet been studied for
Asian Indian and Pakistani American populations (AIPAs)-
one of the fastest-growing major ethnic groups. CONCLUSION AIPA women had better survival than the NHW population
with endometrial cancer when adjusted for covariates. But
there remains a need to define further the unique charac
teristics of endometrial cancer in AIPA women. Disease characteristics and clinical outcomes of endometrial cancer
in Asian Indian and Pakistani American (AIPA) women
Anish Shah, MD
1, Pravash Budhathoki, MD
1, Suman Gaire, MD
2, Ujjwal Karki, MD
3, Siddhartha Yadav, MD
4
1 Bronxcare Health System, Bronx, NY, USA, 2 Mount Sinai Hospital Chicago, Chicago, IL, USA, 3 Beaumont Hospital, Troy, MI, USA, 4 Mayo Clinic,
Rochester, MN, USA Keywords: endometrial cancer, disparities, minorities Keywords: endometrial cancer, disparities, minorities https://doi.org/10.53876/001c.73764 https://doi.org/10.53876/001c.73764 This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License
(CCBY-SA-4.0). View this license’s legal deed at https://creativecommons.org/licenses/by-sa/4.0 and legal code at https://cre
ativecommons.org/licenses/by-sa/4.0/legalcode for more information. Shah A, Budhathoki P, Gaire S, Karki U, Yadav S. Disease characteristics and clinical
outcomes of endometrial cancer in Asian Indian and Pakistani American (AIPA) women.
IJCCD. 2023;3(Supplement 1). doi:10.53876/001c.73764 Shah A, Budhathoki P, Gaire S, Karki U, Yadav S. Disease characteristics and clinical
outcomes of endometrial cancer in Asian Indian and Pakistani American (AIPA) women. IJCCD. 2023;3(Supplement 1). doi:10.53876/001c.73764 Shah A, Budhathoki P, Gaire S, Karki U, Yadav S. Disease characteristics and clinical
outcomes of endometrial cancer in Asian Indian and Pakistani American (AIPA) women. IJCCD. 2023;3(Supplement 1). doi:10.53876/001c.73764 Conference Abstracts RESULTS A total of 116,883 cases were included - among which
115,840 were NHW and 1043 were AIPAs. Among AIPAs, This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License
(CCBY-SA-4.0). View this license’s legal deed at https://creativecommons.org/licenses/by-sa/4.0 and legal code at https://cre
ativecommons.org/licenses/by-sa/4.0/legalcode for more information.
|
https://openalex.org/W3000677234
|
https://www.nature.com/articles/s41598-020-71548-z.pdf
|
English
| null |
Development of humanized mouse and rat models with full-thickness human skin and autologous immune cells
|
bioRxiv (Cold Spring Harbor Laboratory)
| 2,020
|
cc-by
| 9,844
|
Development of humanized mouse
and rat models with full‑thickness
human skin and autologous
immune cells Yash Agarwal1,4, Cole Beatty1,4, Sara Ho1,4, Lance Thurlow2, Antu Das1, Samantha Kelly1,
Isabella Castronova1, Rajeev Salunke1, Shivkumar Biradar1, Tseten Yeshi3,
Anthony Richardson2 & Moses Bility
1* The human skin is a significant barrier for protection against pathogen transmission. Rodent models
used to investigate human-specific pathogens that target the skin are generated by introducing
human skin grafts to immunocompromised rodent strains. Infection-induced immunopathogenesis
has been separately studied in humanized rodent models developed with human lymphoid tissue and
hematopoietic stem cell transplants. Successful co-engraftment of human skin, autologous lymphoid
tissues, and autologous immune cells in a rodent model has not yet been achieved, though it could
provide a means of studying the human immune response to infection in the human skin. Here, we
introduce the human Skin and Immune System (hSIS)-humanized NOD-scid IL2Rγnull (NSG) mouse
and Sprague–Dawley-Rag2tm2hera Il2rγtm1hera (SRG) rat models, co-engrafted with human full-thickness
fetal skin, autologous fetal lymphoid tissues, and autologous fetal liver-derived hematopoietic stem
cells. hSIS-humanized rodents demonstrate the development of human full-thickness skin, along
with autologous lymphoid tissues, and autologous immune cells. These models also support human
skin infection following intradermal inoculation with community-associated methicillin-resistant
Staphylococcus aureus. The co-engraftment of these human skin and immune system components into
a single humanized rodent model could provide a platform for studying human skin infections. The human skin provides the first line of host protection against environmental injury and host defense against
pathogens1, 2. Several emerging pathogens, including community-associated methicillin-resistant Staphylococ-
cus aureus (CA-MRSA), target the skin for infection and disease1, 3–5. Also, vector-borne infectious diseases
such as Lyme disease and dengue fever are transmitted via inoculation into the skin by ticks and mosquitos,
respectively6. Interactions between keratinocytes, skin fibroblasts, and cutaneous immune cells are involved in
initiating the systemic immune response and abrogate pathogen replication and dissemination to other sites
of replication7–10. Thus, the skin provides an ideal vaccination target for inducing immunity against various
pathogens, as reflected by the development of several novel vaccine technologies directed at the skin, including
skin-patch intradermal vaccines11, 12. p
In vivo models for studying environmental insults and pathogens that target the skin and associated cutaneous
immune cells primarily involve mice and rats3. These rodent models have improved mechanistic understanding
of human diseases; however, significant differences exist between the skin and immune system of humans and
rodents3, 13. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2020) 10:14598 Results The hSIS‑humanized NSG mouse model supports the development of full‑thickness human
skin, autologous lymphoid tissues (thymus and spleen), and human immune cells. We previ-
ously demonstrated that NSG mice support the development of human lymphoid tissues (thymus and spleen)
along with autologous immune cells following engraftment of fetal tissues and autologous hematopoietic stem
cells17. Several reports demonstrated that immunodeficient mice support the development of human skin fol-
lowing engraftment of human fetal skin43, 44. Here, we hypothesize that NSG mice will support co-engraftment
of human full-thickness fetal skin, autologous fetal lymphoid tissues, and autologous hematopoietic stem cells. Furthermore, we hypothesize that NSG mice will facilitate human skin and lymphoid tissue development, as
well as enable systemic human immune cell reconstitution in transplanted human tissues and the blood. We pro-
cessed human fetal spleen, thymus, and liver organs into ~ 1 mm3 pieces and isolated autologous human CD34+
hematopoietic stem cells from the fetal liver, then transplanted the tissues and hematopoietic stem cells into
irradiated NSG mice. Human skin tissues were obtained from the scalp and dorsum of donors and were used
in developing human skin engraftments with and without hair in the mouse model, respectively. Full-thickness
human fetal skin was processed via removal of excess fat tissues attached to the subcutaneous layer of the skin,
then engrafted over the rib cage, where the mouse skin was previously excised. In three cohorts, the overall suc-
cess of the human immune system and autologous full-thickness skin development and maintenance for ten
weeks or greater was over 75% (Supplementary Fig. 1). Gross analysis of the human skin xenograft in the hSIS-
NSG mouse model beginning at two weeks post-transplantation demonstrates wound healing and maturation
into adult-like human skin, which is evident at ten weeks post-transplantation (Fig. 1A, Supplementary Fig. 2). A
limitation of the human skin in the hSIS-NSG mouse model is the development of dry skin (resulting in harden-
ing) and early signs of murine hair loss (suggestive of graft-versus-host disease) at 20 weeks post-transplantation
(Supplementary Fig. 2). The human skin in hSIS-NSG mice also develops human skin appendages, with human
hair evident at 12 weeks post-transplantation (Fig. 1B). Gross analysis of human spleen and thymus xenografts
in hSIS-humanized NSG mice at ten weeks post-transplantation demonstrated the growth of those lymphoid
tissues under the kidney capsule (Fig. 1C)17. www.nature.com/scientificreports/ thus highlighting the need for humanized rodent models that can support the engraftment of both human skin
and immune system components1.ti To address the species gap between rodents and humans, researchers have engrafted the immunodeficient
NOD-scid IL2Rγnull (NSG) mouse model, which lacks mature lymphocytes and natural killer (NK) cells and
possesses defects in innate immunity, with various human cells and tissues19, 20. Termed humanized-NSG mice,
these models exhibit both human immune cell reconstitution and human lymphoid tissue growth and have
been used to recapitulate clinical features of human diseases (including skin-associated diseases)21–25. Several
separate reports demonstrate that immunodeficient mice support the engraftment of human skin26, 27. Adult
human skin-derived from either medical wastes (elective plastic surgery)28, 29 or tissue culture-derived engi-
neered skin (keratinocytes and fibroblasts)24 engrafts successfully in immunodeficient mice. Allogeneic adult
human-peripheral blood mononuclear cells (PBMCs) have been introduced into these models to mimic human
immune cell-skin interactions with infectious agents24, 29. Although these mouse models demonstrate successful
engraftment and development of transplanted human skin and are amenable to the transplantation of allogeneic
PBMCs, said platforms are not currently coupled with the engraftment of autologous lymphoid tissues that are
critical for a de novo immune response. Humanized mouse models that combine human skin, human immune
cells, and human lymphoid structures have yet to be established, despite their potential for developing a func-
tional in vivo system, which could enable studies on human skin-immune cell interactions30. Humanized mouse
models with human fetal-derived hematopoietic system and autologous lymphoid tissues are well-established19, 20. Additionally, full-thickness human fetal skin readily engrafts onto immunodeficient mice and develops into adult-
like skin due to its high regenerative capability31, 32. Furthermore, human fetal skin exhibits low levels of major
histocompatibility complex (MHC) I and II expression, which results in reduced immunogenicity compared
to adult skin31, 32. Thus, human fetal-derived tissues and cells provide a feasible means to develop a humanized
mouse model with autologous human skin and immune system. g
y
Rats are a major model specie for long-term (greater than one year) studies33; thus, humanized rat models
that combine human skin, human immune cells, and human lymphoid structures are being explored for use in
long-term in vivo studies of human skin-immune interactions. Reports have demonstrated that nude rats (with
moderate immunodeficiency) support adult human skin (split-thickness skin) engraftment and development,
albeit host-mediated immune rejection occurs within a few months34–36. www.nature.com/scientificreports/ Evidence also suggests that nude rats
support engraftment and development of full-thickness neonatal foreskin37. Recently, an immunodeficient rat,
termed Sprague–Dawley-Rag2tm2hera Il2rγtm1hera (SRG) rat, was developed to support engraftment of human cells
and tissues into a larger and longer-life span rodent model; this model lacks mature T, B and NK cells38, 39. To
date, humanized rat models co-engrafted with human full-thickness skin and autologous lymphoid tissues and
immune cells (including cutaneous immune cells) remain to be developed and established28, 40–42.i Here, we utilize the immunodeficient NSG mouse and SRG rat models to generate rodent models with human
skin, along with autologous lymphoid tissue(s) and autologous immune cells, termed human Skin and Immune
System (hSIS)-NSG mice and SRG rats. Adolescent rodents received co-transplants of human full-thickness
fetal skin, autologous lymphoid tissues, and autologous hematopoietic stem cells. Additionally, the human skin
xenografts were inoculated with CA-MRSA to establish the susceptibility of these hSIS-humanized rodent models
to human skin pathogens. Development of humanized mouse
and rat models with full‑thickness
human skin and autologous
immune cells Rodent skin microanatomy differs from human skin microanatomy due to the rodent skin lacking
a multi-layered epidermis, eccrine and apocrine glands, and the papillary, reticular, and hypo-dermal regions
of the dermal layer14. Human primary and secondary lymphoid tissues microanatomy differs significantly from
that of rodent lymphoid tissues as well, with significant differences in red pulp to white pulp ratio in the spleen
and lobulation of the thymus15–17. It is well-established that lymphoid tissue microenvironment, including stro-
mal cells, plays a significant role in immune cell development18. Interactions between immune cells and stromal
cells in non-lymphoid tissues, such as the skin, play a significant role in modulating tissue-associated immune
responses14. Translational gaps may form between clinical studies performed with traditional rodent models, 1Department of Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, USA. 2Department of
Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA. 3Hera Biolabs, Inc, Lexington,
USA. 4These authors contributed equally: Yash Agarwal, Cole Beatty and Sara Ho. *email: mtbility@pitt.edu | https://doi.org/10.1038/s41598-020-71548-z www.nature.com/scientificreports/ Results The human skin xenograft exhibited multiple layers of human keratinocytes
(AE1/AE3, pan-cytokeratin antibody+ cells) in the epidermis and dermal fibroblasts (Anti-Fibroblasts Anti-
body+ cells) in the dermis (Fig. 2B). Additionally, the human skin exhibited human immune cell repopula-
tion (human CD45+ cells), including Langerhans cells (hCD207+ cells), macrophages (hCD68+), and T cells
(hCD3+) (Fig. 2B, Supplementary Figs. 4 and 5). The human skin in hSIS-humanized NSG mice also exhibited
upregulation of alpha-smooth muscle actin-positive (α-SMA+) cells (i.e., blood vessel cells45) during revascu-
larization and wound healing (~ 2 weeks post-transplantation), followed by a reduction in α-SMA+ cells in the
healed skin (~ 10 weeks post-transplantation) (Fig. 2B). Histochemical analysis demonstrates the development
of the co-transplanted human lymphoid tissues (spleen and thymus) in the renal capsule (~ 10 weeks post-trans-
plantation) (Fig. 2C)17. Human thymus tissue in hSIS-humanized NSG mice exhibits T-cell (human CD3+ cells)
reconstitution, with few B cells in the tissue (Fig. 2C)17, 46, 47. Macrophage reconstitution (human CD68+ cells) in
the human thymus tissue is restricted to the medulla (Fig. 2C)17, 46, 47. Human spleen tissue in hSIS-humanized
NSG mice exhibits macrophage reconstitution, with macrophages predominately in the red-pulp (Fig. 2C)15, 16. The human spleen tissue in hSIS-humanized NSG mice exhibits T and B cell repopulation (human CD3+ and
CD20+ cells), with lymphocytes predominately in the white-pulp (Fig. 2C)15. Analysis of the PBMCs in hSIS-
humanized NSG mice showed human immune cell reconstitution (hCD45+ cells) (Fig. 3A,B). Various human
immune cell (hCD45+) subtypes, namely, αβ T cells (hαβ T cells with a CD4:CD8 ratio average of 1.34:1), γδ T
cells (γδ T), NK cells (hNK), B cells (hB), monocytes (hMo) and granulocytes/polymorphonuclear neutrophils
(hPMN) were reconstituted in the peripheral blood of the hSIS-humanized NSG mouse model (Fig. 3C,D), with
analysis performed using appropriate flow cytometry assay controls (Supplementary Fig. 6). The hSIS‑humanized SRG rat model supports the development of full‑thickness human skin,
thymus tissues, and human immune cells. Although humanized mice have provided in vivo platforms
for modeling human diseases, the short-life span and small tissue size/volume of mice are a major limitation
for long-termed and preclinical studies, respectively. We hypothesized that a larger immunodeficient rodent
model, with a longer life span, namely rat, would support the development of an in vivo model for long-term
studies (> 6 months) and provide large tissue volume/size. Results In addition to supporting the growth of human spleen and thymus
tissues, the hSIS-humanized NSG mouse model supports the reconstitution of the immunodeficient-murine
lymph nodes and spleen (Fig. 1C)17. Histochemical analysis of the human skin in hSIS-humanized NSG mice https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 | www.nature.com/scientificreports/ Figure 1. Development of human skin and lymphoid tissues in the human Skin and Immune System-
humanized NSG mouse model. Transplantation of full-thickness human skin (derived from the scalp for skin
with hair, or derived from dorsum for skin without hair) on the dorsum (A, B) and autologous lymphoid
tissues in the kidney capsule (C) of NSG mice results in the engraftment and development of full-thickness
human skin and lymphoid tissues. (A) Representative gross-photos at 0 (the day of transplantation), 2-, 4- and
10-weeks post-transplantation, with intact mouse skin as control (n = 4 per group). (B) Transplantation of
full-thickness human skin from regions with significant hair follicles (scalp) in the human Skin and Immune
System-humanized NSG mouse model results in the development of human hair, as exhibited in representative
gross-photos at 12 weeks post-transplantation (n = 6). (C) Representative gross-photos of human lymphoid
tissues (spleen and thymus tissues) and humanized lymphoid tissues (reconstituted-immunodeficient murine
lymph node and spleen) at ten weeks post-transplantation (n = 4). Figure 1. Development of human skin and lymphoid tissues in the human Skin and Immune System-
humanized NSG mouse model. Transplantation of full-thickness human skin (derived from the scalp for skin
with hair, or derived from dorsum for skin without hair) on the dorsum (A, B) and autologous lymphoid
tissues in the kidney capsule (C) of NSG mice results in the engraftment and development of full-thickness
human skin and lymphoid tissues. (A) Representative gross-photos at 0 (the day of transplantation), 2-, 4- and
10-weeks post-transplantation, with intact mouse skin as control (n = 4 per group). (B) Transplantation of
full-thickness human skin from regions with significant hair follicles (scalp) in the human Skin and Immune
System-humanized NSG mouse model results in the development of human hair, as exhibited in representative
gross-photos at 12 weeks post-transplantation (n = 6). (C) Representative gross-photos of human lymphoid
tissues (spleen and thymus tissues) and humanized lymphoid tissues (reconstituted-immunodeficient murine
lymph node and spleen) at ten weeks post-transplantation (n = 4). Results demonstrates the development of the human skin xenograft; the microanatomy of the human skin at ten weeks
post-transplantation is comparable to adult human skin, with multiple layers of cells present in the epidermis
(Fig. 2A, Supplementary Fig. 3). The human skin xenograft exhibited multiple layers of human keratinocytes
(AE1/AE3, pan-cytokeratin antibody+ cells) in the epidermis and dermal fibroblasts (Anti-Fibroblasts Anti-
body+ cells) in the dermis (Fig. 2B). Additionally, the human skin exhibited human immune cell repopula-
tion (human CD45+ cells), including Langerhans cells (hCD207+ cells), macrophages (hCD68+), and T cells
(hCD3+) (Fig. 2B, Supplementary Figs. 4 and 5). The human skin in hSIS-humanized NSG mice also exhibited
upregulation of alpha-smooth muscle actin-positive (α-SMA+) cells (i.e., blood vessel cells45) during revascu-
larization and wound healing (~ 2 weeks post-transplantation), followed by a reduction in α-SMA+ cells in the
healed skin (~ 10 weeks post-transplantation) (Fig. 2B). Histochemical analysis demonstrates the development
of the co-transplanted human lymphoid tissues (spleen and thymus) in the renal capsule (~ 10 weeks post-trans-
plantation) (Fig. 2C)17. Human thymus tissue in hSIS-humanized NSG mice exhibits T-cell (human CD3+ cells)
reconstitution, with few B cells in the tissue (Fig. 2C)17, 46, 47. Macrophage reconstitution (human CD68+ cells) in
the human thymus tissue is restricted to the medulla (Fig. 2C)17, 46, 47. Human spleen tissue in hSIS-humanized
NSG mice exhibits macrophage reconstitution, with macrophages predominately in the red-pulp (Fig. 2C)15, 16. The human spleen tissue in hSIS-humanized NSG mice exhibits T and B cell repopulation (human CD3+ and
CD20+ cells), with lymphocytes predominately in the white-pulp (Fig. 2C)15. Analysis of the PBMCs in hSIS-
humanized NSG mice showed human immune cell reconstitution (hCD45+ cells) (Fig. 3A,B). Various human
immune cell (hCD45+) subtypes, namely, αβ T cells (hαβ T cells with a CD4:CD8 ratio average of 1.34:1), γδ T
cells (γδ T), NK cells (hNK), B cells (hB), monocytes (hMo) and granulocytes/polymorphonuclear neutrophils
(hPMN) were reconstituted in the peripheral blood of the hSIS-humanized NSG mouse model (Fig. 3C,D), with
analysis performed using appropriate flow cytometry assay controls (Supplementary Fig. 6). demonstrates the development of the human skin xenograft; the microanatomy of the human skin at ten weeks
post-transplantation is comparable to adult human skin, with multiple layers of cells present in the epidermis
(Fig. 2A, Supplementary Fig. 3). Results The black arrows denote representative IHC+ cells. (C) Representative histological and
immunohistochemical analysis of the human spleen and thymus (both under the kidney capsule) in human
Skin and Immune System-humanized NSG mice demonstrate the development of those lymphoid tissues at ten
weeks post-transplantation, with human macrophages (hCD68+), T cells (hCD3+), B cells (hCD20+) present in
the tissues (n = 4) Scale bars: 200 μm. fetal thymus and liver tissues (~ 1 mm3 pieces) were implanted into the kidney capsules of irradiated SRG rats,
and those rats were immediately transplanted using autologous hematopoietic stem cells. Excess fat tissue was
removed from the human fetal skin, and the tissue was subsequently engrafted over the rib cage, where the rat
skin was previously excised. SRG rats were transplanted using human skin plus CD34+ human hematopoietic
stem cells and thymus tissue, or with human skin only; 100% of the rats successfully engrafted and developed the
transplanted human tissues (Supplementary Fig. 7). However, a human immune cell reconstitution of the blood
in the transplanted rats was not observed in any group using this transplantation methodology (data not shown). Gross analysis of the human skin in rats transplanted with human CD34+ human hematopoietic stem cells plus
thymus and skin demonstrated wound healing, beginning at three weeks post-transplantation, and matura-
tion into adult-like human skin over time (Fig. 4A). The human skin transplanted onto SRG (with or without
lymphoid tissue plus CD34+ human hematopoietic stem cells) also support the development of human skin
appendages (hair) (Fig. 4B). Gross analysis of the lymphoid tissues in hSIS-humanized SRG rats at nine months
post-transplantation showed the growth of the human thymic tissue in the kidney capsule and marginal increase
in the size of the rat spleen (Fig. 4C). The human skin in hSIS-humanized SRG rats exhibited development of
multi-layered human keratinocytes (AE1/AE3, pan-cytokeratin antibody+ cells) in the epidermis, and dermal
fibroblasts (Anti-Fibroblasts Antibody+ cells) in the dermis, both of which are comparable to adult human skin
(Adult-hSkin) and differ from rat skin (Fig. 5A, Supplementary Fig. 8). The human skin in hSIS-humanized SRG
rats exhibited a reconstitution of human immune cells (hCD45+ cells), including Langerhans cells (hCD207+
cells), comparable to adult human skin (Adult-hSkin) (Fig. 5A). Histochemical analysis of human thymus tissue
in hSIS-humanized SRG rats at nine months post-transplantation demonstrates thymus development, including
the presence of thymic lobules (Fig. 5B). Results We further hypothesized that the co-transplantation
of human full-thickness fetal skin, autologous fetal thymus tissues, autologous fetal liver tissues, and autologous
fetal-liver derived hematopoietic stem cells into SRG rat would enable the development of a model containing
human skin, autologous thymus, and autologous human immune cells, termed hSIS-humanized SRG rat. Human https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 | www.nature.com/scientificreports/ Figure 2. Development of human skin and immune cells in the human Skin and Immune System-humanized
NSG mouse model. (A) Representative histological (H&E) analysis of the human skin in human Skin and
Immune System-humanized NSG mice (n = 4) demonstrate the development of human adult-like skin,
including the dermis, multicellular layer (> 5 layers) epidermis, and cornified envelope. Representative
histological (H&E) analysis of the mouse skin demonstrate a thin-layer of epidermal cells, with a thin dermal
layer. (B) Various human skin cells are present in the human skin, including keratinocytes (AE1/AE3+
cells, hCytokeratins+ cells), dermal fibroblasts (TE7+ cells, hFibroblast+ cells), cutaneous immune cells
(hCD45+ cells), and Langerhans cells (hCD207+); alpha-smooth muscle actin-expressing blood vessel cells
(hα-SMA+ cells) are present in the human skin xenograft and expand during wound healing and contract
after healing (n = 4). The black arrows denote representative IHC+ cells. (C) Representative histological and
immunohistochemical analysis of the human spleen and thymus (both under the kidney capsule) in human
Skin and Immune System-humanized NSG mice demonstrate the development of those lymphoid tissues at ten
weeks post-transplantation, with human macrophages (hCD68+), T cells (hCD3+), B cells (hCD20+) present in
the tissues (n = 4) Scale bars: 200 μm. Figure 2. Development of human skin and immune cells in the human Skin and Immune System-humanized
NSG mouse model. (A) Representative histological (H&E) analysis of the human skin in human Skin and
Immune System-humanized NSG mice (n = 4) demonstrate the development of human adult-like skin,
including the dermis, multicellular layer (> 5 layers) epidermis, and cornified envelope. Representative
histological (H&E) analysis of the mouse skin demonstrate a thin-layer of epidermal cells, with a thin dermal
layer. (B) Various human skin cells are present in the human skin, including keratinocytes (AE1/AE3+
cells, hCytokeratins+ cells), dermal fibroblasts (TE7+ cells, hFibroblast+ cells), cutaneous immune cells
(hCD45+ cells), and Langerhans cells (hCD207+); alpha-smooth muscle actin-expressing blood vessel cells
(hα-SMA+ cells) are present in the human skin xenograft and expand during wound healing and contract
after healing (n = 4). Results Human thymus tissues in the hSIS-humanized SRG rat exhibits human
immune cell (human CD45+ cells) reconstitution, including T cell (human CD3+ cells) and macrophage (human
CD68+ cells) reconstitution (Fig. 5B,C). Additionally, human thymic T cells exhibit cytokine response to stimu-
lation using CD3/CD28 beads (Supplementary Fig. 9). Human immune cells in the hSIS-humanized SRG rat
also reconstitute the immunodeficient-rat spleen (Fig. 5D). The human skin xenograft in immunodeficient rodent models supports community‑associated
methicillin‑resistant Staphylococcus aureus infection. Community-associated methicillin-resistant
Staphylococcus aureus (CA-MRSA) infection represents a significant public health threat 5; thus, in vivo models
that enable investigation of human skin infections are of significance. In order to demonstrate that human fetal
skin-derived skin xenografts in immunodeficient rodents provide a means for investigating human skin infec-
tions, we inoculated (intradermal) the adult-like human skin xenografts with CA-MRSA USA300. We observed
lesions in the human skin in SRG rats inoculated with CA-MRSA, which were comparable to lesions in CA-
MRSA patients; those human skin-lesions in the rats exhibited CA-MRSA bacterial growth (Fig. 6). Inoculation
of CA-MRSA in the human skin xenograft in the NSG mouse model at 10–12 weeks post-transplantation, also
supports bacterial growth (Supplementary Fig. 10). https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 www.nature.com/scientificreports/ Figure 3. Development of human peripheral blood mononuclear cells in the human Skin and Immune System-
humanized NSG mouse model. (A) Representative flow cytometry analysis of human immune cell (hCD45+)
reconstitution in peripheral blood mononuclear cells (PBMCs) of human Skin and Immune System-humanized
NSG mice at 10–12 weeks post-transplantation demonstrates high levels (> 10%) of human immune cells in
the blood. (B) Quantification of human immune cell reconstitution (n = 22; 3 independent experiments) in
PBMCs of hSIS-humanized mice at 10 weeks post-transplantation. (C) Representative flow cytometry analysis
of human PBMCs (hCD45+ PBMCs) in human Skin and Immune System-humanized mice at 10–12 weeks
post-transplantation demonstrates readily detectable levels of various human immune cell types (B cells-
hCD19+hCD3- human PBMCs, αβ T cells-hCD3+ human PBMCs, hCD4+ T cells, hCD8+ T cells, hγδTCR+
T cells- hγδ TCR+ CD3+ human PBMCs, natural killer cells (NK)-hCD57+ hCD3- human PBMCs, monocytes
(hMo)-hCD14+ CD3- human PBMCs, and granulocytes (hPMN)-hCD66b+ hCD3- human PBMCs). (D)
Quantification of human immune cell subtypes (n = 4) in human PBMCs (hCD45+ PBMCs) of human Skin and
Immune System-humanized mice at 10–12 weeks post-transplantation. Figure 3. Results Development of human peripheral blood mononuclear cells in the human Skin and Immune System-
humanized NSG mouse model. (A) Representative flow cytometry analysis of human immune cell (hCD45+)
reconstitution in peripheral blood mononuclear cells (PBMCs) of human Skin and Immune System-humanized
NSG mice at 10–12 weeks post-transplantation demonstrates high levels (> 10%) of human immune cells in
the blood. (B) Quantification of human immune cell reconstitution (n = 22; 3 independent experiments) in
PBMCs of hSIS-humanized mice at 10 weeks post-transplantation. (C) Representative flow cytometry analysis
of human PBMCs (hCD45+ PBMCs) in human Skin and Immune System-humanized mice at 10–12 weeks
post-transplantation demonstrates readily detectable levels of various human immune cell types (B cells-
hCD19+hCD3- human PBMCs, αβ T cells-hCD3+ human PBMCs, hCD4+ T cells, hCD8+ T cells, hγδTCR+
T cells- hγδ TCR+ CD3+ human PBMCs, natural killer cells (NK)-hCD57+ hCD3- human PBMCs, monocytes
(hMo)-hCD14+ CD3- human PBMCs, and granulocytes (hPMN)-hCD66b+ hCD3- human PBMCs). (D)
Quantification of human immune cell subtypes (n = 4) in human PBMCs (hCD45+ PBMCs) of human Skin and
Immune System-humanized mice at 10–12 weeks post-transplantation. Scientific Reports | (2020) 10:14598 Discussion
d
d l Rodent models are the primary platforms for investigating human skin-associated infections, injuries, and
diseases10, 43. Although rodent models provide insights into the mechanisms of human diseases and host
response/defense against skin-associated infectious agents, several limitations exist in rodent models10, 43. It is
well established that the human skin exhibits significant structural differences compared to rodent skin10, 43. These
structural differences could play a critical role in skin-microbes interactions and cutaneous immune signaling,
resulting in significant differences in anti-microbial host defense and inflammatory response between human
and rodent skin1, 2. These differences could also significantly impact the translation of mechanistic findings from
traditional rodent models to humans.f In an attempt to address the species differences between traditional rodent models of human skin diseases and
skin diseases in humans, we engrafted human skin, autologous lymphoid tissues, and autologous hematopoietic
stem cells into immunodeficient rodents (SRG rats and NSG mice). The human skin engrafted into immunode-
ficient rodents developed a microanatomical structure that resembled adult human skin. Human keratinocytes
and dermal fibroblasts, which facilitate the development of multi-layered epidermis and dermis, respectively,
were present in the human skin of both the hSIS-humanized rats and mice. To establish that the human skin
xenograft on immunodeficient rats is susceptible to human pathogens, we infected the engrafted human skin with
CA-MRSA via intradermal inoculation. The human skin xenograft in the hSIS rat model supports CA-MRSA-
infection. The human skin in the hSIS-humanized mouse model also supports CA-MRSA-infection, albeit the
infection appears to induce rapid death within three days. Prince et al. reported that human neutrophils and
monocyte-derived macrophages are a target for CA-MRSA-associated Panton-Valentine leucocidin (PVL) in
humanized mice with human immune cells and thymus xenograft 48; thus, enhanced inflammation could be
mediating the rapid deaths. In the hSIS-humanized NSG mouse model, we observed readily detectable levels of
human monocytes/macrophages in the blood and human skin. Future studies will investigate the interactions
between human immune cell perturbations and CA-MRSA in the infected skin. A limitation of the hSIS-human-
ized mouse model is the short experimental window (< 2.5 months) for infection, as drying of the human skin https://doi.org/10.1038/s41598-020-71548-z www.nature.com/scientificreports/ Figure 4. Development of human skin and lymphoid tissues in the human Skin and Immune System-
humanized SRG rat model. Discussion
d
d l Transplantation of full-thickness human fetal skin on the dorsum (A, B)
and autologous fetal lymphoid tissues (thymus and liver) in the kidney capsule (C) of SRG rats results in
engraftment and development of full-thickness human skin and primary lymphoid tissue (thymus) (n = 4). (A) Representative gross-photos at 0- (the day of transplantation), 3-, 20-, and 36-weeks post-transplantation
demonstrate human fetal skin engraftment and development (using donor skin obtained from the dorsum). (B)
Transplantation of full-thickness human fetal skin, derived from regions with significant hair follicles (scalp), on
SRG rats with (left panel) (n = 2) or without (right panel) (n = 2) co-transplantation of thymus and hematopoietic
stem cells results in the development of human hair as exhibited in representative gross-photos at six months
post-transplantation. In the right panel, autologous human skin from dorsum and scalp were co-transplanted to
demonstrate human hair only grows in human skin tissue with preexisting hair follicles (scalp; identified with
black box). (C) Representative gross-photos of lymphoid tissues (human thymus in the kidney capsule and rat
spleen) at nine months post-transplantation demonstrates the development of lymphoid tissues compared to
non-transplanted SRG rat (n = 4). The black circles denote human thymus tissues. Figure 4. Development of human skin and lymphoid tissues in the human Skin and Immune System-
humanized SRG rat model. Transplantation of full-thickness human fetal skin on the dorsum (A, B)
and autologous fetal lymphoid tissues (thymus and liver) in the kidney capsule (C) of SRG rats results in
engraftment and development of full-thickness human skin and primary lymphoid tissue (thymus) (n = 4). (A) Representative gross-photos at 0- (the day of transplantation), 3-, 20-, and 36-weeks post-transplantation
demonstrate human fetal skin engraftment and development (using donor skin obtained from the dorsum). (B)
Transplantation of full-thickness human fetal skin, derived from regions with significant hair follicles (scalp), on
SRG rats with (left panel) (n = 2) or without (right panel) (n = 2) co-transplantation of thymus and hematopoietic
stem cells results in the development of human hair as exhibited in representative gross-photos at six months
post-transplantation. In the right panel, autologous human skin from dorsum and scalp were co-transplanted to
demonstrate human hair only grows in human skin tissue with preexisting hair follicles (scalp; identified with
black box). Discussion
d
d l (C) Representative gross-photos of lymphoid tissues (human thymus in the kidney capsule and rat
spleen) at nine months post-transplantation demonstrates the development of lymphoid tissues compared to
non-transplanted SRG rat (n = 4). The black circles denote human thymus tissues. xenograft (resulting in hardening) and early signs of murine hair loss (suggestive of graft-versus-host disease)
are evident at approximately five months post-transplantation. hSIS-humanized SRG rats do not exhibit signs
of graft versus host disease, even at nine months post-transplantation; thus, hSIS-humanized SRG rats could
provide an in vivo model for studies requiring a wider experimental window (> 3 months). Diverse leukocyte populations mediate human immune response; the hSIS-humanized mouse model supports
the development of both innate and adaptive immune cells. The peripheral blood of hSIS-humanized mice is
reconstituted with lymphocytes, granulocytes, monocytes, dendritic cells, and natural killer cells. Additionally,
the hSIS-humanized mouse model supports the development of human thymus and spleen tissues. The presence
of these human primary and secondary lymphoid tissues, coupled with the development of innate and adaptive
immune cells, may allow this model to recapitulate de novo antigen-specific human immune responses to a skin
infection. Interestingly, the hSIS-humanized rat model exhibited marginal human immune cell reconstitution
in the peripheral blood, despite evidence of human immune cell reconstitution in the human skin and thymus,
as well as the rat spleen. A transgenic variant of the SRG rat model, carrying human signal regulatory protein
alpha, a negative regulator of macrophage phagocytosis, could facilitate human immune cell reconstitution in
the blood49, 50. In summary, we report the development of humanized NSG mouse and SRG rat models that incorporate
human lymphoid tissue(s) and autologous full-thickness human skin with cutaneous immune cells. hSIS-human-
ized NSG mice and SRG rats could provide a means for studying skin infections25, 48, 51–60. Methods Construction of human Skin and Immune System‑humanized rodents. Adult (8–10 weeks) male
and female severely immunodeficient rodents, namely Non-obese Diabetic (NOD) strain mice (Jackson Labo-
ratory, Stock No: 005557) and Sprague Dawley (SD) strain rats (Hera Biolabs), carrying mutations in inter-
leukin-2 receptor subunit gamma (IL2Rγ), as well as in the Protein Kinase, DNA-Activated, Catalytic Subunit
(PRKDC) (mice only) or recombination activating gene 2 (RAG2) (rat only) were obtained from vendor and
bred in the Division of Laboratory Animal Resources (DLAR) facility at the University of Pittsburgh. Human
fetal tissues were obtained from the Health Sciences Tissue Bank at the University of Pittsburgh. Human fetal
tissues for constructing humanized rodents were handled and processed under biosafety level 2 conditions. Male
and female rodents were myoablated via gamma radiation using cesium-137 irradiator, with mice receiving a
dose of 150 rads and rats receiving a dose of 500 rads. Myoablated male and female mice were transplanted with
human fetal-thymus, liver, and spleen in the kidney capsule, autologous CD34+ hematopoietic stem cells (via
retroorbital injection of 0.2 × 106 cells)17, and full-thickness human fetal skin on the panniculus carnosus of the
mouse skin-excised dorsum61–63. Myoablated male and female rats were transplanted with human fetal-thymus
and liver in the renal capsule, autologous CD34+ hematopoietic stem cells (via retroorbital injection of 0.5 × 106
cells), and autologous full-thickness human fetal skin (less than four days old) on the panniculus carnosus of
the rat-skin excised dorsum61–63. In some instances, rodents were only transplanted with full-thickness human https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 | www.nature.com/scientificreports/ Figure 5. Development of human skin and immune cells in the human Skin and Immune System-humanized
SRG rat model. Representative immunohistochemical analysis of the human skin in the human Skin and
Immune System-humanized rat (n = 4) demonstrates the development of human skin, including dermis, multi-
layered (> 5 layers) epidermis, and cornified envelope, which are hallmarks of adult-human skin (Adult-hSkin)
(n = 2) (A). Various human skin cells are present in the human skin, including keratinocytes (AE1/AE3+ cells,
hCytokeratins+ cells), dermal fibroblasts (TE7+ cells, hFibroblast+ cells), cutaneous immune cells (hCD45+
cells), and Langerhans cells (hCD207+; black arrows denote IHC+ cells) (n = 4). Methods (B) Representative histological
and immunohistochemical analysis of the human thymus (under the kidney capsule) in the human Skin and
Immune System-humanized SRG rat demonstrates the development of human thymus tissue at nine months
post-transplantation, with human immune cells (human CD45+), including (C) high levels of T cells (hCD3+)
and macrophages (hCD68+) (n = 4). (D) The rat spleen in the human Skin and Immune System-humanized
SRG rat model (n = 4) is also reconstituted with human immune cells (Humans CD45+); non-transplanted
(NTP) SRG rat (n = 2) was used as a staining control. Scale bars: 200 μm. igure 5. Development of human skin and immune cells in the human Skin and Immune System-humanized Figure 5. Development of human skin and immune cells in the human Skin and Immune System-humanized
SRG rat model. Representative immunohistochemical analysis of the human skin in the human Skin and
Immune System-humanized rat (n = 4) demonstrates the development of human skin, including dermis, multi-
layered (> 5 layers) epidermis, and cornified envelope, which are hallmarks of adult-human skin (Adult-hSkin)
(n = 2) (A). Various human skin cells are present in the human skin, including keratinocytes (AE1/AE3+ cells,
hCytokeratins+ cells), dermal fibroblasts (TE7+ cells, hFibroblast+ cells), cutaneous immune cells (hCD45+
cells), and Langerhans cells (hCD207+; black arrows denote IHC+ cells) (n = 4). (B) Representative histological
and immunohistochemical analysis of the human thymus (under the kidney capsule) in the human Skin and
Immune System-humanized SRG rat demonstrates the development of human thymus tissue at nine months
post-transplantation, with human immune cells (human CD45+), including (C) high levels of T cells (hCD3+)
and macrophages (hCD68+) (n = 4). (D) The rat spleen in the human Skin and Immune System-humanized
SRG rat model (n = 4) is also reconstituted with human immune cells (Humans CD45+); non-transplanted
(NTP) SRG rat (n = 2) was used as a staining control. Scale bars: 200 μm. Figure 6. Human skin xenograft on SRG rats supports CA-MRSA infection. Community-associated
methicillin-resistant Staphylococcus aureus (CA-MRSA) was inoculated into human skin xenograft (intradermal
injection) on humanized (human skin) SRG rats (n = 9) to demonstrate that the human skin xenograft supports
CA-MRSA infection. (A) Gross analysis of the skin tissues were examined in CA-MRSA inoculated humanized
(human skin) SRG rats (n = 9) and compared to CA-MRSA infected human skin (Patient CA-MRSA skin photo
credit: S. Methods Camazine); healthy control-human skin in humanized (human skin) SRG rats (n = 5) were also used as
controls to determine skin lesions. (C) The skin lesions in the CA-MRSA inoculated humanized (human skin)
SRG rats (n = 9) supports high CA-MRSA bacteria load as measured at six weeks post-infection. igure 6. Human skin xenograft on SRG rats supports CA-MRSA infection. Community-associated t Figure 6. Human skin xenograft on SRG rats supports CA-MRSA infection. Community-associated
methicillin-resistant Staphylococcus aureus (CA-MRSA) was inoculated into human skin xenograft (intradermal
injection) on humanized (human skin) SRG rats (n = 9) to demonstrate that the human skin xenograft supports
CA-MRSA infection. (A) Gross analysis of the skin tissues were examined in CA-MRSA inoculated humanized
(human skin) SRG rats (n = 9) and compared to CA-MRSA infected human skin (Patient CA-MRSA skin photo
credit: S. Camazine); healthy control-human skin in humanized (human skin) SRG rats (n = 5) were also used as
controls to determine skin lesions. (C) The skin lesions in the CA-MRSA inoculated humanized (human skin)
SRG rats (n = 9) supports high CA-MRSA bacteria load as measured at six weeks post-infection. Figure 6. Human skin xenograft on SRG rats supports CA-MRSA infection. Community-associated
methicillin-resistant Staphylococcus aureus (CA-MRSA) was inoculated into human skin xenograft (intradermal
injection) on humanized (human skin) SRG rats (n = 9) to demonstrate that the human skin xenograft supports
CA-MRSA infection. (A) Gross analysis of the skin tissues were examined in CA-MRSA inoculated humanized
(human skin) SRG rats (n = 9) and compared to CA-MRSA infected human skin (Patient CA-MRSA skin photo
credit: S. Camazine); healthy control-human skin in humanized (human skin) SRG rats (n = 5) were also used as
controls to determine skin lesions. (C) The skin lesions in the CA-MRSA inoculated humanized (human skin)
SRG rats (n = 9) supports high CA-MRSA bacteria load as measured at six weeks post-infection. https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 | www.nature.com/scientificreports/ fetal skin. Rodents were housed under specific-pathogen-free conditions and fed irradiated chow and autoclaved
water. Immune cell reconstitution and functional assays. For evaluating human immune cell reconstitu-
tion at indicated time points, peripheral blood was collected from animals, and leukocytes were analyzed using
flow cytometry17. Briefly, peripheral blood was collected from rodents and mixed with 20 mM Ethylenediami-
netetraacetic acid (EDTA) at a 1:1 ratio, and single-cell leukocytes were prepared via red blood cells lysis using
Ammonium-Chloride-Potassium (ACK) buffer. Methods CME 324 A, B, C; anti-human CD68-Biocare Medical catalog num-
ber CM 033 A, B, C; anti-human CD20-Biocare Medical catalog number ACR 3004 A, B; anti-human alpha-
smooth muscle actin; anti-pan cytokeratin, Clone AE1/AE3, Biocare Medical catalog number SKU: 011; anti-
human fibroblast, Clone TE7, Millipore Sigma catalog number CBL271; anti-human CD207, Dendritics catalog
number: DDX0362). The immunoreactivity of the antibodies was determined via incubation with DAB substrate
(MACH 2 Detection Kit, Biocare Medical) and counterstaining with hematoxylin. CA‑MRSA infection in the human skin in immunodeficient rodents. The human skin xenografts
on the immunodeficient rodents (SRG rats and NSG mice) were inoculated with CA-MRSA USA30064 via intra-
dermal injection with 1 × 108 CFU for rats and 1 × 106 CFU for mice; non-transplanted rodents were inoculated
via subqutaneous injection at the same dosage. After three days, portions of equal weight human or rodent skin
was excised, and bacterial load was determined based on the number of colony-forming units (CFU); CA-MRSA
bacterial strain was confirmed via polymerase chain reaction (PCR). Ethical approval. De-identified human fetal tissues at the gestational age of 18 to 20 weeks were obtained
from medically or elective indicated termination of pregnancy through Magee-Womens Hospital of the Uni-
versity of Pittsburgh Medical Center (UPMC), with the University of Pittsburgh, Health Sciences Tissue Bank. Written informed consent of the maternal donors was obtained in all cases, under a protocol reviewed and
approved by the Institutional Review Board (IRB) of the University of Pittsburgh; approved guidelines and fed-
eral/state regulations were adhered to for all procedures. The use of de-identified human fetal tissues to con-
struct humanized rodents was reviewed and approved by the University of Pittsburgh IRB Office. The use of
de-identified human fetal tissues did not constitute human subjects research as defined under federal regulations
[45 CFR 46.102(d or f) and 21 CFR 56.102(c), (e), and (l)]. The use of human fetal liver-derived hematopoietic
stem cells was reviewed and approved by the Human Stem Cell Research Oversight (hSCRO) at the University of
Pittsburgh. The use of a biological agent (CA-MRSA), recombinant DNA, and transgenic animals were reviewed
and approved by the Institutional Biosafety Committee (IBC) at the University of Pittsburgh. Methods For evaluating human immune reconstitution in the human
skin xenograft in the hSIS-humanized mouse model, human skin tissue was excised from an hSIS-humanized
mouse. Skin samples were cut into small and digested with collagenase. The dermis was separated from the
epidermis, and a single-cell suspension of dermal tissue was created using a gentleMACS dissociator (Miltenyi
Biotech). Epidermal tissues underwent trypsinization in order to obtain a single-cell suspension. Single-cell sus-
pensions were stained with a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific) and fluo-
rochrome-conjugated antibodies (anti-mouse CD45-BioLegend Cat. No. 103126, anti-human CD45-BioLegend
Cat. No. 304014), fixed with formalin, and analyzed on a BD LSRFortessa™ cell analyzer—flow cytometer (BD
Biosciences). Data were analyzed using FlowJo software (Dako). Leukocytes were selected based on forward,
and side scatter measurements. Single-cell and live leukocytes were selected for further analysis of the percent-
age of human leukocytes (anti-human CD45+, hCD3+, hCD4+, CD8+, hγδTCR+, hCD19+, hCD14+, hCD16+,
hCD66b+) and mouse leukocytes (anti-mouse CD45+). The analysis of the various human immune cell popula-
tions and subsets were gated on human leukocytes. Human T cells were also isolated from the thymus tissue
in humanized SRG rats via immunomagnetic selection using an anti-human CD3 antibody (EasySep™ Human
CD3 Positive Selection, Catalog # 17951, Stemcell Technologies) and treated without (vehicle) or with Gibco™
Dynabeads™ Human T-Activator CD3/CD28 (Cat. No. 111.61D, ThermoFisher Scientific) in the presence of
recombinant IL2 and BD GolgiPlug (BD Biosciences) for 12 h. Human cytokine expression (hTNFα and hIFNγ)
in human T cells were analyzed using BD LSRFortessa™ cell analyzer—flow cytometer (BD Biosciences), and the
data were analyzed using FlowJo software (Dako). Gross/In situ immune cell analysis. Gross analysis of tissues was performed using a camera (8 meg-
apixels), with animals either euthanized or anesthetized prior to photographing. Indicated tissue samples
from humanized rodents or humans (adult human skin from a 77-year-old male, BioChain, catalog number:
T2234218 or adult female breast skin, University of Pittsburgh Biospecimen Repository), were fixed with for-
malin and, subsequently, embedded in paraffin. Paraffin-embedded, fixed sections were stained via hematoxylin
and eosin, or via indicated human antibodies17 (anti-human CD45-Biocare Medical Cat. No. CME PM016AA;
anti-human CD3-Biocare Medical Cat. No. Methods All animal stud-
ies/experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee
at the University of Pittsburgh and were conducted following approved guidelines, which adheres to the NIH
guidelines for housing and care of laboratory animals. References The perifollicular and marginal zones of the human splenic white pulp : do fibroblasts guide
lymphocyte immigration?. Am. J. Pathol. 159, 501–512. https://doi.org/10.1016/S0002-9440(10)61722-1 (2001). 17. Samal, J. et al. Human immunodeficiency virus infection induces lymphoid fibrosis in the BM-liver-thymus-spleen humanized
mouse model. JCI Insight https://doi.org/10.1172/jci.insight.120430 (2018). g
p
g
j
g
8. Roozendaal, R. & Mebius, R. E. Stromal cell-immune cell interactions. Annu. Rev. Immunol. 29, 23–43. https://doi.org/10.1146/
annurev-immunol-031210-101357 (2011). 19. Agarwal, Y. et al. Moving beyond the mousetrap: current and emerging humanized mouse and rat models for investigating
prevention and cure strategies against HIV infection and associated pathologies. Retrovirology 17, 8. https://doi.org/10.1186/
s12977-020-00515-3 (2020).i 0. Akkina, R. et al. Small animal models for human immunodeficiency virus (HIV), hepatitis B, and tuberculosis: proceedings of an
NIAID workshop. Curr. HIV Res. 18, 19–28. https://doi.org/10.2174/1570162X18666191223114019 (2020). 1. Victor Garcia, J. Humanized mice for HIV and AIDS research. Curr. Opin. Virol. 19, 56–64. https://doi.org/10.1016/j.coviro.2016
06.010 (2016). 06.010 (2016). 22. Shultz, L. D., Brehm, M. A., Garcia-Martinez, J. V. & Greiner, D. L. Humanized mice for immune system investigation: progress, 2. Shultz, L. D., Brehm, M. A., Garcia-Martinez, J. V. & Greiner, D. L. Humanized mice for immune system investigation: progress
promise and challenges. Nat. Rev. Immunol. 12, 786–798. https://doi.org/10.1038/nri3311 (2012). g
g
3. Walsh, N. C. et al. Humanized mouse models of clinical disease. Annu. Rev. Pathol. 12, 187–215. https://doi.org/10.1146/annur
ev-pathol-052016-100332 (2017). p
4. Klicznik, M. M. et al. Human CD4(+)CD103(+) cutaneous resident memory T cells are found in the circulation of healthy indi-
viduals. Sci Immunol. https://doi.org/10.1126/sciimmunol.aav8995 (2019). p
g
25. Tseng, C. W. et al. Increased susceptibility of humanized NSG mice to panton-valentine leukocidin and Staphylococcus aureus skin
infection. PLoS Pathog. 11, e1005292. https://doi.org/10.1371/journal.ppat.1005292 (2015). athog. 11, e1005292. https://doi.org/10.1371/journal.ppat.1005292 g
p
g
j
pp
6. Wickersham, M. et al. Metabolic stress drives keratinocyte defenses against Staphylococcus aureus infection. Cell Rep. 18, 2742–2751
https://doi.org/10.1016/j.celrep.2017.02.055 (2017). g
j
7. Soong, G. et al. Methicillin-resistant Staphylococcus aureus adaptation to human keratinocytes. mBio. https://doi.org/10.1128/
mBio.00289-15 (2015). 28. Melican, K., Michea Veloso, P., Martin, T., Bruneval, P. & Dumenil, G. Adhesion of Neisseria meningitidis to dermal vessels leads
to local vascular damage and purpura in a humanized mouse model. PLoS Pathog 9, e1003139. https://doi.org/10.1371/journal.
ppat.1003139 (2013). pp
(
)
29. Schulz, A. et al. Neutrophil recruitment to noninvasive MRSA at the stratum corneum of human skin mediates transient coloniza-
tion. Cell Rep. 29, 1074-1081 e1075. References 1. Balato, A. et al. Human microbiome: composition and role in inflammatory skin diseases. Arch. Immunol. Ther. Exp. (Warsz.) 67,
1–18. https://doi.org/10.1007/s00005-018-0528-4 (2019). p
g
(
)
2. Byrd, A. L., Belkaid, Y. & Segre, J. A. The human skin microbiome. Nat. Rev. Microbiol. 16, 143–155. https://doi.org/10.1038/nrmic
ro.2017.157 (2018).h 3. Reynolds, E. S., Hart, C. E., Hermance, M. E., Brining, D. L. & Thangamani, S. An overview of animal models for arthropod-borne
viruses. Comput. Med. 67, 232–241 (2017). p
4. Rhodes, J. & Fisher, M. C. Global epidemiology of emerging Candida auris. Curr. Opin. Microbiol. 52, 84–89. https://doi.org/10
1016/j.mib.2019.05.008 (2019). j
5. Mediavilla, J. R., Chen, L., Mathema, B. & Kreiswirth, B. N. Global epidemiology of community-associated methicillin resistant
Staphylococcus aureus (CA-MRSA). Curr. Opin. Microbiol. 15, 588–595. https://doi.org/10.1016/j.mib.2012.08.003 (2012).f p y
p
p
g
j
6. Bernard, Q., Jaulhac, B. & Boulanger, N. Skin and arthropods: an effective interaction used by pathogens in vector-borne diseases
Eur. J. Dermatol. 25(Suppl 1), 18–22. https://doi.org/10.1684/ejd.2015.2550 (2015). pp
p
g
j
7. Briant, L., Despres, P., Choumet, V. & Misse, D. Role of skin immune cells on the host susceptibility to mosquito-borne viruses
Virology 464–465, 26–32. https://doi.org/10.1016/j.virol.2014.06.023 (2014). gy
p
g
j
8. Kupper, T. S. & Fuhlbrigge, R. C. Immune surveillance in the skin: mechanisms and clinical consequences. Nat. Rev. Immunol. 4
211–222. https://doi.org/10.1038/nri1310 (2004). p
g
(
)
9. Miller, L. S. & Cho, J. S. Immunity against Staphylococcus aureus cutaneous infections. Nat. Rev. Immunol. 11, 505–518. https://
doi.org/10.1038/nri3010 (2011).ff g
10. Eyerich, S., Eyerich, K., Traidl-Hoffmann, C. & Biedermann, T. Cutaneous barriers and skin immunity: differentiating a connected
network. Trends Immunol. 39, 315–327. https://doi.org/10.1016/j.it.2018.02.004 (2018). 11. Nicolas, J. F. & Guy, B. Intradermal, epidermal and transcutaneous vaccination: from immunology to clinical practice. Expert Rev. Vac. 7, 1201–1214. https://doi.org/10.1586/14760584.7.8.1201 (2008). p
g
2. Combadiere, B. & Liard, C. Transcutaneous and intradermal vaccination. Hum. Vac. 7, 811–827. https://doi.org/10.4161/hv.7.8
16274 (2011). 3. Zomer, H. D. & Trentin, A. G. Skin wound healing in humans and mice: challenges in translational research. J. Dermatol. Sci. 90
3–12. https://doi.org/10.1016/j.jdermsci.2017.12.009 (2018).l 14. Pasparakis, M., Haase, I. & Nestle, F. O. Mechanisms regulating skin immunity and inflammation. Nat. Rev. Immunol. 14, 289–301. https://doi.org/10.1038/nri3646 (2014).fi p
g
5. Steiniger, B. S. Human spleen microanatomy: why mice do not suffice. Immunology 145, 334–346. https://doi.org/10.1111/imm
12469 (2015).hi 16. Steiniger, B., Barth, P. & Hellinger, A. Data availabilityh The datasets generated during and/or analyzed by the authors during this study are available from the corre-
sponding author on reasonable request. https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 |
https://doi.org/10.1038/s41598-020-71548 Scientific Reports | (2020) 10:14598 | www.nature.com/scientificreports/ Received: 9 October 2019; Accepted: 13 August 2020 Received: 9 October 2019; Accepted: 13 August 2020 References Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virio 52. Jaiswal, S. et al. Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize int
humanized BLT-NSG mice. Exp. Biol. Med. (Maywood) 240, 67–78. https://doi.org/10.1177/1535370214546273 (20 humanized BLT-NSG mice. Exp. Biol. Med. (Maywood) 240, 67–78. https://doi.org/10.1177/1535370214546273 (2015). 53. Frias-Staheli, N. et al. Utility of humanized BLT mice for a
2205–2218. https://doi.org/10.1128/JVI.03085-13 (2014). 53. Frias-Staheli, N. et al. Utility of humanized BLT mice for analysis of dengue virus infection and antiviral drug testing. J. Virol. 88,
2205–2218. https://doi.org/10.1128/JVI.03085-13 (2014).i 54. Jaiswal, S. et al. Enhanced humoral and HLA-A2-restricted dengue virus-specific T-cell responses in humanized BLT NSG mice. Immunology 136, 334–343. https://doi.org/10.1111/j.1365-2567.2012.03585.x (2012). gy
p
g
j
5. Mota, J. & Rico-Hesse, R. Dengue virus tropism in humanized mice recapitulates human dengue fever. PLoS ONE 6, e20762. https://
doi.org/10.1371/journal.pone.0020762 (2011). g
j
p
6. Kuruvilla, J. G., Troyer, R. M., Devi, S. & Akkina, R. Dengue virus infection and immune response in humanized RAG2(-/-)
gamma(c)(-/-) (RAG-hu) mice. Virology 369, 143–152. https://doi.org/10.1016/j.virol.2007.06.005 (2007). 57. Bente, D. A., Melkus, M. W., Garcia, J. V. & Rico-Hesse, R. Dengue fever in humanized NOD/SCID mice. J. Virol. 79, 13797–13799. https://doi.org/10.1128/JVI.79.21.13797-13799.2005 (2005).fi p
g
8. Lee, E. K. et al. Effects of lymphocyte profile on development of EBV-induced lymphoma subtypes in humanized mice. Proc. Natl
Acad. Sci. USA 112, 13081–13086. https://doi.org/10.1073/pnas.1407075112 (2015). p
g
58. Lee, E. K. et al. Effects of lymphocyte profile on development p
g
p
9. Whitehurst, C. B. et al. Knockout of Epstein-Barr virus BPLF1 retards B-cell transformation and lymphoma formation in human-
ized mice. mBio 6, e01574-01515 (2015). 0. Wang, L. X. et al. Humanized-BLT mouse model of Kaposi’s sarcoma-associated herpesvirus infection. Proc. Natl. Acad. Sci. USA
111, 3146–3151. https://doi.org/10.1073/pnas.1318175111 (2014). g
1. Denton, P. W. et al. Systemic administration of antiretrovirals prior to exposure prevents rectal and intravenous HIV-1 transmis-
sion in humanized BLT mice. PLoS ONE 5, e8829. https://doi.org/10.1371/journal.pone.0008829 (2010). p
g
j
p
2. Denton, P. W. et al. Generation of HIV latency in humanized BLT mice. J. Virol. 86, 630–634. https://doi.org/10.1128/JVI.06120-11
(2012).i 3. Melkus, M. W. et al. Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat. Med. 12
1316–1322. https://doi.org/10.1038/nm1431 (2006).fii g
4. Mariani, R. et al. Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly. J. Virol. 75
3141–3151. https://doi.org/10.1128/JVI.75.7.3141-3151.2001 (2001). Author contributions M.B., L.T., T.Y., and A.R. conceived and designed experiments in the study. Y.A., C.B., S.H., A.D., L.T., R.S., S.K.,
S.B., I.C., and M.B. performed experiments. M.B., A.R., L.T., Y.A., and C.B. analyzed and interpreted the data. M.B., Y.A., C.B., S.B., I.C. and S.H. prepared the manuscript. Acknowledgments g
We used the UPMC-Hillman Cancer Center and Tissue and Research Pathology within the University of Pitts-
burgh Biospecimen Core, which is supported in part by the NIH award P30CA047904. Miss Vanshika Narala
and Miss Nivitha Periyapatna, Department of Infectious Diseases and Microbiology, University of Pittsburgh
assisted with collecting histological data and analysis. Berthony Deslouches, Department of Microbiology and
Molecular Genetics, University of Pittsburgh provided insightful advice on developing this project. This work
was supported by the National Institutes of Health (NIH)-National Institute of Allergy and Infectious Diseases
(NIAID) (R21AI135412). The National Institute of Health, which funds this work, requires scientists to submit
final peer-reviewed journal manuscripts that arise from NIH funds to the digital archive PubMed Central upon
acceptance for publication. References https://doi.org/10.1016/j.celrep.2019.09.055 (2019). p
p
g
j
p
30. Allen, T. M. et al. Humanized immune system mouse models: progress, challenges and opportunities. Nat. Immunol. 20, 770–
https://doi.org/10.1038/s41590-019-0416-z (2019).t p
g
(
)
1. Erdag, G. & Morgan, J. R. Survival of fetal skin grafts is prolonged on the human peripheral blood lymphocyte reconstituted-severe
combined immunodeficient mouse/skin allograft model. Transplantation 73, 519–528 (2002). i
gt
p
32. Lane, A. T., Scott, G. A. & Day, K. H. Development of human fetal skin transplanted to the nude mouse. J. Invest. Dermatol. 93,
787–791. https://doi.org/10.1111/1523-1747.ep12284423 (1989). p //
g/
/
p
(
)
33. Iannaccone, P. M. & Jacob, H. J. Rats!. Dis. Models Mech. 2, 206–210. https://doi.org/10.1242/dmm.002733 (2009).t 34. Brungger, A., Hubler, M. & Rohr, H. P. Human skin grafts on athymic nude rats. An experimental model for dermatological
research. Exp. Cell Biol. 52, 122–124 (1984).t p
35. Gilhar, A., Etzioni, A. & Krueger, G. G. Hair-growth in human split-thickness skin-grafts transplanted onto nude rats—the role
of ciclosporin. Dermatologica 181, 117–121. https://doi.org/10.1159/000247898 (1990).t p
g
p
g
6. Gilhar, A. et al. Description of and treatment to inhibit the rejection of human split-thickness skin-grafts by congenitally athymic
(nude) rats. Exp. Cell Biol. 54, 263–274 (1986). p
37. Petratos, P. B. et al. Full-thickness human foreskin transplantation onto nude rats as an in vivo model of acute human wound
healing. Plast. Reconstr. Surg. 111, 1988–1997. https://doi.org/10.1097/01.PRS.0000056831.87062.4B (2003). https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 | www.nature.com/scientificreports/ 38. Noto, F. K. et al. Novel immunodeficient rat models capable of supporting the growth of human tumor xenografts. Cancer Res. 78,
72–73 (2018).h 9. Noto, F. K. et al. The SRG (TM) rat: A novel SCID rat for humanization studies. Cancer Res. https://doi.org/10.1158/1538-7445
Am2018-1155 (2018).lf (
)
40. de Oliveira, V. L. et al. Humanized mouse model of skin inflammation is characterized by disturbed keratinocyte differentiation
and influx of IL-17A producing T cells. PLoS ONE 7, e45509 (2012). (
)
0. de Oliveira, V. L. et al. Humanized mouse model of skin inflammation is characterized by disturbed keratinocyte differentiation
d i fl
f IL 17A
d
i
T
ll
PL S ONE 7
45509 (2012) 40. de Oliveira, V. L. et al. Humanized mouse model of skin inflammation is characterized by disturbed keratinocyte differentia
and influx of IL-17A producing T cells. PLoS ONE 7, e45509 (2012). l
g
1. Watanabe, R. et al. References Human skin is protected by four functionally and phenotypically discrete populations of resident and recircu-
lating memory T cells. Sci. Transl. Med. 7, 279ra239 (2015). l
41. Watanabe, R. et al. Human skin is protected by four functio
lating memory T cells. Sci. Transl. Med. 7, 279ra239 (2015) l
41. Watanabe, R. et al. Human skin is protected by four functiona g
y
(
)
42. Hartmann-Fritsch, F., Biedermann, T., Braziulis, E., Meuli, M. & Reichmann, E. A new model for preclinical testing of dermal
substitutes for human skin reconstruction. Pediatr. Surg. Int. 29, 479–488 (2013). g
43. Gudjonsson, J. E., Johnston, A., Dyson, M., Valdimarsson, H. & Elder, J. T. Mouse models of psoriasis. J. Invest. Dermatol. 1292–1308 (2007). 1292 1308 (2007). 4. Lorenz, H. P. et al. Scarless wound repair: a human fetal skin model. Development 114, 253–259 (1992). Lorenz, H. P. et al. Scarless wound repair: a human fetal skin mod
A
d
l
f
h l
d
h 45. Aneiros-Fernandez, J. et al. Expression of smoothelin and smooth muscle actin in the skin. Histol. Histopathol. 26, 673–678. https://
doi.org/10.14670/HH-26.673 (2011).i g
6. Hua, S. et al. Potential role for HIV-specific CD38-/HLA-DR+ CD8+ T cells in viral suppression and cytotoxicity in HIV control-
lers. PLoS ONE 9, e101920. https://doi.org/10.1371/journal.pone.0101920 (2014). g
j
7. Pearse, G. Normal structure, function and histology of the thymus. Toxicol. Pathol. 34, 504–514. https://doi.org/10.1080/01926
230600865549 (2006). 8. Prince, A., Wang, H., Kitur, K. & Parker, D. Humanized mice exhibit increased susceptibility to Staphylococcus aureus pneumonia
J. Infect. Dis. 215, 1386–1395. https://doi.org/10.1093/infdis/jiw425 (2017).i f
g
j
9. Menoret, S. et al. In Vivo Analysis of Human Immune Responses in Immunodeficient Rats. Transplantation 104, 715–723. https://
doi.org/10.1097/TP.0000000000003047 (2020).i g
0. Yang, X. et al. An immune system-modified rat model for human stem cell transplantation research. Stem Cell Rep. 11, 514–521
https://doi.org/10.1016/j.stemcr.2018.06.004 (2018). p
g
j
51. Spengler, J. R. et al. Severity of disease in humanized mice infected with ebola virus or reston virus is associated with magnitude
of early viral replication in liver. J. Infect. Dis. 217, 58–63. https://doi.org/10.1093/infdis/jix562 (2017). y
p
f
p
g
j
52. Jaiswal, S. et al. Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize int
humanized BLT-NSG mice. Exp. Biol. Med. (Maywood) 240, 67–78. https://doi.org/10.1177/1535370214546273 (20 y
p
f
p
g
j
52. Jaiswal, S. et al. Competing interests If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020, corrected publication 2021 © The Author(s) 2020, corrected publication 2021 Competing interests p
g
Yash Agarwal, Cole Beatty, Sara Ho, Lance Thurlow, Antu Das, Samantha Kelly, Rajeev Salunke, Isabella Cas-
tronova, Shivkumar Biradar, Anthony Richardson, and Moses Bility have declared that no conflict of interest
exists. Tseten Yeshi has a financial conflict of interest, as he works for HaraBiolabs, which provides the Sprague–
Dawley-Rag2tm2hera Il2rγtm1hera (SRG) rat commercially. https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 | www.nature.com/scientificreports/ Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-71548-z. Correspondence and requests for materials should be addressed to M.B. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020, corrected publication 2021 Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-71548-z. Correspondence and requests for materials should be addressed to M.B. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-71548-z. Correspondence and requests for materials should be addressed to M.B. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020, corrected publication 2021 https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 |
|
https://openalex.org/W2133255431
|
https://www.scielo.br/j/bjps/a/8NGPZY8wztLjcvYdWMrgHcf/?lang=en&format=pdf
|
English
| null |
Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution
|
Brazilian Journal of Pharmaceutical Sciences
| 2,013
|
cc-by
| 4,558
|
Uniterms: Agar diffusion. Antiseptic. Chlorhexidine. Microbiological assay. Quality control. Validation. Uniterms: Agar diffusion. Antiseptic. Chlorhexidine. Microbiological assay. Quality control. Validation Clorexidina (CHX) é um antisséptico com amplo espectro de ação utilizada em muitos tipos de
preparações farmacêuticas para uso tópico. Uma vez que não há na literatura ensaio microbiológico
oficial para quantificar a clorexidina, este trabalho objetivou o desenvolvimento e validação de um
ensaio microbiológico simples, sensível, exato e reprodutível, por difusão em ágar, para doseamento
de digliconato de clorexidina (CHX-D) em solução aquosa. O ensaio é baseado no efeito da inibição
de Staphylococcus aureus ATCC 25923, utilizado como microorganismo teste, pela CHX-D. Utilizou-
se o delineamento 3x3. Os resultados foram verificados estatisticamente pela análise de variância
(ANOVA) e apresentaram excelente linearidade (r = 0,9999), demonstrando que o método segue
o modelo linear com regressão significativa entre o diâmetro da zona de inibição e o lagaritmo da
concentração no intervalo de 0,5 a 4,5%. Os resultados obtidos foram precisos apresentando desvio
padrão relativo (DPR) para precisão intra-dia de 2,03% e DPR para precisão inter-dias de 2,94%. A
exatidão foi 99,03%. O método provou ser muito útil e apropriado para doseamento microbiológico
da CHX-D em formas farmacêuticas e pode ser empregado para análise desta substância no controle
de qualidade em indústrias farmacêuticas. *Correspondence: H. R. N. Salgado. Universidade Estadual Paulista. Ro
dovia Araraquara-Jaú, km 1, 14801-902 – Araraquara - SP, Brasil. Phone:
55‑16‑33016967, Fax 55-16-33016900. E-mail: salgadoh@fcfar.unesp.br Article Brazilian Journal of
Pharmaceutical Sciences
vol. 49, n. 2, apr./jun., 2013 Unitermos: Difusão em ágar. Antisséptico. Clorexidina. Ensaio microbiológico. Controle de qualidade.
Validação. Flávia Angélica Másquio Fiorentino, Marcos Antonio Corrêa, Hérida Regina Nunes Salgado* partment of Drugs And Medicines, School of Pharmaceutical Sciences, University Estadual Paulista Chlorhexidine (CHX) is a broad-spectrum antiseptic that is used in many topical pharmaceutical
formulations. Because there is no official microbiological assay reported in the literature that is used to
quantify CHX, this paper reports the development and validation of a simple, sensitive, accurate and
reproducible agar diffusion method for the dosage of chlorhexidine digluconate (CHX-D) in an aqueous
solution. The assay is based on the inhibitory effect of CHX-D upon the strain of Staphylococcus aureus
ATCC 25923, which is used as the test microorganism. The design 3x3 parallel-line model was used. The
results were treated statistically by analysis of variance (ANOVA), and they were excellent in terms of
linearity (r = 0.9999), presenting a significant regression between the zone diameter of growth inhibition
and the logarithm of the concentration within the range of 0.5 to 4.5%. The results obtained were precise,
having relative standard deviations (RSD) for intra-day and inter-day precision of 2.03% and 2.94%,
respectively. The accuracy was 99.03%. The method proved to be very useful and appropriate for the
microbiological dosage of CHX-D in pharmaceutical formulations; it might also be used for routine
drug analysis during quality control in pharmaceutical industries. Uniterms: Agar diffusion. Antiseptic. Chlorhexidine. Microbiological assay. Quality control. Validation. INTRODUCTION hands, and surgical sites and are sometimes used to
impregnate catheters (Fiorentino, Corrêa, Salgado, 2010). hands, and surgical sites and are sometimes used to
impregnate catheters (Fiorentino, Corrêa, Salgado, 2010). Chlorhexidine (CHX) is an excellent cationic
antiseptic that belongs to a class of drugs called biguanides
and has the chemical name (1,1’-hexamethylenebis{5-
(p-chlorophenyl)biguanide)} (Buxbaum et al., 2006;
USP, 2008; BP, 2010; EP, 2011). It exists as a variety
of salts, including acetate, gluconate, hydrochloride Chlorhexidine (CHX) is an excellent cationic
antiseptic that belongs to a class of drugs called biguanides
and has the chemical name (1,1’-hexamethylenebis{5-
(p-chlorophenyl)biguanide)} (Buxbaum et al., 2006;
USP, 2008; BP, 2010; EP, 2011). It exists as a variety
of salts, including acetate, gluconate, hydrochloride Antiseptics are products that are used to clean and
disinfect traumatic wounds, burns, mucous membranes, 352 F. A. M. Fiorentino, M. A. Corrêa, H. R. N. Salgado and digluconate, but the digluconate form (Figure 1) is
the most soluble in water and at physiological pH (EP,
2011; Thomas et al., 2000; Martindale, 2009). CHX is
active against Gram-positive bacteria; less active against
Gram-negative bacteria, fungi, and species of Proteus;
and has activity against certain types of enveloped
viruses, including hepatitis virus, herpes simplex, HIV,
cytomegalovirus, influenza and respiratory syncytial virus. For mycobacteria, the CHX exhibits minimal activity;
against endospores and cysts of protozoa the activity is
nil (Fiorentino, Corrêa, Salgado, 2010; Cone et al., 2006;
Martindale, 2009). contain CHX, several physicochemical methods are
presented, such as HPLC (Gavlick, 1992; Há, Cheung,
1996; Izumoto et al., 1997; Havlíková et al., 2007), solid-
phase extraction with UV spectrophotometry (Bonazzi
et al., 1995), gas-liquid chromatography (Miribel et al.,
1983), and capillary electrophoresis (Abad-Villar et al.,
2006). A standard microbiological assay has not been
reported to determine the potency of CHX digluconate in an
aqueous solution using an agar diffusion test. However, in
the literature, there are a large number of reports describing
the use of microbiological assays to assess the potency of
many antibiotics (Marona, Schapoval, 1998; Mendez et al.,
2005; Gomes, Salgado, 2006; Salgado, Lopes, Lucchesi,
2006; Vaugher, Breier, Schapoval, 2006; Souza et al., 2007;
Moreno, Salgado, 2007; Salgado, Tozo, 2007; Schmidt et
al., 2008; Lopes, Salgado, 2010; Cazedey, Salgado, 2011;
Paim et al., 2011; Solano et al., 2011). FIGURE 1 - Chemical structure of chlorhexidine digluconate
(CAS: 18472-51-0). INTRODUCTION Although biological assays have a high variability,
a microbiological assay can reveal subtle changes that are
not demonstrable by chemical methods. A bioassay is an
environmentally friendly technique because it does not
require solvent or produce toxic by-products. Moreover, a
microbiological assay requires no specialised equipment,
is low in cost, is a simple procedure and requires only a
simple facility to obtain results. These factors make the
use of microbiological assays a very good alternative
methodology for a potency assessment of antimicrobial
drugs in a quality control laboratory (Salgado, Lopes,
Lucchesi, 2006; Vaugher, Breier, Schapoval, 2006; Souza
et al., 2007; Schmidt et al., 2008; Lopes, Salgado, 2010;
Paim et al., 2011). Additionally, the excipients used for
physicochemical techniques often cause interference and
require expensive and sophisticated equipment that is not
available in some quality control laboratories (Schmidt
et al., 2008). FIGURE 1 - Chemical structure of chlorhexidine digluconate
(CAS: 18472-51-0). There are many uses for CHX. It is most likely
the most widely used biocide in antiseptic products,
specifically in hand-washing and oral products; it is also
used to impregnate catheters or other medical products, as
a preservative in vaginal gel, as a disinfectant (Cone et al.,
2006; Galice et al., 2006) and as a surgical scrub to clean
the site of surgery (Buxbaum et al., 2006). Standard physicochemical methods for the
determination of CHX are described in the European,
British and United States Pharmacopoeias (USP, 2008;
BP, 2010; EP, 2011). These techniques employ high
performance liquid chromatography (HPLC) (USP, 2008;
BP, 2010; EP, 2011) or titration with 0.1 M perchloric
acid (BP, 2010; EP, 2011). The methods described in the
literature to analyse degradation products of CHX and
related substances are HPLC (USP, 2008) and colorimetric
tests (BP, 2010; EP, 2011). The aim of the present study was to develop
and validate a simple, sensitive, precise and accurate
microbiological assay by agar diffusion using a template
method to quantify CHX-D in aqueous solution as an
ecological alternative to the physicochemical methods
described in the literature. The literature describes several methods for
the determination of CHX in biological fluids, which
include direct UV spectroscopy (Jensen, Christensen,
1971), fluorometry (Vries, Ruben, Arends, 1991), HPLC
(Gaffney, Cooke, 1984; Brougham, Cheng, Pittman, 1986;
Lam et al., 1993; Pesonem, Holmalahti, Pohjola, 1995;
Below, Lehan, Kramer, 2004) and liquid chromatography-
electrospray ionisation mass spectrometry (LC-ESI-MS)
(Usui et al., 2006). For pharmaceutical products that Method validation Aliquots of 250 µL, 750 µL and 2250 µL of a CHX
digluconate aqueous solution were transferred to 10-mL
volumetric flasks, which were filled with water to give
concentrations of 0.5, 1.5 and 4.5% (T1, T2 and T3),
respectively. These standard solutions were tested against
S1, S2 and S3. The method was validated by the determination of
linearity, precision and accuracy (USP, 2008; Marona,
Schapoval, 1998; Gomes, Salgado, 2007; Salgado, Lopes,
Lucchesi, 2006; Moreno, Salgado, 2007; Salgado, Tozo,
2007; Lopes, Salgado, 2010; Cazedey, Salgado, 2011;
Paim et al., 2011; ICH, 2005; AOAC, 2005). Chemicals The CHX digluconate aqueous standard solution
(purity 20.0%, batch number: 07/1306) was kindly donated
by Rioquímica LTDA (Brazil). The CHX digluconate
aqueous solution (Henrifarma®, Brazil) (rotulated purity 353 Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution 20.0%, batch number: CS 0120705) was kindly donated
by the Cosmetology Laboratory of Universidade Estadual
Paulista (UNESP) (Brazil). Purified water was used in all
experiments. 20.0%, batch number: CS 0120705) was kindly donated
by the Cosmetology Laboratory of Universidade Estadual
Paulista (UNESP) (Brazil). Purified water was used in all
experiments. x 20 mm Petri dish as a base layer. After solidification of
this layer, a 5 mL portion of the inoculated BHI agar was
poured onto the base layer. In each plate, a template was placed on the surface
of the inoculated medium. Three alternating holes were
filled with 200 µL of the reference solution (S1, S2 and
S3), and the other three holes were filled with the sample
solutions (T1, T2 and T3). Six plates were used in each
assay. The plates were incubated at 35 ± 1 ºC under aerobic
conditions for 18 hours. The diameters (mm) of the zones
of inhibition were carefully measured using a digital
calliper (Mitutoyo® IP 65) (Figure 2). All experiments
were performed in a biological safety cabinet, and the
infected material was decontaminated before being
discarded. Precision The precision of the method was determined by
repeatability (intra-assay) and the intermediate precision
(inter-assay) and was expressed as the relative standard
deviation (RSD). The repeatability was verified by
assaying samples of CHX digluconate in aqueous solution,
at the same concentration, within one day and under the
same experimental conditions. The intermediate precision
was evaluated by comparing the assay results from three
different days. Chlorhexidine digluclonate reference solutions Chlorhexidine digluclonate reference solutions Aliquots of 250 µL, 750 µL and 2250 µL of the
aqueous CHX digluconate reference solution were
transferred to 10-mL volumetric flasks, which were filled
with water to give concentrations of 0.5, 1.5 and 4.5% (S1,
S2 and S3), respectively. These standard solutions were
used in the bioassay. Linearity The cultures of S. aureus ATCC 25923 were
cultivated on casein-soy agar (Acumedia®) at a temperature
near freezing (2.0 to 8.0 ºC). The microorganism
standardisation was carried out according to the
procedures described in the Brazilian and United States
Pharmacopoeias (USP, 2008; F. BRAS., 2010). Prior to
use, the microorganism was grown in Brain Heart Infusion
(BHI) broth (BD®) in a test tube that was incubated for 24 h
at 35 ºC ± 2 ºC. Using a spectrophotometer (Beckman,
DU® 530) with the wavelength set at 580 nm and a 10 mm
absorption cell, the broth containing the microorganism
was diluted to achieve a suspension turbidity of 25 ± 2%
(transmittance) using the same sterile broth solution as
for the blank. From this standardised suspension, 1.0 mL
aliquots were added to each 100 mL of BHI agar (BD®) at
47 ºC ± 1 ºC and used as the inoculum layer in the plate. To assess the validity of the assay, three doses of
the standard and three doses of the sample were used. The linearity was evaluated by linear regression analysis,
which was calculated using the least squares method. Agar diffusion bioassay Accuracy was determined by adding 3 concentrations,
which covered the specified range. Aliquots of precisely
500, 1000 and 1500 μL of the standard solution (20%)
were transferred into 10 mL volumetric flasks, and diluted
with water to give final concentrations of 1.5, 2.5 and
3.5%, respectively. The percentage recovery of the CHX
digluconate standard was calculated using the equation
proposed by the AOAC (2005). The bioassay described previously followed the 3
x 3 parallel line assay design (three doses of the standard
and three doses of the sample in each plate), with six plates
for each assay, according to the Brazilian Pharmacopoeia
(F. BRAS., 2010). The agar was composed of two separate
layers. The BHI agar (20 mL) was poured into a 100 mm 354 F. A. M. Fiorentino, M. A. Corrêa, H. R. N. Salgado RESULTS AND DISCUSSION The development and validation of analytical
methods for the determination of active ingredients in
medicines is very important for quality control and patient
safety. When choosing an analytical method for use in
routine quality control, the analyst must consider cost,
complexity, required time, availability of equipment and
reagents, purity, quantity of the sample and the generation
of residues (ICH, 2005; Salgado, Lopes, Lucchesi, 2006;
Moreno, Salgado, 2007). Calculation of potency TABLE I - Tested conditions to establish the parameters for
microbiological assay of CHX digluconate microbiological assay of CHX digluconate
Parameter
Condition
Standard curve (μg/mL)
1.5, 3.0, 6.0%; 0.25, 1.0, 4.0%
and 0.5, 1.5, 4.5%
Diluents
Buffer solution pH 6.0 and
pH 8.0
Purified water
Inoculum (%)
0.5 and 1.0
Microorganism
Bacillus subtilis ATCC 9372
Staphylococcus aureus ATCC
25923
Culture media
Mueller Hinton agar
Brain Heart Infusion (BHI)
agar To calculate the activity of CHX digluconate in
aqueous solution, the Hewitt [38] equation was used. The
assays were statistically analysed using the linear parallel
model and regression analysis and verified using analysis
of variance (ANOVA) (USP, 2008; Marona, Schapoval,
1998; Salgado, Gomes, Salgado, 2006; Lopes, Lucchesi,
2006; Moreno, Salgado, 2007; Salgado, Tozo, 2007; F. BRAS., 2010; Lopes, Salgado, 2010; Cazedey, Salgado,
2011; Paim et al., 2011). Method validation In this experimental work, a 3 x 3 design was
used, using three concentrations of both a standard and
sample solution, according to the procedure described
in the Brazilian Pharmacopoeia (F. BRAS., 2010). The
calculation assumes a direct relationship between the
observed diameter of the zone of inhibition and the
logarithm of the applied dose. The growth inhibition zones
of aqueous solutions of CHX digluconate and the reference
substance are shown in Figure 2. g
Moreover, the development and validation
of analytical methods to determine potency, such
as microbiological assays, have recently received
considerable attention, mainly from regulatory agencies,
because of their importance in pharmaceutical analysis
(Mendez et al., 2005; Moreno, Salgado, 2007; Salgado,
Tozo, 2007; Lopes, Salgado, 2010; Cazedey, Salgado,
2011). Microbiological assays have the potential to prevent
the possible loss of activity, while physicochemical
methods do not prevent the loss of the antimicrobial
potency (USP, 2008). FIGURE 2 - An agar diffusion assay using a strain of S. aureus
ATCC 25923 as the test microorganism. The chlorhexidine
digluconate reference solutions are at concentrations of 0.5 (S1),
1.5 (S2) and 4.5% (S3) and the chlorhexidine digluconate samples
are at concentrations of 0.5 (T1), 1.5 (T2) and 4.5% (T3). For this reason, a microbiological assay was
proposed as a suitable method for the determination of
CHX digluconate in aqueous solution. The experimental conditions were tested and
adjusted to accurately determine the performance of the
assay. Some parameters were tested previously to establish
the described conditions and shown in Table I. The strain
of Staphylococcus aureus that was used, ATCC 25923,
appears to be an appropriate test microorganism because
of its susceptibility to CHX-D and its capacity to form
sharply defined zones of growth inhibition, allowing for
precise measurements. FIGURE 2 - An agar diffusion assay using a strain of S. aureus
ATCC 25923 as the test microorganism. The chlorhexidine
digluconate reference solutions are at concentrations of 0.5 (S1),
1.5 (S2) and 4.5% (S3) and the chlorhexidine digluconate samples
are at concentrations of 0.5 (T1), 1.5 (T2) and 4.5% (T3). The potency of an antibiotic may be demonstrated
under suitable conditions by comparing the inhibition
of growth of a microorganism caused by known
concentrations of the antibiotic and a reference standard
(USP, 2008; F. BRAS., 2010; EP, 2011). Table II gives the results, represented as the
diameters of the zones of inhibition, for CHX digluconate
in aqueous solution. Method validation 355 Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution TABLE II - Diameters of the zones of inhibition for CHX digluconate in aqueous solution and the reference solution, obtained using
a standard curve TABLE II - Diameters of the zones of inhibition for CHX digluconate in aqueous solution and the reference solution, obtained using
a standard curve
Concentration (%)
Range of zone size, mm
Means diameters of growth
inhibition zonesa, mm
RSD%
0.5
19.27 – 19.32
19.30
0.9
1.5
20.79 – 20.84
20.81
1.0
4.5
22.33 – 22.39
22.37
1.1
aMean of 3 assays with 6 plates in each. aMean of 3 assays with 6 plates in each. The method precision in terms of repeatability
(intra-assay) shows a mean R.S.D. of 2.03%, indicating
good intra-day precision for the method. The intermediate
precision shows a mean R.S.D. of 2.94%. The calibration curve for CHX digluconate in
aqueous solution was constructed by plotting the zone
diameter (mm) versus the log of the concentration (%) and
showed good linearity in the 0.5 to 4.5% range. The mean accuracy was 99.03%, and the R.S.D. was 1.32% (Table IV), which confirms the ability of the
method to accurately determine the concentration of
CHX digluconate in aqueous solution and shows that the
results obtained from the bioassay were close to the true
concentrations of the samples. The representative linear equation for CHX
digluconate was y = 1.3972 Ln(x) + 20.26, where x is
the log dose and y is the zone diameter. The coefficient
of regression was 0.9999, which is highly significant for
the method. According to the Brazilian and the United
States Pharmacopoeias (USP, 2008; F. BRAS., 2010), if a
parallel-line model is chosen, the two log dose-response
lines of the preparations to be examined, as well as that of
the reference preparation, must be parallel, and they must
be linear over the range of doses used in the calculation. These conditions must be verified by validity tests for
a given probability, usually p = 0.05. The assays were
validated by analysis of variance (ANOVA), as described
in those official codes. There were no deviations from
parallelism or linearity in the obtained results (p < 0.05)
(Table III). Calculation of potency The CHX digluconate activity ranged from 92.52
to 92.61%, with an R.S.D. value of 0.1%. This value is
acceptable because the American Pharmacopoeia (USP,
2008) allows for variations of up to 10% for mouth rinse. Moreover, CHX digluconate is a non-sterile product that
is indicated for external use only, which allows a 10%
variation (USP, 2008; EP, 2011). The low R.S.D. values
that were obtained confirm that the proposed method can
generate reproducible results with little variation between
independent assays. TABLE III - Analysis of variance of determination of CHX
digluconate by bioassay
Parameters
F calc
F tab
Linear regression
154264.9*
4.24
Linearity deviation
3.159
3.38
Parallelism
0.182
4.24
* p < 0.05). TABLE III - Analysis of variance of determination of CHX
digluconate by bioassay The quantification of ingredients in antiseptics
using chemical methods, such as HPLC and UV
spectrophotometry, although precise, cannot provide a true
indication of biological activity. Therefore, bioassays play
an essential role in the manufacturing and quality control
of antibiotics. This study confirms the validity of using
bioassays for the quality control of antiseptic medicines Parameters
F calc
F tab
Linear regression
154264.9*
4.24
Linearity deviation
3.159
3.38
Parallelism
0.182
4.24
* p < 0.05). TABLE IV - Accuracy of the microbiological assay of CHX digluconate TABLE IV - Accuracy of the microbiological assay of CHX digluconate
Run
Amount of standard (%)
Added (%)
Recovered (%)
Recovery (%)a
RSD (%)
R1
5.00
4.93
98.6
R2
10.00
9.80
98.0
1.32
R3
15.00
15.07
100.5
aMean of 3 assays 356 F. A. M. Fiorentino, M. A. Corrêa, H. R. N. Salgado BONAZZI, D.; ANDRISANO, V.; GATTI, R.; CAVRINI, V. Analysis of pharmaceutical creams: a useful approach base
on solid-phase extraction (SPE) and UV spectrophotometry. J. Pharm. Biomed. Anal., v.13, p.1321-1329, 1995. this for antiseptic medicines, and this technique still
demands considerable skill and expertise to ensure success
(Vaugher, Breier, Schapoval, 2006; Lopes, Salgado, 2010;
Cazedey, Salgado, 2011; Paim et al., 2011). Although
biological assays might have a high variability, the analysis
of the results demonstrates that the proposed method might
be very useful for determination of the potency of CHX
digluconate in aqueous solution. BRITISH PHARMACOPOEIA. London: Her Majesty’s
Stationery Office, 2010. p.467-470, p.2485-2488. BROUGHAM, L.R.; CHENG, H.; PITTMAN, K.A. Sensitive
high-performance liquid chromatographic method for the
determination of chlorhexidine in human serum and urine. J. Chromatogr., v.383, p.365-373, 1986. ACKNOWLEDGEMENTS EUROPEAN PHARMACOPOEIA. 7.ed. Strasbourg: Council
of Europe, 2011. p.1659-1663. The authors are grateful to Maria de Fátima
Rodrigues for technical support and to Dr. Greici C. G. Tozo, Rioquimica Ltda (São José Rio Preto, Brazil) for
providing the standard of CHX. CNPq – Brazil, CAPES –
Brazil and PADC – FCF – Araraquara Program supported
this work. FARMACOPEIA BRASILEIRA. 5.ed. Brasília: ANVISA,
2010. p.258-270. FIORENTINO, F.A.M.; CORRÊA, M.A.; SALGADO, H.RN. Analytical methods for the determination of chlorhexidine:
a review. Critical Rev. Anal. Chem., v.40, p.89-101, 2010. CONCLUSION The results indicated that the microbiological assay
demonstrated excellent linearity, precision and accuracy
at concentrations ranging from 0.5 to 4.5%. Therefore, it
is an acceptable alternative method for the routine quality
control of CHX digluconate in aqueous solution. The
method uses simple reagents, requires minimal sample
preparation, produces no toxic residues and there is no
sophisticated equipment required, supporting its use in
routine analysis. CAZEDEY, E.C.L.; SALGADO, H.R.N. Development and
validation of a microbiological agar assay for determination
of orbifloxacin in pharmaceutical preparations. Pharmaceutics, v.3, p.572-580, 2011. CONE, R.A.; HOEN, T.; WONG, X.; ABUSUWWA, R.;
ANDERSON, D.J; MOENCH, T.R. Vaginal microbicides:
detecting toxicities in vivo that paradoxically increase
pathogen transmission. BMC Infect. Dis., v.6, p.1-16, 2006. Calculation of potency The results obtained in this study were very satisfactory,
and the validation demonstrated that microbiological assays
are a good alternative methodology for the pharmaceutical
analysis of CHX digluconate in aqueous solution. It is a
useful analytical tool in conjunction with or substitution for
a physicochemical method. BUXBAUM, A.; KRATZER, C.; GRANINGER, W.;
GEORGOPOULOS, A. Antimicrobial and toxicological
profile of the new biocide Akacid plus. J. Antimicrob. Chemother.,v.58, p.193-197, 2006. REFERENCES GAFFNEY, M.H.; COOKE, M.J. Improved method for the
determination of chlorhexidine in urine. Chromatogr.,
v.306, p.303-313, 1984. ABAD-VILLAR, E.M.; ETTER, S.F.; THIEL, M.A.; HAUSER,
P.C. Determiantion of chlorhexidine digluconate and
polyhexamethylene biguanide in eye drops by capillary
electrophoresis with contactless conductivity detection. Anal. Chem. Acta., v.561, p.133-137, 2006. GALICE, M.D.; BONACORSI, C.; SOARES, V.C.G.;
RADDI, M.S.G.; FONSECA, L.M. Effect of subinhibitory
concentration of chlorhexidine on Streptococcus agalactiae
virulence factor expression. Int. J. Antimicrob. Agents., v.28,
p.143-146, 2006. AOAC INTERNATIONAL. Official methods of analysis. 18ed. Gaithersburg: AOAC INTERNATIONAL, 2005. AOAC INTERNATIONAL. Official methods of analysis. 18ed.
Gaithersburg: AOAC INTERNATIONAL, 2005. BELOW, H.; LEHAN, N.; KRAMER, A. HPLC determination
of the antiseptic agent chlorhexidine and its degradation
products 4-chloroaniline and 1-chloro-4-nitrobenzene in
serum and urine. Microchim. Acta, v.146, p.129-135, 2004. 357 Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution GAVLICK, W.K. High-performance liquid chromatographic
analysis of chlorhexidine and p-chloroaniline using a
specialty column and a photodiode-array detector. J. Chromatogr., v.623, p.375-380, 1992. MARONA, H.R.N.; SCHAPOVAL, E.E.S. Desarrollo
de análisis microbiológico para la determinación de
esparfloxacino en polvo y en comprimidos de 200 mg. Inf. Tecnol., v.9, p.251-254, 1998. GOMES, G.C.; SALGADO, H.R.N. Microbiological assay for
determination of lomefloxacin in coated tablets. J. AOAC
Int., v.89, p.1077-1079, 2006. MARTINDALE: the complete drug reference. 36ed. Pharmaceutical Press, 2009. p.1635-1638. MENDEZ, A.S.L.; WEISHEIMER, V.; OPPE, T.P.; STEPPE,
M.; SCHAPOVAL, E.E.S. Microbiological assay for the
determination of meropenem in pharmaceutical dosage
form. J. Pharm. Biomed. Anal., v.37, p.649-653, 2005. HÁ, Y.; CHEUNG, A.P. New stability-indicating high
performance liquid chromatography assay and proposed
hydrolytic pathways of chlorhexidine. J. Pharm. Biomed. Anal., v.14, p.1327-1334, 1996. MIRIBEL, L.; BRAZIER, J.L.; COMET, F.; LECOMPTE, D. Gas-liquid chromatographic determination of chlorhexidine
in pharmaceutical formulations. J. Chromatogr., v.268,
p.321-328, 1983. HAVLÍKOVÁ, L.; MATYSOVÁ, L.; NOVÁKOVÁ, L.;
HAJKOVÁ, R.; SOLICH, P. HPLC determination of
chlorhexidine gluconate and p-chloroaniline in topical
ointment. J. Pharm. Biomed. Anal., v.43, p.1169-1173,
2007. MORENO, A.H.; SALGADO, H.R.N. Microbiological assay
for ceftazidime injection. J. AOAC Int., v.90, p.1379-1382,
2007. HEWITT, W. Microbiological assay for pharmaceutical
analysis: a rational approach. Boca Raton: Interpharm/CRC
Press, 2003. p.97-115. PAIM, C.S.; F’UHR, F.; BARTH, A.B.; GONÇALVES,
C.E.I.; NARDI, N.; STEPPE, M.; SCHAPOVAL, E.E.S. Gemifloxacin mesylate (GFM) stability evaluation applying
a validated bioassay method and in vitro cytotoxic study. Talanta, v.83, p.1774-1779, 2011. INTERNATIONAL CONFERENCE ON HARMONIZATION. ICH. Topic Q2(R1) validation of analytical procedures: text
and methodology. Geneva: ICH Steering Committee, 2005. PESONEM, T.; HOLMALAHTI, J.; POHJOLA, J. Determination of chlorhexidine in saliva using high-
performance liquid chromatography. J. Chromatogr. B. Biomed. Appl., v.665, p.222-225, 1995. IZUMOTO, S.; MACHIDA, Y.; NISHI, H.; NAKAMURA, K.;
NAKAI, H.; SATO, T. Chromatography of crotamiton and
its applications to the determination of active ingredients
in ointment. J. Pharm. Biomed. Anal., v.15, p.1457-1466,
1997. SALGADO, H.R.N.; LOPES, C.C.G.O.; LUCCHESI, M.B.B. Microbiological assay of gatifloxacin in pharmaceutical
formulations. J. Pharm. Biomed. Anal., v.40, p.443-446,
2006. JENSEN, J.E.; CHRISTENSEN, F. AOAC INTERNATIONAL. Official methods of analysis. 18ed.
Gaithersburg: AOAC INTERNATIONAL, 2005. A study of the elimination
of chlorhexidine from the oral cavity using a new
spectrophotometric method. J. Periodont. Res., v.6, p.306-
311, 1971. SALGADO, H.R.N.; TOZO, G.C.G. Microbiological assay for
cefoxitin sodium in dosage form. J. AOAC Int., v.90, n.2,
p.452-455, 2007. LAM, Y.W.F.; CHAN, D.C.N.; RODRIGUEZ, S.;
LINTAKOON, J.H.; LAM, T-H. Sensitive high-performance
liquid chromatographic assay for the determination of
chlorhexidine in saliva. J. Chromatogr., v.612, p.166-171,
1993. SCHMIDT, C.A.; CARAZZO, M.; LAPORTA, L.V.;
BITTENCOURT, C.F.; SANTOS, M.R.; FRIEDRICH,
M. Development and validation of an agar diffusion
assay for determination of ceftazidime in pharmaceutical
preparations. J. AOAC Int., v.91, p.59-66, 2008. LOPES, C.C.G.O.; SALGADO, H.R.N. Development and
validation of a stability-indicative agar diffusion assay to
determine the potency of linezolid in tablets in the presence
of photodegradation products. Talanta, v.82, p.918-922,
2010. 358 F. A. M. Fiorentino, M. A. Corrêa, H. R. N. Salgado SOLANO, A.G.R.; PREIRA, L.M.C.S.; LEONEL, M.F.V.;
NUNAN, E.A. Development of agar diffusion method for
dosage of gramididin. Braz. J. Pharm. Sci., v. 47, p. 555-
572, 2011. USUI, K.; HISHINUMA, T.; YAMAMGUCHI, H.; TACHIIRI,
N.; GOTO, J. Determination of chlorhexidine (CHD)
and nonylphenolethoxylates (NPEOn) using LC-ESI-MS
method and application to hemolyzed blood. J. Chromatogr. B, v.831, p.105-109, 2006. SOUZA, M.J.E.; ROLIM, C.M.B.; MELO, J.; SOUZA-FILHO,
P.S.; BERGOLD, A.M. Development of a microbiological
assay to determine the potency of ceftiofur sodium powder. J. AOAC Int., v.90, p.1724-1728, 2007. VAUGHER, L.C.; BREIER, A.R.; SCHAPOVAL, E.E.S. Microbiological assay for the determination of telithromycin
in tablets. J. AOAC Int., v.89, p.1398-1402, 2006. VRIES, J.; RUBEN, J.; ARENDS, J. Determination of
chlorhexidine in saliva and in aqueous solutions. Caries
Res., v.25, p.410-414, 1991. Received for publication on: 08th May 2012
Accepted for publication on: 12th December 2012 VRIES, J.; RUBEN, J.; ARENDS, J. Determination of
chlorhexidine in saliva and in aqueous solutions. Caries
Res., v.25, p.410-414, 1991. UNITED STATES PHARMACOPOEIA. 31ed. Rockville: The
United States Pharmacopeia Convention, 2008. p.1731-
1733. Received for publication on: 08th May 2012
Accepted for publication on: 12th December 2012 THOMAS, L.; MAILLARD, J.Y.; LAMBERT, R.J.W.;
RUSSELL, A.D. Development of resistance to chlorhexidine
diacetate in Pseudomonas aeruginosa and the effect of a
residual concentration. J. Hosp. Infect., v.46, p.297-303,
2000.
|
https://openalex.org/W3190928269
|
https://journal.unnes.ac.id/nju/index.php/beaj/article/download/30142/11595
|
Indonesian
| null |
Profitabilitas dalam Memediasi Pengaruh Kepemilikan Institusional, Komite Audit, dan Ukuran Perusahaan terhadap Pengungkapan Sustainability Report
|
Business and Economic Analysis Journal
| 2,021
|
cc-by
| 5,476
|
BEAJ Vol 1 (1) (April) 2021 : 14-26 BEAJ Vol 1 (1) (April) 2021 : 14-26 Permalink/DOI: http://dx.doi.org/10.15294/beaj.v1i1.30142 Permalink/DOI: http://dx.doi.org/10.15294/beaj.v1i1.30142 Permalink/DOI: http://dx.doi.org/10.15294/beaj.v1i1.30142 Corresponding author :
E-mail: muh_khafid@mail.unnes.ac.id Kata Kunci : Kepemilikan Institusional; Komite Audit; Profitabilitas; Sustainability Report; Ukuran
Perusahaan Devi Istiani Roviqoh, Muhammad Khafid Devi Istiani Roviqoh, Muhammad Khafid Jurusan Akuntansi, Fakultas Ekonomi, Universitas Negeri Semarang, Gedung L2, Kampus Sekaran,
Gunungpati, Semarang, Jawa Tengah, 50229, Indonesia Abstrak Tujuan penelitian dilakukan adalah menguji pengaruh kepemilikan institusional, komite audit, dan
ukuran perusahaan terhadap pengungkapan sustainability report dengan variabel intervening
profitabilitas. Populasi dalam penelitian ini adalah perusahaan yang termasuk kategori indeks LQ45
periode 2013-2017 sebanyak 70 perusahaan. Teknik sampel yang digunakan pada penelitian ini yaitu
purposive sampling. Sampel yang diperoleh sebanyak 14 perusahaan sehingga diperoleh total 70 unit
analisis. Teknik analisis yang digunakan adalah analisis statistik deskriptif dan path analysis dengan
software IBM SPSS Statistics 23. Penelitian ini menunjukkan adanya pengaruh positif profitabilitas
terhadap pengungkapan sustainability report, komite audit berpengaruh positif terhadap profitabilitas,
dan ukuran perusahaan berpengaruh negatif terhadap profitabilitas. Sedangkan kepemilikan
institusional, ukuran perusahaan, dan komite audit tidak mempengaruhi pengungkapan sustainability
report, serta kepemilikan institusional tidak berpengaruh terhadap profitabilitas. Hasil Sobel Test
menunjukkan profitabilitas mampu memediasi pengaruh ukuran perusahaan dan komite audit terhadap
pengungkapan sustainability report, namun tidak mampu memediasi pengaruh kepemilikan institusional
terhadap pengungkapan sustainability report. Simpulan penelitian yaitu pengungkapan sustainability
report akan lebih tinggi ketika ukuran perusahaan dan komite audit yang dimiliki, ditunjang dengan
tingginya tingkat profitabilitas perusahaan. Hal tersebut menunjukkan bahwa manajemen hendaknya
menjaga profitabilitas perusahaan karena terbukti berpengaruh langsung dan mampu memediasi
terhadap pengungkapan sustainability report. Kata Kunci : Kepemilikan Institusional; Komite Audit; Profitabilitas; Sustainability Report; Ukuran
Perusahaan Corresponding author :
E-mail: muh_khafid@mail.unnes.ac.id 15
BEAJ Vol 1 (1) (2021): 14-26 15 Kurangnya
antusias
dan
pemahaman
perusahaan
pada
pentingnya
peran
dari
lingkungan
dan
sosial
menjadi
penyebab
rendahnya
angka
pengungkapan
tersebut. Alasan lainnya yaitu peraturan yang mengatur
tentang sustainability report di Indonesia baru
hadir
di
tahun
2017
melalui
POJK
51/POJK.03/2017. PENDAHULUAN Haladu & Salim (2016), Afsari et
al., (2017), dan Nurleni et al., (2017) menyatakan
ada
dampak
positif
dari
kepemilikan
institusional
pada
pengungkapan
laporan
keberlanjutan. H1:
Kepemilikan
institusional
berpengaruh
positif terhadap pengungkapan sustainability
report proporsi
inverstor
institusional
mampu
mempengaruhi
pengungkapan
laporan
keberlanjutan. Haladu & Salim (2016), Afsari et
al., (2017), dan Nurleni et al., (2017) menyatakan
ada
dampak
positif
dari
kepemilikan
institusional
pada
pengungkapan
laporan
keberlanjutan. profitabilitas
mencerminkan
kehandalan
suatu organisasi dalam menciptakan profit
berdasarkan
aktivitas
normal
bisnisnya. Seiring meningkatnya profit maka suatu
organisasi memiliki sumber modal untuk
menjalankan
kegiatan
sosial
kepada
masyarakat maupun lingkungan sebagai
tanggung
jawab
perusahaan. Sehingga,
kegiatan tersebut harus diinformasikan dan
diungkapkan
kepada
para
pemangku
kepentingan
perusahaan
melalui
sustainability report. Dalam teori agensi, struktur kepemilikan
mayoritas dianggap mampu mempengaruhi
mekanisme
corporate
governance. Pihak
institusi sebagai investor berperan signifikan
dalam memonitor manajemen seiring besarnya
investasi yang diberikan kepada perusahaan. Monitoring
ditujukan
untuk
memastikan
kesejahteraan para stakeholder. Jika performa
manajer dirasa kurang maksimal, akibatnya
investor akan berhenti menanamkan modalnya
pada perusahaan. Meminimalisir hal tersebut,
manajer akan bersikap lebih akuntabel dalam
menjalankan aktivitas perusahaan. Akibatnya,
kinerja keuangan perusahaan akan meningkat
melalui
tingginya
profitabilitas. Sehingga
besarnya proporsi kepemilikan institusional,
akan
membuat
tingkat
profitabilitas
juga
mengalami peningkatan. Nurkhin et al., (2017)
dan Kumai et al., (2014) menyatakan ada
dampak positif dari kepemilikan institusional
pada profitabilitas. Penelitian
ini
berlandaskan
pada
agency
theory,
legitimacy
theory,
dan
stakeholders theory. Agency theory secara
implisit menyatakan bahwa manajer perlu
mengungkapkan sustainability report untuk
menjamin
akuntabilitas
manajemen
terhadap stakeholder, sehingga harapannya
dapat memperkecil konflik
kepentingan
antara agen dan principal. Legitimacy theory
menjelaskan bahwa suatu organisasi harus
mendapatkan dukungan dari masyarakat
dengan cara beraktivitas sesuai dengan
aturan yang ditetapkan. Sedangkan teori
stakeholder menunjukkan bahwa hubungan
perusahaan dengan stakeholdernya juga
harus dijaga, salah satu caranya dengan
mengungkapkan
informasi
secara
transparan. Kepemilikan institusional merupakan
banyaknya pihak lembaga eksternal yang
memiliki saham di perusahaan. Besarnya
kepemilikan
institusional
mampu
mempengaruhi
pengendalian
investor
kepada manajemen agar mengungkapkan
sustainability report. Hal ini penting sebagai
bentuk
transparansi
perusahaan
atas
informasi yang di dalamnya berisi tindakan
yang telah dilakukan untuk menjaga kondisi
perusahaan dari segi ekonomi, lingkungan,
dan
sosial. Atas
dasar
teori
agensi,
pengungkapan sustainability report bisa
memperkecil ketidakseimbangan informasi
antara agen dan pemilik. Sehingga, besarnya H2: Kepemilikan institusional berpengaruh
positif terhadap profitabilitas Tingkat
kepercayaan
investor
salah
satunya ditentukan oleh ukuran perusahaan. Perusahaan besar memiliki konsekuensi lebih
diperhatikan publik seiring besarnya pengaruh
perusahan
terhadap
masyarakat
dan
lingkungan. PENDAHULUAN Seiring dengan perkembangan zaman,
perlahan konsep maksimalisasi laba bukan
menjadi satu-satunya tujuan perusahaan. Selain berfokus pada laba yang tinggi,
perusahaan juga harus memenuhi tanggung
jawab sosial dan lingkungan akibat aktivitas
operasi yang dilakukannya (Racelia et al.,
2017). Sejalan
dengan
perkembangan
tersebut,
perusahaan
diharapkan
dapat
memberikan
kontribusinya
pada
sektor
ekonomi, sosial, dan lingkungan. Berdasarkan
penelusuran
riset-riset
sebelumnya
mengenai
pengungkapan
sustainability report, masih ditemukan adanya
research gap. Penelitian Haladu & Salim (2016),
Nurleni et al., (2017), dan Afsari et al., (2017)
memperoleh bukti kepemilikan institusional
mempengaruhi
pengungkapan
laporan
keberlanjutan. Sementara Pratama & Yulianto
(2015) serta Setyawan et al., (2018) memperoleh
bukti
kepemilikan
institusional
tidak
mempengaruhi
pengungkapan
laporan
keberlanjutan. Bhatia
&
Tuli
(2017)
dan
Karaman et al., (2018) membuktikan ukuran
perusahaan
mempengaruhi
pengungkapan
laporan keberlanjutan. Sedangkan Nasir et al.,
(2014)
dan
Aliniar
&
Wahyuni
(2017)
memperoleh bukti ukuran perusahaan tidak
mempengaruhi
pengungkapan
laporan
keberlanjutan. Aniktia & Khafid (2015) dan
Racelia et al., (2017) dalam penelitiannya
berhasil memperoleh bukti komite audit bisa
mempengaruhi
laporan
keberlanjutan. Sedangkan Pratama & Yulianto (2015) dan
Aliniar & Wahyuni (2017) membuktikan komite
audit
tidak
mempengaruhi
pengungkapan
laporan keberlanjutan. Namun
kenyataannya,
kerusakan
lingkungan
akibat
aktivitas
bisnis
perusahaan masih sering terjadi. Hal tersebut
menandakan bahwa perusahaan-perusahaan
di Indonesia kurang mempedulikan dampak
negatif dari kegiatan operasional perusahaan
tersebut
yang
berimbas
pada
ruginya
masyarakat
di
sekitar.Hal
ini
tentunya
akanmembuat
timbulnya
citra
negatif
perusahaan di mata masyarakat dan para
pemangku kepentingan. Sehingga diperlukan
suatu informasi yang transparan mengenai
cara
perusahaan
mengelola
aktivitas
bisnisnya. Informasi
mengenai
dampak
aktivitas
bisnis
perusahaan
dapat
diungkapkan melalui laporan keberlanjutan
(sustainability report). Saat
ini
di
Indonesia,
jumlah
pengungkapan
sustainability
reportnya
masih terbilang rendah. Terbukti menurut
OJK (2017) hingga Maret 2017, jumlah
perusahaan
terdaftar
BEI
yang
telah
menerbitkan
sustainability
report
baru
sekitar 9%. Setyawan et al., (2018) dalam
penelitiannya juga menunjukkan rendahnya
tingkat pengungkapan sustainability report,
dapat dilihat dari rata-ratanya pada objek
penelitian yaitu 37,31%. Selain itu, penelitian
Kusuma
&
Priantinah
(2018)
juga
menemukan
rendahnya
pengungkapan
sustainability report yaitu sebesar 33,20%. Tujuan dilakukannya riset adalah menguji
pengaruh kepemilikan institusional, komite
audit,
serta
ukuran
perusahaan
terhadap
pengungkapan laporan keberlanjutan dengan
variabel intervening profitabilitas. Orisinalitas
dalam
penelitian
yaitu
menghadirkan
profitabilitas sebagai variabel intervening untuk
memediasi pengaruh kepemilikan institusional,
komite audit, serta ukuran perusahaan terhadap
pengungkapan laporan keberlanjutan. Rasio Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan 16 proporsi
inverstor
institusional
mampu
mempengaruhi
pengungkapan
laporan
keberlanjutan. PENDAHULUAN Berdasarkan
teori
legitimasi,
perusahaan yang ingin berkembang secara
berkelanjutan haruslah memperoleh izin dan
keberpihakan dari masyarakat, sebagai pemilik
sumber daya yang akan digunakan perusahaan. Sehingga, perusahaan dengan ukuran makin
besar, berdampak pada makin banyaknya item 17
BEAJ Vol 1 (1) (2021): 14-26 17 akan melakukan tugasnya dalam mengawasi
kegiatan
pelaporan
yang
dilakukan
oleh
manajemen. Berdasarkan teori stakeholders,
pihak
manajemen
perusahaan
dapat
dimaksimalkan
fungsinya
melalui
adanya
dorongan dari komite audit agar melakukan
transparansi
informasi
sesuai
kebutuhan
stakeholder. Salah
satu
informasi
yang
dibutuhkan stakeholder adalah pengungkapan
laporan keberlanjutan. Aniktia & Khafid (2015),
Racelia et al., (2017), serta Buallay & Aldhaen
(2018) membuktikan adanya pengaruh antara
komite audit terhadap pengungkapan laporan
keberlanjutan. yang harus diungkap pada sustainability
report. Hal
tersebut
penting
untuk
meyakinkan masyarakat bahwa kegiatan dan
kinerja perusahaan sudah sesuai dan tidak
melanggar aturan yang berlaku. Bhatia & Tuli
(2017), Khafid et al., (2018), Karaman et al.,
(2018) menyatakan adanya dampak positif
dari
kepemilikan
institusional
pada
pengungkapan laporan keberlanjutan. H3: Ukuran perusahaan berpengaruh positif
terhadap pengungkapan sustainability report Besar kecilnya aktivitas operasional di
dalam perusahaan dapat tercermin melalui
ukuran
perusahaan. Perusahaan
besar
memiliki kemampuan berproduksi pada
skala ekonomis, akibatnya produk yang
dihasilkan memiliki harga satuan yang
rendah. Harga satuan yang rendah tersebut
membuat perusahaan memiliki daya saing
yang tinggi di pasaran. Akibatnya terjadi
peningkatan penjualan perusahaan yang
pada
akhirnya
meningkatkan
laba/keuntungan perusahaan. Keunggulan
lain
dari
perusahaan-perusahaan
besar
adalah dimilikinya akses pasar yang lebih
baik dibanding perusahaan dengan skala
kecil (Purnomosidi et al., 2014). Dengan
demikian, perusahaan dengan skala besar
lebih berkesempatan untuk meningkatkan
profitabilitasnya. Atas
dasar
teori
stakeholder,
peningkatan
profitabilitas
merupakan
tanggung
jawab
perusahaan
untuk
memenuhi
ekspektasi
seluruh
pemangku kepentingan. Penelitian John &
Adebayo, (2013), Wikardi & Wiyani (2017),
serta Giriyani & Diyani (2018) menemukan
bukti adanya dampak positif dari ukuran
perusahaan pada profitabilitas. H5: Komite audit berpengaruh positif terhadap
pengungkapan sustainability report Komite
audit
dalam
membahas
permasalahan yang ada di internal perusahaan
perlu mengadakan pertemuan secara berkala. Atas dasar teori agensi, pertemuan secara
berkala
tersebut
mampu
membantu
memperkecil ketidak seimbangan informasi
yang dapat menimbulkan masalah, seperti
kemungkinan adanya manipulasi data-data
keuangan dan prosedur akuntansi. Akibatnya,
kinerja keuangan perusahaan akan meningkat. Kinerja
keuangan
yang
meningkat
dapat
ditandai dengan tingginya rasio profitabilitas
yang ada. Sehingga semakin sering rapat komite
audit dilakukan, mampu menciptakan tren
positif
dengan
tingginya
profitabilitas
perusahaan. Abernathy et al., (2015), Racelia et
al., (2017), serta Buallay & Aldhaen (2018)
menunjukkan
hasil
komite
audit
mempengaruhi profitabilitas perusahaan. PENDAHULUAN H6: Komite audit berpengaruh positif terhadap
profitabilitas Profitabilitas menjadi hal penting dalam
mengungkapkan
sustainability
report. Alasannya,
perusahaan
yang
memiliki
kemampuan lebih dalam memperoleh profit
menandakan sehatnya kondisi keuangan yang
dimiliki
perusahaan. Sehingga
perusahaan
mampu menghasilkan sumber daya keuangan H4: Ukuran perusahaan berpengaruh positif
terhadap profitabilitas Komunikasi
dan
koordinasi
antar
anggota komite audit merupakan suatu
keharusan
dalam
menjalankan
tanggungjawab pekerjaannya. Komite audit Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan 18 H8: Kepemilikan institusional berpengaruh
terhadap pengungkapan sustainability report
melalui profitabilitas yang besar untuk membuat komitmen
pengungkapan sustainability report dengan
kualitas
tinggi
(Kuzey
&
Uyar,
2017). Menurut teori stakeholder, dalam rangka
mewujudkan kondisi senantiasa harmonis
antara perusahaan dan stakeholdernya, maka
perusahaan
akan
berupaya
memenuhi
kebutuhan stakeholder termasuk kebutuhan
informasi
yang
diwujudkan
melalui
pengungkapan laporan keberlanjutan. Nazari
et al. (2015), dan Martínez-Ferrero et al.,
(2015) membuktikan adanya pengaruh
antara profitabilitas terhadap pengungkapan
laporan keberlanjutan. Perusahaan yang besar akan semakin
disorot para pemangku kepentingan untuk
melakukan
transparansi
insformasi
dalam
mewujudkan legitimasi perusahaan. Atas dasar
teori legitimasi, perusahaan dengan skala besar
mengindikasikan makin luasnya item yang
mampu perusahaan tersebut ungkapkan pada
laporan keberlanjutan yang diterbitkan sebagai
laporan
pertanggungjawaban. Khafid
&
Mulyaningsih (2015), Bhatia & Tuli (2017), serta
Karaman et al., (2018) membuktikan ukuran
perusahaan
mampu
mempengaruhi
pengungkapan pada laporan keberlanjutan. Besarnya perusahaan biasanya diiringi dengan
tingginya perolehan laba. Perusahaan yang
mempunyai
kemampuan
tinggi
dalam
memperoleh laba akan memiliki kemampuan
lebih untuk memenuhi semua kepentingan para
pemangku
kepentingan,
termasuk
pengungkapan informasi melalui sustainability
report. Nazari et al. (2015) serta Martínez-
Ferrero et al., (2015) dalam penelitiannya
berhasil membuktikan profitabilitas mampu
mempengaruhi pengungkapan pada laporan
keberlanjutan. H7:
Profitabilitas
berpengaruh
positif
terhadap pengungkapan sustainability report terhadap pengungkapan sustainability report
Dalam
teori
legitimasi,
organisasi
harus
mendapatkan
dukungan
dari
masyarakat dengan cara beraktivitas sesuai
dengan aturan yang ditetapkan. Investor
institusional sebagai pemilik saham dalam
perusahaan tentunya memerlukan informasi
untuk
memperoleh
keyakinan
bahwa
perusahaan telah memenuhi aspek legitimasi
tersebut
melalui
pengungkapan
sustainability report. Akibatnya, investor
institusi
cenderung
lebih
mendukung
manajemen
untuk
menerbitkan
sustainability report. Haladu & Salim (2016),
Afsari et al., (2017), serta Nurleni et al., (2017)
menunjukkan dampak positif kepemilikan
institusional pada pengungkapan laporan
keberlanjutan. Besarnya proporsi investor
institusional dapat mendorong organisasi
untuk mengoptimalkan kinerja keuangannya
yang
tercermin
melalui
tingginya
profitabilitas. Dengan tingginya profitabilitas
bisa disimpulkan bahwa besarnya biaya-
biaya
dalam
rangka
mengungkapkan
sustainability report mampu perusahaan
tanggung. Nazari et al. (2015) dan Martínez-
Ferrero et al., (2015) menunjukkan peran
positif
antara
profitabilitas
dan
pengungkapan laporan keberlanjutan. PENDAHULUAN H9: Ukuran perusahaan berpengaruh terhadap
pengungkapan sustainability report melalui
profitabilitas Komite audit memiliki tanggung jawab
tugas pada dewan komisaris untuk mengawasi
proses pelaporan yang dilakukan manajemen. Atas dasar teori stakeholders disebutkan bahwa
manajer
perusahaan
dapat
dimaksimalkan
fungsinya melalui adanya dorongan dari komite
audit supaya mengungkapkan semua informasi
yang dibutuhkan secara transparan. Informasi
yang dibutuhkan stakeholder salah satunya
diwujudkan dalam pengungkapan sustainability
report
untuk
mendapat
legitimasi
dari
masyarakat. Racelia et al., (2017) dan Buallay &
Aldhaen (2018) berhasil menemukan bukti
komite
audit
dapat
mempengaruhi 19
BEAJ Vol 1 (1) (2021): 14-26 19 pengungkapan
laporan
keberlanjutan. Seringnya
pertemuan
komite
audit
diselenggarakan, berdampak pada semakin
baiknya pengawasan terhadap manajemen
perusahaan. Pengawasan yang baik mampu
membuat
meningkatnya
performa
perusahaan yang tercermin melalui tingginya
profitabilitas. Dengan
tingginya
profitabilitas,
bisa
disimpulkan
bahwa
besarnya
biaya-biaya
dalam
rangka
mengungkapkan
sustainability
report
mampu perusahaan tanggung. Nazari et al. (2015) serta Martínez-Ferrero et al., (2015)
menunjukkan
peran
positif
antara
profitabilitas dan pengungkapan laporan
keberlanjutan. H10:
Komite
audit
berpengaruh
terhadap
pengungkapan sustainability report melalui
profitabilitas METODE PENELITIAN (Khafid, 2012)
(Pratama & Yulianto, 2015)
Ukuran
Perusahaan
(SIZE)
Besar kecilnya perusahaan yang tercermin dari
jumlah aset perusahaan pada neraca akhir periode
(Mahardika et al., 2014)
Ln(Total Asset)
(Adhipradana & Daljono, 2014) Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan 20 20
Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan
Variabel
Definisi
Pengukuran
Komite Audit
(KA)
Komite bentukan dewan komisaris dalam rangka
membantu melaksanakan tugas dan fungsinya
(Khafid, 2012)
∑ rapat komite audit dalam satu
periode
(Pratama & Yulianto, 2015)
Profitabilitas
(ROA)
kehandalan suatu organisasi dalam menciptakan
profit berdasarkan aktivitas normal bisnisnya. (Agustina & Rusmana, 2016)
(Aniktia & Khafid, 2015) Variabel
Definisi
Pengukuran
Komite Audit
(KA)
Komite bentukan dewan komisaris dalam rangka
membantu melaksanakan tugas dan fungsinya
(Khafid, 2012)
∑ rapat komite audit dalam satu
periode
(Pratama & Yulianto, 2015)
Profitabilitas
(ROA)
kehandalan suatu organisasi dalam menciptakan
profit berdasarkan aktivitas normal bisnisnya. (Agustina & Rusmana, 2016)
(Aniktia & Khafid, 2015) Definisi Sumber: Berbagai referensi diolah, 2019 Sumber: Berbagai referensi diolah, 2019 Teknik
pengambilan
data
menggunakan
teknik
dokumenter. Sedangkan
model
penelitian
dianalisis
dengan analisis jalur menggunakan aplikasi
SPSS
versi
23. Analisis
jalur
dengan
meregresikan KI, SIZE, dan KA terhadap
ROA sehingga diperoleh persamaan 1:
ROA
= α + β1KI + β2SIZE + β3KA + e1 (1) Teknik
pengambilan
data
menggunakan
teknik
dokumenter. Sedangkan
model
penelitian
dianalisis
dengan analisis jalur menggunakan aplikasi
SPSS
versi
23. Analisis
jalur
dengan
meregresikan KI, SIZE, dan KA terhadap
ROA sehingga diperoleh persamaan 1:
ROA
= α + β1KI + β2SIZE + β3KA + e1 (1) menunjukkan nilai yang cukup tinggi yaitu
masing-masing 66.5% dan 18 kali. Selanjutnya,
rata-rata ukuran perusahaan menunjukkan nilai
sebesar 32.04. Sedangkan untuk profitabilitas
menunjukkan rata-rata sebesar 6.4%. Hasil uji normalitas Kolmogorov-Smirnov
menunjukkan nilai signifikansi 0.081 di atas
0.050, maka dapat diambil kesimpulan data
residual penelitian terdistribusi secara normal. Uji multikolinearitas menunjukkan VIF<10 dan
tolerance>0.01 maka artinya seluruh variabel
yang
digunakan
terbebas
dari
gejala
multikolinearitas. Selanjutnya dilakukan uji
white dengan c2 hitung<c2 tabel (17.29<89.39). Maka disimpulkan tidak terjadi adanya gejala
heteroskedastisitas. Durbin-Watson
Test
digunakan
untuk
menguji
ada
tidaknya
autokorelasi,
dimana
diperoleh
nilai
DW
sebesar 1.742 berada diantara 1.735 dan 2.265,
sehingga
disimpulkan
model
penelitian
terbebas dari autokorelasi. Pengaruh KI, SIZE,
KA dan ROA terhadap SR ditunjukkan pada R2
yaitu 0.104 atau 10,4%, sedangkan 89,6% karena
adanya
variabel
independen
lain. METODE PENELITIAN Secara
ringkas, pengujian hipotesis hasilnya dapat
dilihat pada Tabel 3 Langkah selanjutnya yaitu meregresikan KI,
SIZE, KA serta ROA terhadap variabel
dependen SR sehingga diperoleh persamaan 2:
SR = α + β1KI + β2SIZE + β3KA + β4ROA + e2
(2) (2) Pengujian pengaruh variabel intervening
menggunakan uji Sobel Daniel Soper dengan
tingkat signifikansi 5%. METODE PENELITIAN Jenis penelitian ini adalah kuantitatif
diambil dari laporan tahunan dan laporan
keberlanjutan yang diakses melalui laman BEI
dan website perusahaan terkait sebagai sumber
data sekunder. 70 perusahaan LQ45 periode
2013 hingga 2017 menjadi populasi dalam riset
ini. Teknik
pemilihan
sampel
dipilih
menggunakan purposive sampling dan didapat
14 perusahaan. Pengamatan dilakukan selama 5
tahun sehingga total unit analisis sebanyak 70. j
gg
y
7
Tabel 1. Kriteria Pemilihan Sampel
No
Kriteria
Tidak Sesuai Kriteria
Jumlah
Populasi
70
1
Perusahaan BEI yang terdaftar secara konsisten dalam indeks
LQ45 dalam kurun waktu 2013-2017
(39)
31
2
Perusahaan LQ45 yang menerbitkan sustainability report
secara terpisah dari annual report dalam kurun waktu 2013-
2017
(17)
14
3
Perusahaan yang memberikan informasi lengkap terkait
variabel penelitian. -
14
Sisa (Sampel Penelitian)
14
Periode pengamatan
5
Total unit analisis
70
Sumber: Data sekunder diolah, 2019
Adapun penjelasan variabel penelitian dapat
dijelaskan pada Tabel 2. Tabel 2. Definisi Operasional Variabel Penelitian
Variabel
Definisi
Pengukuran
Pengungkapan
Sustainability
Report (SR)
Media perwujudan transparansi perusahaan yang
mencakup 3 aspek pelaporan, yakni sosial, ekonomi
dan lingkungan (Khafid & Mulyaningsih, 2015). (Doktoralina et al., 2018)
Kepemilikan
Institusional
(KI)
Banyaknya pihak lembaga eksternal yang memiliki
saham di perusahaan. (Khafid, 2012)
(Pratama & Yulianto, 2015)
Ukuran
Perusahaan
(SIZE)
Besar kecilnya perusahaan yang tercermin dari
jumlah aset perusahaan pada neraca akhir periode
(Mahardika et al., 2014)
Ln(Total Asset)
(Adhipradana & Daljono, 2014) Tabel 1. Kriteria Pemilihan Sampel Tabel 1. Kriteria Pemilihan Sampel
No
Kriteria
Tidak Sesuai Kriteria
Jumlah
Populasi
70
1
Perusahaan BEI yang terdaftar secara konsisten dalam indeks
LQ45 dalam kurun waktu 2013-2017
(39)
31
2
Perusahaan LQ45 yang menerbitkan sustainability report
secara terpisah dari annual report dalam kurun waktu 2013-
2017
(17)
14
3
Perusahaan yang memberikan informasi lengkap terkait
variabel penelitian. -
14
Sisa (Sampel Penelitian)
14
Periode pengamatan
5
Total unit analisis
70
Sumber: Data sekunder diolah, 2019 j
p
Tabel 2. Definisi Operasional Variabel Penelitian
Variabel
Definisi
Pengukuran
Pengungkapan
Sustainability
Report (SR)
Media perwujudan transparansi perusahaan yang
mencakup 3 aspek pelaporan, yakni sosial, ekonomi
dan lingkungan (Khafid & Mulyaningsih, 2015). (Doktoralina et al., 2018)
Kepemilikan
Institusional
(KI)
Banyaknya pihak lembaga eksternal yang memiliki
saham di perusahaan. Tabel 3. Hasil Pengujian Hipotesis
Hipotesis
β
Sig
Hasil
H1
Kepemilikan Institusional berpengaruh positif terhadap
Pengungkapan Sustainability Report
0,156
0,535
Ditolak
H2
Kepemilikan Institusional berpengaruh positif terhadap
Profitabilitas
-0,132
0,058
Ditolak
H3
Ukuran
Perusahaan
berpengaruh
positif
terhadap
Pengungkapan Sustainability Report
0,000
0,993
Ditolak
H4
Ukuran
perusahaan
berpengaruh
positif
terhadap
profitabilitas
-0,021
0,000
Ditolak HASIL DAN PEMBAHASAN Pengujian
statistik
deskriptif
menunjukkan
bahwa
pengungkapan
sustainability
report
selama
tahun
pengamatan menunjukkan rata-rata yang
rendah
yaitu
sebesar
32.5%. Rata-rata
kepemilikan institusional dan komite audit Pengujian
statistik
deskriptif
menunjukkan
bahwa
pengungkapan
sustainability
report
selama
tahun
pengamatan menunjukkan rata-rata yang
rendah
yaitu
sebesar
32.5%. Rata-rata
kepemilikan institusional dan komite audit Tabel 3. Hasil Pengujian Hipotesis
Hipotesis
β
Sig
Hasil
H1
Kepemilikan Institusional berpengaruh positif terhadap
Pengungkapan Sustainability Report
0,156
0,535
Ditolak
H2
Kepemilikan Institusional berpengaruh positif terhadap
Profitabilitas
-0,132
0,058
Ditolak
H3
Ukuran
Perusahaan
berpengaruh
positif
terhadap
Pengungkapan Sustainability Report
0,000
0,993
Ditolak
H4
Ukuran
perusahaan
berpengaruh
positif
terhadap
profitabilitas
-0,021
0,000
Ditolak Tabel 3. Hasil Pengujian Hipotesis
Hipotesis
β
Sig
Hasil
H1
Kepemilikan Institusional berpengaruh positif terhadap
Pengungkapan Sustainability Report
0,156
0,535
Ditolak
H2
Kepemilikan Institusional berpengaruh positif terhadap
Profitabilitas
-0,132
0,058
Ditolak
H3
Ukuran
Perusahaan
berpengaruh
positif
terhadap
Pengungkapan Sustainability Report
0,000
0,993
Ditolak
H4
Ukuran
perusahaan
berpengaruh
positif
terhadap
profitabilitas
-0,021
0,000
Ditolak 21
BEAJ Vol 1 (1) (2021): 14-26 21 Hipotesis
β
Sig
Hasil
H5
Komite audit berpengaruh positif terhadap pengungkapan
sustainability report
0,000
0,913
Ditolak
H6
Komite audit berpengaruh positif terhadap profitabilitas
0,060
0,032
Diterima
H7
Profitabilitas berpengaruh positif terhadap pengungkapan
sustainability report
1,275
0,005
Diterima
H8
Kepemilikan institusional berpengaruh positif terhadap
pengungkapan sustainability report melalui profitabilitas
-0,168
0,053
Ditolak
H9
Ukuran
perusahaan
berpengaruh
positif
terhadap
pengungkapan sustainability report melalui profitabilitas
-0,027
0,005
Ditolak
H10
Komite audit berpengaruh positif terhadap pengungkapan
sustainability report melalui profitabilitas
0,001
0,029
Diterima
S
b
D
k
d
di l h Hipotesis Hipotesis saham
di
perusahaan. Akibatnya,
tidak
diperlukan monitoring pihak institusi sebagai
pemilik saham mayoritas yang dapat mendorong
peningkatan kinerja manajemen, dalam rangka
meningkatkan
kemampuan
perusahaan
memperoleh
profit
yang
diukur
dengan
profitabilitas. Pemikiran tersebut mendukung
konsep teori stewardship, dimana manajer
berupaya memaksimalkan kinerja perusahaan
demi mencapai tujuan organisasi sesuai dengan
kepentingan investor. Riset ini mendukung
temuan Wiranata & Nugrahanti (2013) dan Hu et
al., (2018). Pengaruh Kepemilikan Institusional terhadap
Pengungkapan Sustainability Report Kepemilikan
institusional
tidak
mempengaruhi pengungkapan sustainability
report. Kondisi ini dapat diamati pada salah
satu perusahaan sampel selama lima tahun
pengamatan
menunjukkan
tingkat
kepemilikan institusional yang tinggi yaitu
sekitar
84-90%. Namun
kenyataannya,
tingginya investor institusi tersebut belum
mampu memberi dampak peningkatan pada
pengungkapan
sustainability
report,
dibuktikan
dengan
rendahnya
rata-rata
pengungkapan sustainability report sebesar
32.5% pada objek penelitian. HASIL DAN PEMBAHASAN Hal ini diduga
karena pihak institusi di Indonesia lebih
berfokus pada pencapaian jangka pendek dan
kurang
sadar
akan
pentingnya
keberlangsungan masa depan perusahaan
yang hanya bisa tercapai apabila dilakukan
pengungkapan
laporan
keberlanjutan. Penelitian
ini
mendukung
penelitian
Setyawan et al., (2018) serta Pratama &
Yulianto (2015). Pengaruh
Ukuran
Perusahaan
terhadap
Pengungkapan Sustainability Report Pengaruh
Ukuran
Perusahaan
terhadap
Pengungkapan Sustainability Report Riset ini tidak berhasil menemukan adanya
pengaruh dari ukuran perusahaan terhadap
pengungkapan laporan keberlanjutan. Kondisi
tersebut dapat terjadi karena ukuran perusahaan
sampel yang didasarkan pada pengklasifikasian
distribusi frekuensi menunjukkan nilai yang
besar
yaitu
32.05. Namun
kenyataannya,
besarnya ukuran perusahaan tersebut tidak
diimbangi dengan peningkatan item dalam
sustainability
report,
dibuktikan
dengan
rendahnya nilai pengungkapan sustainability
report yang didasarkan pada pengklasifikasian
distribusi frekuensi sebesar 32.5%. Hasil riset
tidak sejalan dengan teori legitimasi, yang
menyebutkan
perusahaan
besar
seharusnya
mengungkapkan
sustainability report
secara
lebih luas. Hal ini karena adanya pemikiran Pengaruh Kepemilikan Institusional terhadap
Profitabilitas Pengaruh Kepemilikan Institusional terhadap
Profitabilitas Kepemilikan
institusional
tidak
mempengaruhi profitabilitas. Hal ini karena
manajemen perusahaan diyakini akan terus
berupaya
memenuhi
kehendak
para
pemegang
saham,
tanpa
memperhatikan
berapa jumlah institusi yang berinvestasi Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan 22 bahwa
perusahaan
besar
dapat
mempertahankan eksistensi usahanya dan
telah diakui oleh publik, selain itu perusahaan
juga terlalu fokus dalam memperoleh profit
sehingga
mengesampingkan
tanggung
jawabnya
mengungkapkan
laporan
keberlanjutan. Riset ini mendukung temuan
Nasir et al., (2014) dan Aliniar & Wahyuni
(2017). diperhatikan pula efektivitas rapat yang dilihat
dari seberapa banyak masalah perusahaan yang
dapat diselesaikan dan kebijakan-kebijakan
yang diambil dalam rapat sehingga manajemen
dapat mengambil keputusan untuk melakukan
pengungkapan laporan keberlanjutan. Riset ini
sejalan dengan Aliniar & Wahyuni (2017) serta
Pratama & Yulianto (2015). bahwa perusahaan Pengaruh Komite Audit terhadap Profitabilitas g
p
f
Hasil riset membuktikan komite audit
mempengaruhi profitabilitas positif signifikan. Dalam melaksanakan perannya, komite audit
telah
berhasil
memonitor
dengan
baik
pengendalian
internal
perusahaan
untuk
membantu dewan komisaris. Menurut teori
agensi, pada hakikatnya konflik kepentingan
dapat
menimbulkan
masalah,
seperti
kemungkinan
adanya
manipulasi
data-data
keuangan dan prosedur akuntansi. Namun,
pertemuan secara berkala komite audit mampu
membantu
memperkecil
peluang
terjadinya
masalah tersebut, Akibatnya kinerja keuangan
perusahaan akan meningkat (Mamun, 2014). Hasil riset ini sesuai dengan riset terdahulu
Racelia et al., (2017) dan Buallay & Aldhaen
(2018). Pengaruh
Ukuran
Perusahaan
terhadap
Profitabilitas Ukuran
perusahaan
mempengaruhi
negatif signifikan profitabilitas. Hal ini sesuai
dengan teori critical resources (Grossman &
Hart, 1986) yang menyatakan bahwa ukuran
perusahaan pada jumlah tertentu akhirnya
akan menurunkan laba perusahaan. Alasan
yang mendasarinya yakni semakin besar
perusahaan dengan kepemilikan aset yang
banyak
pada
akhirnya
kurang
bisa
memanfaatkan aset tersebut secara optimal. Karena
seiring
meningkatnya
ukuran
perusahaan juga berpengaruh pada tingginya
pembiayaan
operasional
yang
harus
dicurahkan
untuk
membiayai
karyawan,
perawatan aset, dan lain-lain. Dimana nilai
tersebut dapat melebihi profit yang didapat
perusahaan
dari
memaksimalkan
ukuran
perusahaan tersebut. Riset ini sejalan dengan
Anindito (2015) dan Marwanti & Yulianti
(2015). Pengaruh Profitabilitas terhadap Pengungkapan
Sustainability Report Profitabilitas mempengaruhi secara positif
signifikan pengungkapan laporan keberlanjutan. Beradasrkan teori stakeholders dinyatakan bahwa
hubungan perusahaan dengan stakeholdernya
juga harus dijaga, salah satu caranya dengan
melakukan
pertanggungjawaban
sosial
dan
lingkungan beserta pengungkapannya. Tinggi
profitabilitas
membuat
perusahaan
dapat
melakukan
hal
tersebut. Dengan
diungkapkannya
sustainability
report,
maka
image dan reputasi perusahaan akan lebih baik di
mata masyarakat, serta memiliki risiko yang
lebih minim atas peristiwa sosial negatif yang
bisa menciderai reputasi perusahaan. Hasil riset
ini sesuai dengan riset terdahulu Pratama &
Yulianto (2015) serta Agustina & Rusmana (2016). Pengaruh Komite audit terhadap Pengungkapan
Sustainability Report melalui Profitabilitas Profitabilitas mampu memediasi positif
signifikan komite audit pada pengungkapan
laporan keberlanjutan melalui profitabilitas. Berdasarkan teori agensi, konflik keagenan akan
lebih rentan terjadi pada perusahaan yang
membedakan
antara
fungsi
pemilik
dan
manajer,
sehingga
perusahaan
perlu
menerapkan tata kelola yang baik. Salah
satunya melalui penerapan rapat rutin komite
audit. Keefektifan rapat dapat menghasilkan
keputusan-keputusan
terbaik yang mampu
membawa peningkatan
kinerja perusahaan
melalui tingginya profitabilitas. Profitabilitas
yang tinggi menandakan semakin baiknya
kinerja keuangan perusahaan, sehingga mampu
mendorong pengungkapan sustainability report. Dimana laporan tersebut akan memberikan
pemahaman untuk para pemangku kepentingan
tentang dampak keberlanjutan bagi kegiatan
bisnis
perusahaan. Sehingga,
profitabilitas
berhasil
menunjukkan
perannya
sebagai
pemediasi. Pengaruh
Ukuran
Perusahaan
terhadap
Pengungkapan Sustainability Report melalui
Profitabilitas Profitabilitas
mampu
memediasi
pengaruh negatif ukuran perusahaan terhadap
pengungkapan sustainability report. Besarnya
perusahaan akan berdampak pada semakin
besarnya
biaya
yang
harus
perusahaan
keluarkan
untuk
menjalankan
aktivitas
bisnisnya. Sehingga, menjadikan perusahaan
kurang
menghasilkan
profit
akibat
dari
tingginya biaya yang ditanggung perusahaan. Perusahaan
dengan
profitabilitas
rendah
cenderung
mengungkapkan
informasi
seperlunya saja. Hal ini karena pengungkapan
informasi membutuhkan biaya yang cukup
besar sementara beban yang ditanggung
perusahaan
sudah
terlalu
banyak. Data Pengaruh
Komite
Audit
terhadap
Pengungkapan Sustainability Report Pengaruh
Komite
Audit
terhadap
Pengungkapan Sustainability Report Hasil riset membuktikan komite audit
tidak mempengaruhi pengungkapan laporan
keberlanjutan. Berdasarkan
statistik
deskriptif,
komite
audit
mealksanakan
pertemuan kurun waktu satu periode rata-
ratanya mencapai 18 kali, dimana sebesar
56% data penelitian berada di bawah nilai
rata-rata. Artinya
perusahaan
sampel
cenderung jarang melakukan rapat komite
audit. Hal tersebut diduga menjadi penyebab
ditolaknya
hipotesis. Selain
itu,
perlu 23
BEAJ Vol 1 (1) (2021): 14-26 23 Pengaruh Kepemilikan Institusional terhadap
Pengungkapan Sustainability Report melalui
Profitabilitas penelitian menunjukkan profitabilitas padasalah
satu
sampel
mempunyai
penurunan
nilai
profitabilitas tiap tahunnya. Sedangkan untuk
total
aset
perusahaan
justru
mengalami
peningkatan tiap tahunnya. Sementara item yang
diungkapkan dalam sustainability report terus
mengalami
penurunan
sebanding
dengan
menurunnya profitabilitas. Dengan demikian,
penelitian ini memberikan bukti bahwa secara
positif ukuran perusahaan memberikan pengaruh
terhadap pengungkapan laporan keberlanjutan
melalui profitabilitas. Riset ini tidak berhasil membuktikan
pengaruh
kepemilikan
institusional
pada
pengungkapan laporan keberlanjutan melalui
profitabilitas. Berdasarkan data salah satu
sampel penelitian selama periode pengamatan
diketahui persentase proporsi kepemilikan
institusional yang cenderung tetap, yaitu
sebesar 50% namun tingkat profitabilitas yang
diperoleh
perusahaan
berubah-ubah
dan
cenderung rendah. Hal ini dapat ditafsirkan
bahwa
keterlibatan
investor
institusional
relatif
kecil
di
dalam
mendorong
dan
mengawasi kinerja manajemen perusahaan. Akibatnya, kendali investor institusi sebagai
pemegang saham mayoritas tidak menjamin
kinerja manajer agar dapat menghasilkan
profitabilitas
untuk
mengungkapkan
informasi
secara
transparan
melalui
sustainability report. Sehingga profitabilitas
gagal
dalam
menjadi
pemediasi
antara
kepemilikan
institusional
dengan
pengungkapan laporan keberlanjutan. Pengaruh Komite audit terhadap Pengungkapan
Sustainability Report melalui Profitabilitas KESIMPULAN Hasil riset ini dapat ditarik kesimpulan
bahwa pengungkapan laporan keberlanjutan
dapat lebih tinggi ketika ukuran perusahaan dan
komite audit yang dimiliki, ditunjang dengan
tingginya tingkat profitabilitas perusahaan. Hal Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan 24 Influence Of Corporate Governance And
Compay
Characteristics
To
The
Disclosure
Of
Corporate
Sosial
Responsibility
(Empirical
Study
In
Mining
Companies
That
Publish
Sustainability
Report). Simposiun
Nasional Akuntansi Xix Lampung, 1-22. Influence Of Corporate Governance And
Compay
Characteristics
To
The
Disclosure
Of
Corporate
Sosial
Responsibility
(Empirical
Study
In
Mining
Companies
That
Publish
Sustainability
Report). Simposiun
Nasional Akuntansi Xix Lampung, 1-22. tersebut
karena
profitabilitas
terbukti
berpengaruh langsung dan mampu menjadi
pemediasi antara ukuran perusahaan dan
komite audit pada pengungkapan laporan
keberlanjutan. Hasil
statistik
deskriptif
memperlihatkan
bahwa
pengungkapan
sustainability report di Indonesia terbilang
rendah, dilihat dari rata-rata pengungkapan
sebesar
32.5%. Manajemen
perusahaan
hendaknya
menjaga
profitabilitas
perusahaan yang tercermin melalui ROA
karena
variabel
tersebut
terbukti
mempengaruhi secara positif pengungkapan
sustainability
report
dan
mampu
menunjukkan perannya sebagai pemediasi. Bagi
penelitian
selanjutnya
diharapkan
mempertimbangkan indeks referensi POJK
sebagai
pedoman
pengungkapan
sustainability report seiring dikeluarkannya
peraturan OJK nomor 51/POJK.03/2017. Aliniar, D., & Wahyuni, S. (2017). Pengaruh
Mekanisme Good Corporate Governance
(GCG) dan Ukuran Perusahaan terhadap
Kualitas
Pengungkapan
Sustainability
Report pada Perusahaan Terdaftar di
BEI. Kompartemen, 15(1), 26-41. Aniktia, R., & Khafid, M. (2015). Pengaruh
Mekanisme Good Corporate Governance
dan
Kinerja
Keuangan
terhadap
Pengungkapan Sustainability Reporting. Accounting Analysis Journal, 4(3), 1-10. Anindito,
B. (2015). Analisis
Pengaruh
Perputaran Modal Kerja , Struktur Modal
,
Umur
Perusahaan
dan
Ukuran
Perusahaan terhadap Profitabilitas Yang
Tergabung dalam LQ45 Bursa Efek
Indonesia
Tahun
2010-2012. Skripsi. Semarang:Universitas Diponegoro. REFERENSI Abernathy, J. L., Beyer, B., Masli, A., &
Stefaniak, C (2015). How The Source
Of
Audit
Committee
Accounting
Expertise
Influences
Financial
Reporting Timeliness. Current Issues
In Auditing, 9(1). Bhatia, A., & Tuli, S. (2017). Corporate Attributes
Affecting Sustainability Reporting: An
Indian Perspective. International Journal
Of Law And Management, 59(3), 322-340. Buallay, A. M., & Aldhaen, E. (2018). The
Relationship Between Audit Committee
Characteristics
And
The
Level
Of
Sustainability Report Disclosure. 2(3). Adhipradana, F., & Daljono. (2014). Pengaruh
Kinerja
Keuangan,
Ukuran
Perusahaan,
dan
Corporate
Governance terhadap Pengungkapan
Sustainability
Report. Diponegoro
Journal Of Accounting, 3(1), 1-12. Doktoralina, C. M., Anggraini, D., & Melzatia, S. (2018). The Importance Of Sustainability
Reports In Non-Financial Companies. Jurnal Akuntansi, Xxii(03), 368-384. Afsari, R., Purnamawati,. A., & Prayudi, M. (2017). Pengaruh Leverage, Ukuran
Perusahaan,
Komite
Audit
dan
Kepemilikan Institusional terhadap
Luas
Pengungkapan
Sustainability
Report. Jurnal
Imiah
Mahasiswa
Akuntansi Undiksha, 8(2), 1-12. Giriyani, N. L. P. W., & Diyani, L. A. (2018). Pengaruh
Cash
Conversion
Cycle,
Likuiditas
dan
Firm
Size
terhadap
Profitabilitas. Jurnal Akuntansi Fakultas
Ekonomi dan Bisnis Universitas Telkom,
12(1). Grossman, S. J., & Hart, O. (1986). The Costs and
Benefits of Ownership: A Theory of Agustina, U., & Rusmana, O. (2016). The 25
BEAJ Vol 1 (1) (2021): 14-26 25 Vertical
and
Lateral
Integration. Journal of Political Economy, 94(4),
691-719. Ownership Structure On The Financial
Performance Of Listed Insurance Firms
In Nigeria. International Journal Of
Academic
Research
In
Accounting,
Finance And Management Sciences, 4(1),
409-416. Haladu, A., & Salim, B. (2016). Corporate
Characteristics
and
Sustainability
Reporting Environmental Agencies’
Moderating
Effects. International
Journal of Economics and Financial
Issues, 6(4), 1784-1790 Kusuma, W., & Priantinah, D. (2018). Pengaruh
Pengungkapan Sustainability Report dan
Ukuran
Perusahaan
terhadap
Nilai
Perusahaan dengan Profitabilitas sebagai
Variabel Pemoderasi pada Perusahaan
yang
Bergabung
di
ISSI
dan
Konvensional Periode 2014-2016. Jurnal
Nominal, 7(2). Hu, Y. Y., Zhu, Y., Tucker., & Hu, Y. (2018). Ownership
Influence
and
CSR
Disclosure
in
China. Accounting
Research Journal, 31(1), 8-21. John, A. O., & Adebayo, O. (2013). Effect of
Firm Size on Profitability : Evidence
from Nigerian Manufacturing Sector. Prime
Journal
of
Business
Administration
and
Management,
3(9), 1171–1175. Kuzey, C., & Uyar, A. (2017). Determinants Of
Sustainability Reporting And Its Impact
On Firm Value: Evidence From The
Emerging Market Of Turkey. Journal Of
Cleaner Production, 143, 27-39. Mahardika, E. S. S., Khafid, M., & Agustina, L. (2014). Pengaruh Struktur Kepemilikan,
Ukuran
Perusahaan
dan
Umur
Perusahaan terhadap Kinerja Intellectual
Capital. Kumai, L., & Isa, K. (2014). The Impact Of REFERENSI Accounting Analysis Journal, 3(1),
100-108. Karaman, A. S., Kilic, M., & Uyar, A. (2018). Sustainability
Reporting
In
The
Aviation
Industry:
Worldwide
Evidence. Sustainability Accounting,
Management And Policy Journal, 9(4),
362-391. Khafid, M, Baroroh, N., & Firmansyah, M. (2018). The
Role
Of
Corporate
Governance
In
Moderating
The
Influence Of Company Growth And
Size
On
Corporate
Social
Responsibility Disclosure. Kne Social
Sciences, 3(10). Mamun,
A. Al. (2014). Investigating
The
Development And Effects Of Social
Capital Through Participation In Group-
Based
Microcredit
Programme
In
Peninsular
Malaysia. Journal
Of
Interdisciplinary Economics, 26(12), 33-
59. Khafid, M. (2012). Pengaruh Tata Kelola
Perusahaan (Corporate Governance)
dan Struktur Kepemilikan terhadap
Persistensi Laba. Jurnal Dinamika
Akuntansi, 4(2), 139-148. Martínez-Ferrero, J., Garcia-Sanchez, M., &
Cuadrado-Ballesteros, B. (2015). Effect of
financial
reporting
quality
on
sustainability
information
disclosure. Corporate
Social
Responsibility
and
Environmental Management, 22(1), 45–
64. Khafid,
M.,
&
Mulyaningsih. (2015). Kontribusi Karakteristik Perusahaan
Dan Corporate Governance terhadap
Publikasi Sustainability Report. Jurnal
Ekonomi dan Keuangan, 19(3), 340-
359. Marwati, C. P., & Yulianti. (2015). Analisis
Pengungkapan
Sustainability
Report
pada Perusahaan Non-Keuangan Tahun
2009-2013. Jurnal Dinamika Akuntansi,
7(2), 167-181. Kumai, L., & Isa, K. (2014). The Impact Of Kumai, L., & Isa, K. (2014). The Impact Of Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan 26 Of Company Size, Capital Structure,
Good Corporate Governance, Inflation,
Interest Rate, And Exchange Rate Of
Financial Performance And Value Of The
Company. Interdisciplinary Journal Of
Contemporary Research In Business,
5(10), 26-33. Nasir, A., Ilham, E., & Utara, V. (2014). Pengaruh Karakteristik Perusahaan
dan Corporate Governance terhadap
Pengungkapan Sustainability Report
pada
Perusahaan
LQ45. Jurnal
Ekonomi, 22(1). Nazari, J. A., Herremans, I. M., & Warsame,
H. A. (2015). Sustainability Reporting:
External Motivators And Internal
Facilitators. Corporate
Governance
Journal, 15(3). Racelia, D. D., Adri, R., & Diyanto, V. (2017). Pengaruh Karakteristik Perusahaan dan
Corporate
Governance
terhadap
Publikasi Sustainability Report (Studi
Empiris Pada Perusahaan Indeks Kompas
100 di Bursa Efek Indonesia Tahun 2014-
2015). Jom Fekon, 4(2). Nurkhin, A., Wahyudin, A., & Fajriah, A. (2017). Relevansi
Struktur
Kepemilikan terhadap Profitabilitas
dan
Nilai
Perusahaan
Barang
Konsumsi. Jurnal
Akuntansi
Multiparadigma, 8(1), 35-46. Setyawan, S. H., Yuliandari, W. S., & Aminah, W. (2018). Pengaruh
Kepemilikan
Manajerial, Kepemilikan Institusional,
dan
Dewan
Komisaris
Independen
terhadap Pengungkapan Sustainability
Report (Studi pada Perusahaan Non
Perbankan dan Non Keuangan yang
Listing di Bursa Efek Indonesia. E-
Proceeding Of Management, 5(1), 670-
677. Nurleni, Darmawati, B., & Amiruddin, A. (2017). REFERENSI The effect of managerial and
institutional ownership on corporate
social
responsibility
disclosure. International Journal of Law and
Management, 3(4). Otoritas Jasa Keuangan. 2017. Peraturan
Nomor
51/POJK.03/2017
tentang
Penerapan Keuangan Berkelanjutan
bagi Lembaga Jasa Keuangan, Emiten
dan Perusahaan Publik. Wikardi, D., & Wiyani, N (2017). Pengaruh Debt
To Equity Ratio, Firm Size, Inventory
Turnover
,
Assets
Turnover
dan
Pertumbuhan
Penjualan
terhadap
Profitabilitas. Jurnal
Online
Insan
Akuntan, 2(1), 99-118. Pratama, A., & Yulianto, A. (2015). Faktor
Keuangan dan Corporate Governance
Sebagai
Penentu
Pengungkapan
Sustainability
Report. Accounting
Analysis Journal, 4(2), 1-10. Wiranata & Nugrahanti, Y. (2013). Pengaruh
Struktur
Kepemilikan
terhadap
Profitabilitas Perusahaan Manufaktur di
Indonesia. Jurnal
Akuntansi
dan
Keuangan, 15(1), 15-26. Purnomosidi, L., Suhadak, Siregar, H., &
Dzulkirom, M. (2014). The Influences
|
https://openalex.org/W3204381417
|
https://europepmc.org/articles/pmc8482691?pdf=render
|
English
| null |
The expression landscape of JAK1 and its potential as a biomarker for prognosis and immune infiltrates in NSCLC
|
BMC bioinformatics
| 2,021
|
cc-by
| 8,566
|
© 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 mate-
rial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or
exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://
creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publi
cdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Janus-activated kinase-1 (JAK1) plays a crucial role in many aspects of
cell proliferation, differentiation, apoptosis and immune regulation. However, cor-
relations of JAK1 with prognosis and immune infiltration in NSCLC have not been
documented. Methods: We analyzed the relationship between JAK1 expression and NSCLC progno-
sis and immune infiltration using multiple public databases. Results: JAK1 expression was significantly decreased in NSCLC compared with that in
paired normal tissues. JAK1 overexpression indicated a favourable prognosis in NSCLC. In subgroup analysis, high JAK1 expression was associated with a preferable prognosis
in lung adenocarcinoma (OS: HR, 0.74, 95% CI from 0.58 to 0.95, log-rank P = 0.017),
not squamous cell carcinoma. In addition, data from Kaplan–Meier plotter revealed
that JAK1 overexpression was associated with a preferable prognosis in male and stage
N2 patients and patients without distant metastasis. Notably, increased levels of JAK1
expression were associated with an undesirable prognosis in patients with stage 1 (OS:
HR, 1.46, 95% CI from 1.06 to 2.00, P = 0.02) and without lymph node metastasis (PFS:
HR, 2.18, 95% CI from 1.06 to 4.46, P = 0.029), which suggests that early-stage NSCLC
patients with JAK1 overexpression may have a bleak prognosis. Moreover, multiple
immune infiltration cells, including NK cells, CD8 + T and CD4 + T cells, B cells, mac-
rophages, neutrophils, and dendritic cells (DCs), in NSCLC were positively correlated
with JAK1 expression. Furthermore, diverse immune markers are associated with JAK1
expression. Conclusions: JAK1 overexpression exhibited superior prognosis and immune infiltra-
tion in NSCLC. Keywords: JAK1, Immune infiltrating, Prognosis, NSCLC The expression landscape of JAK1 and its
potential as a biomarker for prognosis
and immune infiltrates in NSCLC Kaikai Shen1†, Yuqing Wei2†, Tangfeng Lv3, Yong Song3, Xiaogan Jiang1, Zhiwei Lu2, Ping Zhan3,
Xianghai Wang2, Meng Fan4 and Weihua Lu1* *Correspondence:
lwh683@126.com
†Kaikai Shen and Yuqing Wei
contributed equally to this
work
1 Department of Critical Care
Medicine, The First Affiliated
Hospital of Wannan Medical
College (Yijishan Hospital
of Wannan Medical College),
Wuhu 241000, China
Full list of author information
is available at the end of the
article Open Access Introduction Lung cancer, as a malignant tumour with high morbidity and mortality, poses a serious
threat to people’s physical and mental health [1]. Non-small-cell lung cancer (NSCLC)
accounts for approximately 80% of all lung cancer cases [2]. With the advent of precision Shen et al. BMC Bioinformatics (2021) 22:471
https://doi.org/10.1186/s12859-021-04379-y Shen et al. BMC Bioinformatics (2021) 22:471
https://doi.org/10.1186/s12859-021-04379-y Shen et al. BMC Bioinformatics (2021) 22:471 Page 2 of 14 therapy, lung cancer treatment has entered molecular therapy, including targeted ther-
apy, anti-angiogenesis therapy and immunotherapy [3]. However, the prognosis has not
improved significantly, and the 5-year survival rate remains poor [1]. In recent years,
clinical studies have shown that immunotherapy (PD-1/L1 monoclonal antibody, CTLA-
4 inhibitor) has great potential in the treatment of lung cancer patients without epider-
mal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutations
[4]. Nevertheless, immunotherapy only activates immune cells in a subset of patients. With the continuous exploration of the tumour immune microenvironment (TME),
which can directly or indirectly affect the development of tumours, including promoting
tumour angiogenesis, changing the biological characteristics of the tumour, promoting
immune escape, and even regulating the activity of cancer stem cells (CSCs) [5, 6]. Many
studies have found that TAMs (tumour-associated macrophages), TILs (tumour infil-
trating lymphocytes) and TINs (tumour-infiltrating neutrophils) in the TME can affect
the efficacy of immunotherapy [7, 8]. Hence, it is imperative to find immune infiltration-
related biomarkers that are related to the prognosis of NSCLC. p
g
Janus-activated kinase (JAK) is an inactive tyrosine protein kinase that consists of four
family members, including JAK1, JAK2, TYK2, and JAK3 [9]. JAKs approach each other
and are activated by interactive tyrosine phosphorylation, ultimately leading to signal
transducer and activator of transcription (STAT) proteins forming a homo/heterodimer
that is incorporated into the nucleus and binding to the target gene promoter to activate
transcription and expression [10]. Previous studies have shown that the JAK1/STAT3
pathway is widely involved in many significant biological processes, such as cell prolif-
eration, differentiation, apoptosis and immune regulation [11–13]. JAK family kinases
play an essential role in cytokine signalling. Functionally acquired JAK1 mutations can
encourage the development of cancers, especially leukaemia. Abnormal JAK1 expression
either promotes or suppresses tumour growth [10, 14, 15]. Chen et al. [10] showed that
high expression of JAK1 mRNA was associated with TNM (Tumor, Node, Metastasis)
stage and superior prognosis of breast cancer. In addition, infiltration and enrichment of
immunoregulatory cells were significantly positively correlated with JAK1 expression. In
contrast, Zhang et al. [16] showed that JAK1 signal activation could promote the prolif-
eration of bladder cancer cells and lead to a poor prognosis. Hu et al. also indicated that
JAK1/STAT3 plays a crucial role in ovarian cancer as a pro-oncogenic signalling path-
way [17]. Whether JAK1 expression is involved in the prognosis and the level of immune
infiltration in NSCLC still needs to be further explored. i
In our descriptive study, we explored the expression landscape of JAK1 in NSCLC
and its relationship with prognosis using shared databases, including TIMER, GEPIA,
Kaplan–Meier Plotter and PrognoScan. We also visualized the relationship between
JAK1 and immune infiltration using TIMER and TISIDB. Moreover, correlations
between JAK1 expression and multiple gene marker sets related to immune infiltrates
were also analysed via TIMER and GEPIA. TISIDB analysis TISIDB is also a web portal for tumour and immune system interaction, which inte-
grates multiple heterogeneous data types. (http://cis.hku.hk/TISIDB/index.php) [19]. We explored the correlation between JAK1 expression in NSCLC and the abundance of
multiple immune cells, including activated CD4 T cells (Act_CD4), activated dendritic
cells (Act_DCs), immature dendritic cells (iDCs), neutrophils, natural killer cells (NKs),
plasmacytoid dendritic cells (pDCs), central memory CD4 cells (Tcm_CD4), and effec-
tor memory CD8 cells (Tem_CD8). The relative abundance of each immune cell was
inferred by using gene set variation analysis (GSVA) based on the gene expression pro-
file. JAK1 expression was drawn on the x-axis, while the abundance of immune cells was
drawn on the y-axis. A scatterplot was used to display the correlation between the abun-
dance of each immune cell and JAK1 expression. GEPIA database analysis To further verify the gene marker associated with immune infiltration in NSCLC. We
used the public database Gene Expression Profiling Interactive Analysis (GEPIA) (http://
gepia.cancer-pku.cn/index.html) [20], which analyses the RNA sequencing expression
from the TCGA and GTEx projects of 9736 tumours and 8587 normal samples. The
correlation coefficient was determined by the Spearman method. The tumour and nor-
mal tissue datasets were used for analysis. JAK1 expression profiles across LUAD (lung
adenocarcinoma) and LUSC (lung squamous cell carcinoma) samples and paired normal
tissues from GEPIA were also analysed. Materials and methods
TIMER database analysis The TIMER (Tumour Immune Estimation Resource) web server is a comprehensive
resource for the systematic analysis of immune infiltrates across diverse cancer types. Shen et al. BMC Bioinformatics (2021) 22:471 Page 3 of 14 (https://cistrome.shinyapps.io/timer/) [18]. The abundances of six immune infiltrates (B
cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells) were
estimated by the TIMER algorithm. We evaluated the correlation between JAK1 expres-
sion levels and various immune infiltrating cells via the TIMER algorithm. In addition,
JAK1 expression profiles across various tumour samples and paired normal tissues from
the TCGA data in TIMER were also determined. Finally, to further identify other poten-
tial subtypes of immune cell infiltration, we also analysed the correlation between JAK1
expression and diverse immune cell markers, including monocytes, tumour-associated
macrophages (TAMs), M1 macrophages, M2 macrophages, CD8+ T cells, B cells, neu-
trophils, dendritic cells, natural killer (NK) cells, T-helper 1 (Th1) cells, T-helper 2 (Th2)
cells, Tregs and exhausted T cells (https://www.rndsystems.com/cn/resources/cell-
markers/immune-cells). Tumour purity was also determined. The gene expression level
was described in terms of log2TAM. JAK1 expression was drawn in the x-axis, while
marker genes were drawn in the y-axis. A scatterplot was used to describe the specific
connection between every immune gene marker and JAK1 expression. (https://cistrome.shinyapps.io/timer/) [18]. The abundances of six immune infiltrates (B
cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells) were
estimated by the TIMER algorithm. We evaluated the correlation between JAK1 expres-
sion levels and various immune infiltrating cells via the TIMER algorithm. In addition,
JAK1 expression profiles across various tumour samples and paired normal tissues from
the TCGA data in TIMER were also determined. Finally, to further identify other poten-
tial subtypes of immune cell infiltration, we also analysed the correlation between JAK1
expression and diverse immune cell markers, including monocytes, tumour-associated
macrophages (TAMs), M1 macrophages, M2 macrophages, CD8+ T cells, B cells, neu-
trophils, dendritic cells, natural killer (NK) cells, T-helper 1 (Th1) cells, T-helper 2 (Th2)
cells, Tregs and exhausted T cells (https://www.rndsystems.com/cn/resources/cell-
markers/immune-cells). Tumour purity was also determined. The gene expression level
was described in terms of log2TAM. JAK1 expression was drawn in the x-axis, while
marker genes were drawn in the y-axis. A scatterplot was used to describe the specific
connection between every immune gene marker and JAK1 expression. Prognostic analysis We used public databases including Kaplan–Meier Plotter (https://kmplot.com/analy
sis/) [21] and PrognoScan (http://dna00.bio.kyutech.ac.jp/PrognoScan/index.html) [22]
to examine the relationship between JAK1 expression level and NSCLC prognosis. The
Kaplan–Meier plotter is competent for assessing the effect of 54,000 genes on prognosis
in 21 cancer types. Sources included the GEO, EGA, and TCGA databases. The hazard Shen et al. BMC Bioinformatics (2021) 22:471 Page 4 of 14 ratio (HR) and its 95% confidence interval (95% CI) for OS (overall survival) and PFS
(progression-free survival) in NSCLC were calculated. The log-rank P value was likewise
computed. Similarly, the prognostic database PrognoScan was designed to analyse the correlation
between JAK1 expression and overall survival (OS). The threshold was set as a Cox P
value < 0.05. Statistical analysish The results examined in TIMER and GEPIA are displayed with P values determined by
t tests, fold changes, and gene ranks. Survival outcomes were presented with Kaplan–
Meier plots and PrognoScan, and the results are displayed with HR and Cox P values
from a log-rank test. The correlation between JAK1 expression and each gene marker
was assessed by Spearman’s correlation test and statistical significance. The strength of
the correlation was defined as follows: 0.00–0.19 “very weak”, 0.20–0.39 “weak”, 0.40–
0.59 “moderate”, 0.60–0.79 “strong”, and 0.80–1.0 “very strong”. For all analyses, a P value
less than 0.05 indicates statistical significance. JAK1 expression in multiple human tumours We evaluated the differences in JAK1 expression in various human tumour tissues and
paired normal tissues using RNA sequencing data from the TCGA. The detailed expres-
sion of JAK1 in the tumour and adjacent tissues is shown in Fig. 1A. JAK1 expression
was significantly decreased in BLCA (bladder urothelial carcinoma), BRCA (breast inva-
sive carcinoma), COAD (colon adenocarcinoma), KICH (kidney chromophobe), LUAD,
LUSC, PRAD (prostate adenocarcinoma), READ (rectum adenocarcinoma), and UCEC
(uterine corpus endometrial carcinoma) compared to that in adjacent normal tissues,
while the expression of JAK1 was significantly higher in CHOL (cholangiocarcinoma),
ESCA (oesophageal carcinoma), HNSC (head and neck squamous cell carcinoma), KIRC
(kidney renal clear cell carcinoma), KIRP (kidney renal papillary cell carcinoma), LIHC
(liver hepatocellular carcinoma), STAD (stomach adenocarcinoma), and THCA (thyroid
carcinoma) than that in adjacent normal tissues. Fig. 1 JAK1 expression profile in various types of human tumours. A Human JAK1 expression profile across
various tumour samples and paired normal tissues from the TCGA data in TIMER. B Human JAK1 expression
profile in NSCLC from the GEPIA database. The ordinate of B indicates the expression level of JAK1 (log2 TPM). LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, NSCLC non-small-cell lung cancer, TPM
transcripts per million, N normal, T tumour. *P < 0.05, **P < 0.01, ***P < 0.001 Fig. 1 JAK1 expression profile in various types of human tumours. A Human JAK1 expression profile across
various tumour samples and paired normal tissues from the TCGA data in TIMER. B Human JAK1 expression
profile in NSCLC from the GEPIA database. The ordinate of B indicates the expression level of JAK1 (log2 TPM). LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, NSCLC non-small-cell lung cancer, TPM
transcripts per million, N normal, T tumour. *P < 0.05, **P < 0.01, ***P < 0.001 Shen et al. BMC Bioinformatics (2021) 22:471 Page 5 of 14 To further evaluate the expression patterns of JAK1 in NSCLC, the GEPIA database
was further selected. Similar results were likewise obtained, namely, JAK1 expression in
LUAD and LUSC was significantly lower than that in the paired normal tissues (Fig. 1B). JAK1 expression predicts the prognosis of NSCLC Next, we explored the prognostic value of JAK1 for NSCLC by adopting two public
databases. First, we investigated JAK1 expression and the prognosis of NSCLC, LUAD
and LUSC using Kaplan–Meier Plotter, which principally focused on the strength of the
information from the GEO, EGA and TCGA miRNA gene chips. The results showed that
high JAK1 expression indicated a favourable prognosis in NSCLC (OS: HR, 0.62, 95% CI
from 0.53 to 0.74, log-rank P < 0.001; PFS: HR, 0.65, 95% CI from 0.50 to 0.86, log-rank
P = 0.002). In the subgroup analysis, the high expression of JAK1 in LUAD lasted longer
in OS (HR: 0.74, 95% CI from 0.58 to 0.95, log-rank P = 0.017), but there was no benefit
in PFS (HR: 0.83, 95% CI from 0.60 to 1.14, log-rank P = 0.24). In LUSC, high expres-
sion of JAK1 was associated with longer duration of PFS (HR: 0.65, 95% CI from 0.39 to
1.09, log-rank P = 0.097), while the difference was not statistically significant. In addi-
tion, there was no benefit in OS (HR: 0.95, 95% CI from 0.69 to 1.29, log-rank P = 0.73). (Fig. 2). Next, we investigated the association of JAK1 expression and prognosis with distinct
clinicopathological features in NSCLC (Table 1). JAK1 overexpression related to supe-
rior OS and PFS in males (HR: 0.64, 0.62, 95% CI from 0.52 to 0.79, P < 0.001) rather than
females. In addition, the higher expression of JAK1 is associated with preferable OS in
patients with N2 lymph node metastasis (HR: 0.39, 95% CI from 0.17 to 0.86, P = 0.016) Fig. 2 Kaplan–Meier survival curves comparing high and low expression of JAK1 in NSCLC. A, B OS and PFS
survival curves of NSCLC (n = 1144, n = 596). C, D OS and PFS survival curves of LUAD (n = 672, n = 443). E, F
OS and PFS survival curves of LUSC (n = 271, n = 141). OS overall survival, PFS progression-free survival, LUAD
lung adenocarcinoma, LUSC lung squamous cell carcinoma, NSCLC non-small-cell lung cancer, HR hazard ratio Fig. 2 Kaplan–Meier survival curves comparing high and low expression of JAK1 in NSCLC. A, B OS and PFS
survival curves of NSCLC (n = 1144, n = 596). C, D OS and PFS survival curves of LUAD (n = 672, n = 443). JAK1 expression predicts the prognosis of NSCLC E, F
OS and PFS survival curves of LUSC (n = 271, n = 141). OS overall survival, PFS progression-free survival, LUAD
lung adenocarcinoma, LUSC lung squamous cell carcinoma, NSCLC non-small-cell lung cancer, HR hazard ratio Page 6 of 14 Shen et al. BMC Bioinformatics (2021) 22:471 Table 1 Association between JAK1 expression and prognosis with different clinicopathological
features of NSCLC by Kaplan–Meier plotter
N Number, HR Hazard Ratio
Clinicopathological
characteristics
Overall survival (n = 1144)
Progression-free survival (n = 596)
N
HR
P value
N
HR
P value
Gender
Male
659
0.64 (0.52–0.79)
2.20E−05
343
0.61 (0.43–0.86)
0.0043
Female
374
0.86 (0.61–1.21)
0.38
253
0.77 (0.49–1.20)
0.25
Stage
1
449
1.46 (1.06–2.00)
0.02
596
1.26 (0.81–1.95)
0.31
2
161
0.99 (0.63–1.55)
0.95
125
0.64 (0.38–1.11)
0.108
3
44
1.11 (0.55–2.25)
0.77
17
–
–
AJCC stage T
1
224
0.86 (0.57–1.28)
0.44
54
0.95 (0.23–3.53)
0.94
2
190
0.86 (0.58–1.27)
0.44
121
1.75 (0.94–3.27)
0.073
3
29
0.83 (0.38–1.85)
0.65
2
–
–
4
23
0.74 (0.30–1.84)
0.52
0
–
–
AJCC stage N
0
324
0.87 (0.63–1.19)
0.38
126
2.18 (1.06–4.46)
0.029
1
102
0.64 (0.38–1.08)
0.09
51
0.98 (0.40–2.41)
0.96
2
32
0.39 (0.17–0.86)
0.016
0
–
–
AJCC stage M
0
462
0.73 (0.56–0.93)
0.013
177
1.66 (0.95–2.9)
0.07
Smoking history
Yes
300
1.59 (1.04–2.24)
0.029
297
0.73 (0.48–1.08)
0.11
No
141
2.24 (0.96–5.25)
0.056
141
1.39 (0.75–2.58)
0.29
Chemotherapy
Yes
34
0.82 (0.26–2.64)
0.74
34
1.18 (0.45–3.11)
0.74
No
21
0.18 (0.02–1.52)
0.075
21
0.63 (0.19–2.07)
0.44 Table 1 Association between JAK1 expression and prognosis with different clinicopathological
features of NSCLC by Kaplan–Meier plotter without distant metastasis (HR: 0.73, 95% CI from 0.56 to 0.93, P = 0.013) of NSCLC. Notably, overexpression of JAK1 is associated with undesirable prognosis in patients
with stage 1 NSCLC (OS: HR, 1.46, 95% CI from 1.06 to 2.00, P = 0.02) and without
lymph node metastasis (PFS: HR, 2.18, 95% CI from 1.06 to 4.46, P = 0.029), which
implicit early NSCLC patients with JAK1 overexpression may have a poor prognosis. Regrettably, there were no statistically significant differences between JAK1 expression
and prognosis in females, stage 2 to 3, stage T1 to T4, N1 lymph node metastasis or
prior chemotherapy. The exact survival time is shown in Additional file 1: Table S1. JAK1 expression predicts the prognosis of NSCLC hi
Finally, we selected the PrognoScan database to further verify the relationship
between JAK1 expression and prognosis in NSCLC. Five cohorts containing a total of
530 patients with NSCLC and LUAD showed that high expression of JAK1 was associ-
ated with favourable OS (Table 2). Correlation of JAK1 expression and immune infiltration Tumour infiltrating lymphocytes (TILs) are closely related to prognosis and sub-
sequent immunotherapy in lung cancer [23, 24]. We investigated the correlation
between JAK1 expression level and immune cell infiltration in LUAD and LUSC Shen et al. BMC Bioinformatics (2021) 22:471 Page 7 of 14 Table 2 Survival analysis of JAK1 mRNA in NSCLC from the PrognoScan database
Dataset
Subtype
Endpoint
Number
Ln
(HR-high/
HR-low)
COX P value
ln HR
HR [95% CIlow–
CIup]
jacob-00182-
CANDF
LUAD
OS
82
− 1.11
0.002460
− 1.37
0.25 [0.10–0.62]
GSE31210
LUAD
OS
204
− 1.18
0.026306
− 1.13
0.32 [0.12–0.88]
GSE11117
NSCLC
OS
41
− 1.55
0.034669
− 0.78
0.46 [0.22–0.95]
MICHIGAN-LC
LUAD
OS
86
− 1.25
0.094138
− 0.85
0.43 [0.16–1.16]
GSE13213
LUAD
OS
117
− 0.73
0.057336
− 0.43
0.65 [0.42–1.01] Table 2 Survival analysis of JAK1 mRNA in NSCLC from the PrognoScan database f JAK1 mRNA in NSCLC from the PrognoScan database Fig. 3 TIMER database showing the relationship between JAK1 expression level and immune infiltration in
LUAD and LUSC. A JAK1 expression was negatively correlated with tumour purity and significantly positively
correlated with infiltrating levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic
cells in LUAD. B JAK1 expression was negatively correlated with tumour purity and significantly positively
correlated with infiltrating levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic
cells in LUSC. LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma. A P value less than 0.05
indicated statistical significance Fig. 3 TIMER database showing the relationship between JAK1 expression level and immune infiltration in
LUAD and LUSC. A JAK1 expression was negatively correlated with tumour purity and significantly positively
correlated with infiltrating levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic
cells in LUAD. B JAK1 expression was negatively correlated with tumour purity and significantly positively
correlated with infiltrating levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic
cells in LUSC. LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma. A P value less than 0.05
indicated statistical significance Fig. 3 TIMER database showing the relationship between JAK1 expression level and immune infiltration in
LUAD and LUSC. A JAK1 expression was negatively correlated with tumour purity and significantly positively
correlated with infiltrating levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic
cells in LUAD. Correlation of JAK1 expression and immune infiltration B JAK1 expression was negatively correlated with tumour purity and significantly positively
correlated with infiltrating levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic
cells in LUSC. LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma. A P value less than 0.05
indicated statistical significance Fig. 3 TIMER database showing the relationship between JAK1 expression level and immune infiltration in
LUAD and LUSC. A JAK1 expression was negatively correlated with tumour purity and significantly positively
correlated with infiltrating levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic
cells in LUAD. B JAK1 expression was negatively correlated with tumour purity and significantly positively
correlated with infiltrating levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic
cells in LUSC. LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma. A P value less than 0.05
indicated statistical significance from TIMER. The results showed that JAK1 expression was negatively correlated
with tumour purity (r = − 0.229, P = 2.73e-07) and significantly positively correlated
with infiltrating levels of B cells (r = 0.155, P = 6.20e−04), CD8+ T cells (r = 0.307,
P = 4.41e−12), CD4+ T cells (r = 0.422, P = 2.42e−22), macrophages (r = 0.342,
P = 9.91e−15), neutrophils (r = 0.459, P = 1.41e−26), and dendritic cells (r = 0.479,
P = 2.25e−29). Similar results were also observed in LUSC. JAK1 expression was
negatively correlated with tumour purity (r = − 0.309, P = 5.34e−12) and signifi-
cantly positively correlated with infiltrating levels of B cells (r = 0.224, P = 8.82e−07),
CD8+ T cells (r = 0.26, P = 9.26e−09), CD4+ T cells (r = 0.517, P = 7.50e−34), mac-
rophages (r = 0.405, P = 2.92e−20), neutrophils (r = 0.522, P = 1.36e−34), and den-
dritic cells (r = 0.5, P = 2.20e−31) (Fig. 3). In addition, the public database TISIDB also explored the correlation between the
abundance of multiple immune cells and JAK1 expression in NSCLC. The enrich-
ment of diversified immune cells, such as Act_CD4, Act_DCs, iDCs, neutrophils,
NK cells, pDCs, Tcm_CD4 and Tem_CD8, was positively correlated with JAK1
expression in LUAD and LUSC. What needs illustration is that JAK1 expression has Shen et al. BMC Bioinformatics (2021) 22:471 Page 8 of 14 Page 8 of 14 Fig. Correlation of JAK1 expression and immune infiltration 4 Association between JAK1 expression level and immune cell infiltration in LUAD from the TISIDB web
portal (517 samples). A–H JAK1 expression was significantly positively correlated with infiltrating levels of
Act_CD4, Act_DCs, iDCs, neutrophils, NK cells, pDCs, Tcm_CD4 and Tem_CD8. LUAD lung adenocarcinoma,
Act_CD4 activated CD4 T cells, Act_DCs activated dendritic cells, iDCs immature dendritic cells, NK cells natural
killer cells, pDCs plasmacytoid dendritic cells, Tcm_CD4 central memory CD4 cells, Tem_CD8 effector memory
CD8 cells. A P value less than 0.05 indicated statistical significance Fig. 4 Association between JAK1 expression level and immune cell infiltration in LUAD from the TISIDB web ii portal (517 samples). A–H JAK1 expression was significantly positively correlated with infiltrating levels of
Act_CD4, Act_DCs, iDCs, neutrophils, NK cells, pDCs, Tcm_CD4 and Tem_CD8. LUAD lung adenocarcinoma,
Act_CD4 activated CD4 T cells, Act_DCs activated dendritic cells, iDCs immature dendritic cells, NK cells natural
killer cells, pDCs plasmacytoid dendritic cells, Tcm_CD4 central memory CD4 cells, Tem_CD8 effector memory
CD8 cells. A P value less than 0.05 indicated statistical significance no significant corrections with infiltrating levels of Act_CD4 in LUSC. For details,
please refer to Fig. 4 and Additional file 1: Fig S1. Correlations between JAK1 expression and immune gene markers To further understand the interaction between JAK1 expression and TME in NSCLC. We further explored the potential correlation between JAK1 and immune gene markers
in the public databases TIMER and GEPIA (Tables 3, 4). These gene markers depicted
diverse immune infiltration cells, including monocytes, TAMs, M1 macrophages, M2
macrophages, CD8+ T cells, B cells, neutrophils, dendritic cells and NK cells. In addi-
tion, various T cells, including Th1, Th2, Tregs, and T cell exhaustion, which play dif-
ferent functions in the TME, were included. Although they were adjusted for tumour
purity, most immune markers remained significantly related to JAK1 expression levels in
LUAD and LUSC. Interestingly, the results from TIMER and GEPIA showed that most gene sets of
monocytes, M1 macrophages, and TAMs were significantly associated with JAK1
expression levels in LUAD. However, we discovered that JAK1 expression was also
associated with most gene sets of monocytes and TAMs rather than M1 macrophages. Notably, the majority chemokine ligand, which induced cells of the immune system to
enter the site of infection, CCL-2, CD80 and CD68 of TAMs, IRF5 and NOS2 of M1,
CD163 and MS4A4A of M2 were strongly related to JAK1 expression in LUAD (all P
value < 0.0001). These consequences suggest that JAK1 may play a vital role in the TME
by regulating the function of macrophages. In addition, some of the gene markers, such
as MPO, CCR7 and CD11b (ITGAM), of neutrophils and CD8A of CD8+ T cells were
associated with JAK1 expression in LUAD and LUSC. Page 9 of 14 Shen et al. Correlations between JAK1 expression and immune gene markers BMC Bioinformatics (2021) 22:471 Shen et al. BMC Bioinformatics LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, TAM tumour-associated macrophage, M1 M1 macrophage,
M2 M2 macrophage, Th T helper cell, Treg regulatory T cell, Cor R value of Spearman’s correlation, None correlation without
adjustment, Purity correlation adjusted by purity
*P < 0.01; **P < 0.001; ***P < 0.0001
Table 3 (continued)
Description
Gene markers
LUAD
LUSC
None
Purity
None
Purity
Cor
P
Cor
P
Cor
P
Cor
P
Treg
FOXP3
0.354
***
0.297
***
0.481
***
0.402
***
STAT5B
0.46
***
0.45
***
0.274
***
0.288
***
TGFβ (TGFB1)
0.487
***
0.451
***
0.312
***
0.266
***
CCR8
0.422
***
0.378
***
0.442
***
0.358
***
CD25(IL2RA)
0.357
***
0.305
***
0.368
***
0.273
***
T cell exhaustion
PD-1(PDCD1)
0.225
***
0.144
*
0.357
***
0.267
***
CTLA4
0.274
***
0.197
***
0.331
***
0.221
***
LAG3
0.173
***
0.104
0.021
0.257
***
0.181
***
TIM-3 (HAVCR2)
0.371
***
0.314
***
0.391
***
0.292
*** Table 4 Correlation analysis between JAK1 and relevant genes and markers of monocytes and
macrophages in GEPIA Table 4 Correlation analysis between JAK1 and relevant genes and markers of monocytes and
macrophages in GEPIA
LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, TAM tumour-associated macrophage, M1 M1 macrophage,
M2 M2 macrophage
*P < 0.01; **P < 0.001; ***P < 0.0001
Description
Gene markers
LUAD
LUSC
Tumor
Normal
Tumor
Normal
R
P
R
P
R
P
R
P
Monocyte
CD14
0.3
***
− 0.051
0.7
0.27
***
0.027
0.85
CD115
0.51
***
0.2
0.13
0.43
***
0.32
0.024
CD86
0.4
***
− 0.22
0.093
0.28
***
− 0.035
0.81
TAM
CCL2
0.23
***
0.16
0.24
0.23
***
− 0.02
0.89
CD80
0.35
***
0.21
0.11
0.21
***
0.22
0.13
CD68
0.4
***
0.01
0.94
0.28
***
0.14
0.32
M1
IRF5
0.25
***
− 0.1
0.43
0.0053
0.91
0.11
0.45
ROS
0.2
***
0.51
***
0.31
***
0.34
0.015
NOS2
0.0033
0.94
0.56
***
− 0.042
0.36
0.49
**
M2
CD163
0.33
***
− 0.18
0.18
0.29
***
0.074
0.61
ARG1
0.05
0.27
0.27
0.036
− 0.027
0.55
0.14
0.34
MS4A4A
0.33
***
− 0.29
0.024
0.22
***
− 0.038
0.79 Moreover, the vast majority of gene sets of dendritic cells, including HLA-DPB1,
HLA-DQB1, HLA-DRA, HLA-DPA1, BDCA-1, BDCA-4 and CD11C, were positively
correlated with JAK1 expression levels in LUAD and LUSC. Correlations between JAK1 expression and immune gene markers BMC Bioinformatics (2021) 22:471 from the TIMER database
Description
Gene markers
LUAD
LUSC
None
Purity
None
Purity
Cor
P
Cor
P
Cor
P
Cor
P
Monocyte
CD14
0.262
***
0.2
***
0.425
***
0.323
***
CSF1R
0.503
***
0.463
***
0.553
***
0.482
***
CD86
0.395
***
0.344
***
0.444
***
0.349
***
TAM
CCL2
0.254
***
0.19
***
0.368
***
0.296
***
CD80
0.385
***
0.331
***
0.383
***
0.294
***
CD68
0.383
***
0.339
***
0.382
***
0.29
***
M1
IRF5
0.323
***
0.277
***
0.068
0.126
0.049
0.281
NOS2
0.22
***
0.194
***
0.062
0.165
0.071
0.122
M2
CD163
0.45
***
0.414
***
0.458
***
0.378
***
ARG1
0.113
*
0.11
0.015
− 0.069
0.125
− 0.074
0.107
MS4A4A
0.352
***
0.3
***
0.345
***
0.24
***
CD8+ T cell
CD8A
0.248
***
0.177
***
0.262
***
0.175
**
CD8B
0.118
*
0.051
0.257
0.188
***
0.135
*
B cell
CD19
0.121
*
0.02
0.653
0.277
***
0.146
*
CD79A
0.137
*
0.047
0.295
0.323
***
0.198
***
Neutrophils
CEACAM8
0.259
***
0.252
***
0.072
0.106
0.048
0.293
MPO
0.22
***
0.183
***
0.342
***
0.29
***
CCR7
0.361
***
0.295
***
0.39
***
0.291
***
CD11b(ITGAM)
0.472
***
0.435
***
0.578
***
0.517
***
Dendritic cell
HLA-DPB1
0.313
***
0.249
***
0.457
***
0.371
***
HLA-DQB1
0.253
***
0.192
***
0.328
***
0.245
***
HLA-DRA
0.281
***
0.215
***
0.387
***
0.292
***
HLA-DPA1
0.353
***
0.3
***
0.454
***
0.371
***
BDCA-1(CD1C)
0.294
***
0.243
***
0.341
***
0.227
***
BDCA-4(NRP1)
0.386
***
0.377
***
0.523
***
0.473
***
CD11C(ITGAX)
0.383
***
0.327
***
0.513
***
0.427
***
CD141(THBD)
0.366
***
0.344
***
0.032
0.469
− 0.006
0.89
NK cell
KIR2DL1
0.046
0.296
0.023
0.612
0.143
*
0.091
0.047
KIR2DL3
0.104
0.019
0.06
0.187
0.158
**
0.117
0.010
KIR2DL4
0.062
0.163
0.015
0.737
0.113
0.011
0.051
0.265
KIR3DL1
0.064
0.15
0.024
0.601
0.241
***
0.187
***
KIR3DL2
0.139
*
0.087
0.053
0.141
*
0.069
0.134
KIR3DL3
0.002
0.964
− 0.019
0.67
0.029
0.52
0.002
0.962
KIR2DS4
0.119
*
0.082
0.068
0.122
*
0.093
0.043
CD7
0.168
**
0.091
0.043
0.339
***
0.237
***
XCL1
0.036
0.416
0.004
0.931
− 0.015
0.742
0.017
0.705
Th1
T-bet (TBX21)
0.322
***
0.263
***
0.363
***
0.273
***
STAT4
0.336
***
0.274
***
0.49
***
0.415
***
STAT1
0.398
***
0.364
***
0.298
***
0.251
***
IFN-γ (IFNG)
0.129
*
0.06
0.181
0.121
*
0.057
0.216
TNF-α (TNF)
0.271
***
0.229
***
0.402
***
0.345
***
Th2
GATA3
0.435
***
0.387
***
0.551
***
0.517
***
STAT6
0.293
***
0.318
***
0.265
***
0.284
***
STAT5A
0.53
***
0.495
***
0.549
***
0.491
***
IL13
0.079
0.072
0.032
0.479
0.152
**
0.083
0.072 Page 10 of 14 Shen et al. Discussionh The JAK1/STAT signalling pathway, as a stimulant that is intimately related to the
physiological function of interferon, plays a significant role in cell growth, differen-
tiation, immune regulation and other aspects [11, 25, 26]. The exhaustive function
of JAK1 in NSCLC has not yet been clarified. Here, we report the expression pro-
file of JAK1 and its association with prognosis and immune infiltration in NSCLC. We found that JAK1 was expressed at low levels in NSCLC, and its expression level
was positively correlated with the prognosis of NSCLC, especially in LUAD. Interest-
ingly, JAK1 overexpression was associated with preferable survival in males, stage N2
patients and patients without distant metastasis. In addition, increased levels of JAK1
expression are associated with undesirable survival in patients with earlier stages
(stage 1 and N0), suggesting that early-stage NSCLC patients with JAK1 overexpres-
sion may have a bleak prognosis. Moreover, diverse immune infiltration cells and gene
sets were positively correlated with JAK1 expression level. Hence, to the best of our
knowledge, our study is the first to reveal the potential mechanism by which JAK1
functions in the TME and acts as a prognostic biomarker of NSCLC. The TME plays a crucial role in the gene expression and clinical efficacy of tumour
tissues, which are prerequisites and guarantees tumour immune escape [27]. The TME
refers to the sum of various immune-related factors, mainly consisting of immune
cells and immune-related molecules. In our study, we found that JAK1 expression was
significantly positively correlated with the infiltration of various immune cells (mono-
cytes, neutrophils, B cells, dendritic cells, TAMs) in LUAD and LUSC. Presently, the
antitumour function of manifold cells has been extensively recognized, especially
CD8+ T cells [28], whose number reflects the immune system’s ability to kill tumour
cells to some extent. Moreover, CD8+ T cell density was positively correlated with the
efficacy of immune checkpoint inhibitors (ICIs) in NSCLC and melanoma [29, 30]. This finding may provide an early indication for the efficacy of immunotherapy for
NSCLC. Another significant part of our study is that diverse gene sets were positively corre-
lated with JAK1 expression levels. First, M1 macrophage-related gene markers, such as
IRF5 and NOS2, and the gene marker CD163 of M2 macrophages were strongly corre-
lated with JAK1 expression. These findings suggested that JAK1 may play a role in regu-
lating TAM polarization in the TME. Correlations between JAK1 expression and immune gene markers These results indicated
that LAYN may regulate DCs to play a major role in the TME. Regretfully, nearly all
of the gene markers of NK cells had no correlation with JAK1 expression levels. Fur-
thermore, we investigated the relationship between JAK1 expression and gene sets of
Tregs and T cell exhaustion. All gene sets suggested a positive correlation with JAK1 Shen et al. BMC Bioinformatics (2021) 22:471 Page 11 of 14 expression. Finally, immune checkpoints such as PD-1, CTLA4, LAG3 and TIM3 were
strongly connected with the level of JAK1 expression, which suggested that JAK1 may
play a role in immunotherapy for NSCLC. Further molecular biology experiment ver-
ification is needed. Discussionh Second, overexpression of JAK1 is associated with
a variety of T helper cells (Th1, Th2). This intense correlation may indicate that JAK1
regulates T cell function in the immune microenvironment of NSCLC. Third, our study
showed a significant correlation between Treg activation (FOXP3, STAT5B, TGFB1,
CCR8, CD25 in LUAD and LUSC) and induced T cell exhaustion (PD-1, CTLA-4, TIM-3
in LUAD and LUSC) and JAK1 overexpression. PD-1 (programmed death receptor 1) is
a vital immunosuppressive molecule expressed on the surface of T cells that regulates
the immune system and promotes tolerance by downregulating the immune system’s Shen et al. BMC Bioinformatics (2021) 22:471 Page 12 of 14 response to human cells and by suppressing the inflammatory activity of T cells [31]. Additionally, CTLA-4 and Tim-3 are expressed on regulatory T cells and exhausted T
cells as crucial receptor proteins, respectively [32, 33], and both are significantly posi-
tively correlated with JAK1 expression. These results suggest that JAK1 plays a potential
role in recruiting immune-infiltrating cells in the TME of NSCLC. i
Recent studies provide possible mechanisms which explains why JAK1 overexpression
correlates with immune infiltration and superior prognosis. Previous studies have shown
that JAK1 overexpression can lead to the activation of downstream interferon-stimulated
genes, which can eventually exert a range of antitumour effects [34, 35]. These include
increased antigen presentation by inducing proteasome subunits, activating transport-
ers associated with antigen processing (TAP), stimulating major histocompatibility com-
plex (MHC) molecules to be involved in antigen recognition and promoting chemokine
production to exploit a first-hand antitumour role [36]. Remarkably, numerous studies
have revealed that loss-of-function JAK1 mutations are insinuative of immune evasion
[11, 37, 38]. Research by Shin et al. [35] showed that JAK1 mutations could induce pri-
mary resistance to PD-1 inhibitors in melanoma and colon cancer patients. Rodig et al. [39] also indicated that loss of JAK1 caused perinatal death in mice. Luo et al. [40] have
shown that the response of melanoma to PD-L1 inhibitor immunotherapy requires JAK1
signaling, which may be related to its potentiated IFN-γ response in vivo and in vitro. Besides, researchers also point out that human melanoma cell lines are insensitive to
interferon (IFN)-induced antitumor effects after JAK1/2 knockout [41]. Consequently,
JAK1 may regulate immune-related pathways that affect the prognosis and immune infil-
trates of NSCLC. Concrete mechanisms have yet to be explored. However, the shortcomings of our descriptive study should be noted. Discussionh First, the
sequencing data and tumour tissue chips are based on a variety of platforms and data-
bases, and systematic errors and bias are inevitable. Second, our study analysed only
JAK1 expression and immune cell infiltration using a variety of databases, which still
needs to be verified by specific in vitro experiments. Finally, the precise regulatory path-
way of JAK1 in the TME of NSCLC still needs to be further explored. In summary, the elevated expression of JAK1 is associated with superior prognosis and
abundant immune cell infiltration in NSCLC. These findings may lay the foundation for
immunotherapy for NSCLC. pp
y
The online version contains supplementary material available at https://doi.org/10.1186/s12859-021-04379-y. Additional file 1: Table S1. Association between JAK1 expression and prognosis with different clinicopathological
features of NSCLC by Kaplan-Meier plotter (specific survival data). Fig. S1. Correlation between JAK1 expression level
and immune cell infiltration in LUSC from the TISIDB web portal (501 samples). (A-H) JAK1 expression had no signifi-
cant correlation with infiltrating levels of Act_CD4 and was significantly positively correlated with infiltrating levels of
Act_DCs, iDCs, neutrophils, NK cells, pDCs, Tcm_CD4 and Tem_CD8. LUSC, lung squamous cell carcinoma; Act_CD4,
activated CD4 T cells; Act_DCs, activated dendritic cells; iDCs, immature dendritic cells; NK cells, natural killer cells;
pDCs, plasmacytoid dendritic cells; Tcm_CD4, central memory CD4 cells; Tem_CD8, effector memory CD8 cells. A P
value less than 0.05 indicated statistical significance. Supplementary Information y
The online version contains supplementary material available at https://doi.org/10.1186/s12859-021-04379-y. Acknowledgements
Not applicable. Acknowledgements
Not applicable. Page 13 of 14 Shen et al. BMC Bioinformatics (2021) 22:471 Shen et al. BMC Bioinformatics Availability of data and materials All data generated or analysed during this study are included in this article. Funding g
This work was supported by Anhui Provincial Special Project of Central Government Guiding Local Science and Technol-
ogy Development of China (Grant No. 201907d07050001); Funding of “Peak” Training Program for Scientific Research of
Yijishan Hospital, Wannan Medical College (Grant No. GF2019J03). Authors’ contributions KS and YW wrote the main manuscript text. WL, TL and YS mainly involved in the design of articles and financial support. XJ, ZL, PZ, XW, and MF help with the making of charts and diagrams for the article. All authors read and approved the
final manuscript. Author details
1 1 Department of Critical Care Medicine, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wan-
nan Medical College), Wuhu 241000, China. 2 Department of Respiratory Medicine, The First Affiliated Hospital of Wannan
Medical College (Yijishan Hospital of Wannan Medical College), Wuhu 241000, China. 3 Department of Respiratory
Medicine, Jinling Hospital, Nanjing University School of Medicine, Nanjing 210002, China. 4 Department of Radiology
Medicine, The No. 2 People’s Hospital, Hefei 230000, China. Received: 24 March 2021 Accepted: 17 September 2021 Received: 24 March 2021 Accepted: 17 September 2021 Received: 24 March 2021 Accepted: 17 September 2021 References 1. William CB, Caroline C, Timothy RC, et al. Lung cancer incidence and mortality with extended follow-up in the 1. William CB, Caroline C, Timothy RC, et al. Lung cancer incidence and
national lung screening trial. J Thorac Oncol. 2019;14(10):1732–42. . a
CB, Ca o
e C,
ot y C, et a . u g ca ce
c de ce a d
o ta ty
t e te de
national lung screening trial. J Thorac Oncol. 2019;14(10):1732–42. g
g
2. Shen K, Cui J, Wei Y, et al. Effectiveness and safety of PD-1/PD-L1 or CTLA4 inhibitors combined with chemotheri 2. Shen K, Cui J, Wei Y, et al. Effectiveness and safety of PD-1/PD-L1 or CTLA4 inhibitors combined w
as a first-line treatment for lung cancer: a meta-analysis. J Thorac Dis. 2018;10(12):6636–52. s a first-line treatment for lung cancer: a meta-analysis. J Thorac Dis i
g
y
3. Camidge DR, Doebele RC, Kerr KM. Comparing and contrasting predictive biomarkers for immunotherapy and
targeted therap of NSCLC Nat Re Clin Oncol 2019 16(6) 341 55 i
3. Camidge DR, Doebele RC, Kerr KM. Comparing and contrasting predictive biomarkers for immunotherapy and Camidge DR, Doebele RC, Kerr KM. Comparing and contrasting pr 3. Camidge DR, Doebele RC, Kerr KM. Comparing and contrasting predictive biomarkers f targeted therapy of NSCLC. Nat Rev Clin Oncol. 2019;16(6):341–55. 4. Soukalas N, Kiakou M, Tsapakidis K, et al. PD-1 and PD-L1 as immunotherapy targets and biomarkers in non-small
cell lung cancer. J BUON. 2019;24(3):883–8. 4. Soukalas N, Kiakou M, Tsapakidis K, et al. PD-1 and PD-L1 as immunotherapy targets and biomarkers in non-small
cell lung cancer. J BUON. 2019;24(3):883–8. 5. Khan MAW, Ologun G, Arora R, et al. Gut microbiome modulates response to cancer immunotherapy. Dig Dis Sci. 2020;65(3):885–96. ;
( )
6. Lei X, Lei Y, Li JK, et al. Immune cells within the tumor microenvironment: biological functions and roles in cancer
immunotherapy. Cancer Lett. 2020;470:126–33. 6. Lei X, Lei Y, Li JK, et al. Immune cells within the tumor microenvironment: biological functions and roles in cancer
immunotherapy. Cancer Lett. 2020;470:126–33. 7. De Palma M, Biziato D, Petrova TV. Microenvironmental regulation of tumour angiogenesis. Nat Rev Cancer. 2017;17(8):457–74. 8. Li X, Yao W, Yuan Y, et al. Targeting of tumour-infiltrating macrophages via CCL2/CCR2 signalling as a therapeutic
strategy against hepatocellular carcinoma. Gut. 2017;66(1):157–67. strategy against hepatocellular carcinoma. Gut. 2017;66(1):157– gy g
p
9. Competing interests The authors declare no competing financial interests. Ethics approval and consent to participate
Not applicable. Ethics approval and consent to participate
Not applicable. References Nucleic Acids Res. 2017;45(W1):W98–102. analyses. Nucleic Acids Res. 2017;45(W1):W98–102. y
21. Lánczky A, Nagy Á, Bottai G, et al. miRpower: a web-tool to validate survival-associated miRNAs utilizing expression
data from 2178 breast cancer patients. Breast Cancer Res Treat. 2016;160(3):439–46. 22. Mizuno H, Kitada K, Nakai K, et al. PrognoScan: a new database for meta-analysis of the prognostic value of genes. BMC Med Genom. 2009;2:18. ;
23. Oja AE, Piet B, van der Zwan D, et al. Functional heterogeneity of CD4+ tumor-infiltrating lymphocytes with a resi-
dent memory phenotype in NSCLC. Front Immunol. 2018;9:2654. y p
yp
24. Castaneda CA, Castillo M, Aliaga K, et al. Level of tumor-infiltrating lymphocytes and density of infiltrating immune
cells in different malignancies Biomark Med 2019 13(17) 1481 91 y
y
24. Castaneda CA, Castillo M, Aliaga K, et al. Level of tumor-infiltrating lym
cells in different malignancies. Biomark Med. 2019;13(17):1481–91. 24. Castaneda CA, Castillo M, Aliaga K, et al. Level of tumor-infiltrating lymphocytes and density of infiltrating
cells in different malignancies. Biomark Med. 2019;13(17):1481–91. cells in different malignancies. Biomark Med. 2019;13(17):1481 f
g
25. Danziger O, Pupko T, Bacharach E, Ehrlich M. Interleukin-6 and interferon-α signaling via JAK1-STAT differentially f
g
25. Danziger O, Pupko T, Bacharach E, Ehrlich M. Interleukin-6 and interferon-α signaling via JAK1-STAT differentially
regulate oncolytic versus cytoprotective antiviral states. Front Immunol. 2018;9:94. 25. Danziger O, Pupko T, Bacharach E, Ehrlich M. Interleukin-6 and interferon-α signaling via JAK1-STAT
regulate oncolytic versus cytoprotective antiviral states. Front Immunol. 2018;9:94. g
,
p
,
,
g
gf
regulate oncolytic versus cytoprotective antiviral states. Front Immunol. 2018;9:94. regulate oncolytic versus cytoprotective antiviral states. Front Immunol. 2018;9:94. g
y
y
p
;
26. Stark GR, Darnell JE Jr. The JAK-STAT pathway at twenty. Immunity. 2012;36(4):503–14. 26. Stark GR, Darnell JE Jr. The JAK-STAT pathway at twenty. Immu 27. Osipov A, Saung MT, Zheng L, Murphy AG. Small molecule immunomodulation: the tumor microenvironment and
overcoming immune escape. J Immunother Cancer. 2019;7(1):224. overcoming immune escape. J Immunother Cancer. 2019;7(1):2 28. Tumeh PC, Harview CL, Yearley JH, et al. PD-1 blockade induces responses by inhibiting adaptive immune resist-
ance. Nature. 2014;515(7528):568–71. 29. Jansen CS, Prokhnevska N, Master VA, et al. An intra-tumoral niche maintains and differentiates stem-like CD8 T cells. Nature. 2019;576(7787):465–70. 30. Althammer S, Tan TH, Spitzmüller A, et al. Automated image analysis of NSCLC biopsies to predict response to anti-
PD-L1 therapy. J Immunother Cancer. 2019;7(1):121. 31. References Kleppe M, Kwak M, Koppikar P, et al. JAK-STAT pathway activation in malignant and nonmalignant cells contributes
to MPN pathogenesis and therapeutic response. Cancer Discov. 2015;5(3):316–31. 9. Kleppe M, Kwak M, Koppikar P, et al. JAK-STAT pathway activation in malignant and nonmalig
to MPN pathogenesis and therapeutic response. Cancer Discov. 2015;5(3):316–31. 10. Chen B, Lai J, Dai D, et al. JAK1 as a prognostic marker and its correlation with immune infiltrates in breast cance
Aging (Albany NY). 2019;11(23):11124–35. 10. Chen B, Lai J, Dai D, et al. JAK1 as a prognostic marker and its correlation with immune infiltrates in breast cancer. Aging (Albany NY). 2019;11(23):11124–35. y
11. Witalisz-Siepracka A, Klein K, Prinz D, et al. Loss of JAK1 drives innate immune deficiency. Front Immunol. 2019;9:3108. y
11. Witalisz-Siepracka A, Klein K, Prinz D, et al. Loss of JAK1 drives innate immune deficiency. Front Immunol. 2019;9:3108. 12. Wehde BL, Rädler PD, Shrestha H, et al. Janus kinase 1 plays a critical role in mammary cancer progression. Cell Rep. 2018;25(8):2192-2207.e5. 12. Wehde BL, Rädler PD, Shrestha H, et al. Janus kinase 1 plays a critical role in mammary cancer progression. Cell R
2018;25(8):2192-2207.e5. 13. Chen W, Wu G, Zhu Y, et al. HOXA10 deteriorates gastric cancer through activating JAK1/STAT3 signaling pathway. Cancer Manag Res. 2019;11:6625–35. 14. Orpaczy E, Tripolt S, Hoelbl-Kovacic A, et al. Aggressive B-cell lymphomas in patients with myelofibrosis receiving
JAK1/2 inhibitor therapy. Blood. 2018;132(7):694–706. 15. Mohrherr J, Haber M, Breitenecker K, et al. JAK-STAT inhibition impairs K-RAS-driven lung adenocarcinoma progres-
sion. Int J Cancer. 2019;145(12):3376–88. 16. Zhang N, Li F, Gao J, et al. Osteopontin accelerates the development and metastasis of bladder cancer via activating
JAK1/STAT1 pathway. Genes Genom. 2020;42(4):467–75. JAK1/STAT1 pathway. Genes Genom. 2020;42(4):467–75. p
y
17. Wen W, Liang W, Wu J, et al. Targeting JAK1/STAT3 signaling suppresses tumor progression and metastasis in a
peritoneal model of human ovarian cancer. Mol Cancer Ther. 2014;13(12):3037–48. Page 14 of 14 Shen et al. BMC Bioinformatics (2021) 22:471 18. Li T, Fan J, Wang B, et al. TIMER: a web server for comprehensive analysis of tumor-infiltrating immune cells. Cancer
Res. 2017;77(21):e108–10. 19. Ru B, Wong CN, Tong Y, et al. TISIDB: an integrated repository portal for tumor-immune system interactions. Bioinfor-
matics. 2019;35(20):4200–2. ;
(
)
20. Tang Z, Li C, Kang B, et al. GEPIA: a web server for cancer and normal gene expression profiling and interactive
analyses. References Rodig SJ, Meraz MA, White JM, et al. Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles
of the Jaks in cytokine-induced biologic responses. Cell. 1998;93(3):373–83. y
40. Luo N, Formisano L, Gonzalez-Ericsson PI, et al. Melanoma response to anti-PD-L1 immunotherapy requires JAK1
signaling, but not JAK2. Oncoimmunology. 2018;7(6):e1438106. y
40. Luo N, Formisano L, Gonzalez-Ericsson PI, et al. Melanoma res 40. Luo N, Formisano L, Gonzalez-Ericsson PI, et al. Melanoma response to anti-PD-L1 immunotherapy requires JAK1
signaling, but not JAK2. Oncoimmunology. 2018;7(6):e1438106. 40. Luo N, Formisano L, Gonzalez-Ericsson PI, et al. Melanoma response to anti-PD-
signaling, but not JAK2. Oncoimmunology. 2018;7(6):e1438106. g
g
gy
41. Torrejon DY, Abril-Rodriguez G, Champhekar AS, et al. Overcoming genetically based resistance mechanisms to
PD-1 blockade. Cancer Discov. 2020;10(8):1140–57. 41. Torrejon DY, Abril-Rodriguez G, Champhekar AS, et al. Overcoming genetically based resistance mechanisms to
PD-1 blockade. Cancer Discov. 2020;10(8):1140–57. References Wang L, Ma Q, Yao R, et al. Current status and development of anti-PD-1/PD-L1 immunotherapy for lung cancer. Int
Immunopharmacol. 2020;79:106088. 32. Formenti SC, Rudqvist NP, Golden E, et al. Radiotherapy induces responses of lung cancer to CTLA-4 blockade. Nat
Med. 2018;24(12):1845–51. 33. Datar I, Sanmamed MF, Wang J, et al. Expression analysis and significance of PD-1, LAG-3, and TIM-3 in human
non-small cell lung cancer using spatially resolved and multiparametric single-cell analysis. Clin Cancer Res. 2019;25(15):4663–73. 33. Datar I, Sanmamed MF, Wang J, et al. Expression analysis and significance of PD-1, LAG-3, and TIM-3 in human
non-small cell lung cancer using spatially resolved and multiparametric single-cell analysis. Clin Cancer Res. 34. Kaleviste E, Saare M, Leahy TR, et al. Interferon signature in patients with STAT1 gain-of-function mutation is epige-
netically determined. Eur J Immunol. 2019;49(5):790–800. y
35. Shin DS, Zaretsky JM, Escuin-Ordinas H, et al. Primary resistance to PD-1 blockade mediated by JAK1/2 mutations. Cancer Discov. 2017;7(2):188–201. 36. Bach EA, Aguet M, Schreiber RD. The IFN gamma receptor: a paradigm for cytokine receptor signaling. Annu Rev
Immunol. 1997;15:563–91. ;
37. Albacker LA, Wu J, Smith P, et al. Loss of function JAK1 mutations occur at high frequency in cancers with microsat- 37. Albacker LA, Wu J, Smith P, et al. Loss of function JAK1 mutations occur at high frequency in cancers with microsat
ellite instability and are suggestive of immune evasion PLoS ONE 2017;12(11):e0176181 37. Albacker LA, Wu J, Smith P, et al. Loss of function JAK1 mutations occur at high frequency in
ellite instability and are suggestive of immune evasion. PLoS ONE. 2017;12(11):e0176181. 37. Albacker LA, Wu J, Smith P, et al. Loss of function JAK1 mutations occur at high frequency in cancers with microsat 37. Albacker LA, Wu J, Smith P, et al. Loss of function JAK1 mutations occur at high frequency
ellite instability and are suggestive of immune evasion. PLoS ONE. 2017;12(11):e0176181. y
38. Zaretsky JM, Garcia-Diaz A, Shin DS, et al. Mutations associated with acquired resistance to PD-1 blockade in mela-
noma. N Engl J Med. 2016;375:819–29. 38. Zaretsky JM, Garcia-Diaz A, Shin DS, et al. Mutations associated with acquired resistance to PD-1 blockade in mela-
noma. N Engl J Med. 2016;375:819–29. g
39. Rodig SJ, Meraz MA, White JM, et al. Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles
of the Jaks in cytokine-induced biologic responses. Cell. 1998;93(3):373–83. g
39. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from: •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from:
|
https://openalex.org/W2120188690
|
https://publikationen.bibliothek.kit.edu/1000065774/7300679
|
English
| null |
Aerosol microphysics simulations of the Mt.~Pinatubo eruption with the UM-UKCA composition-climate model
|
Atmospheric chemistry and physics
| 2,014
|
cc-by
| 22,144
|
Aerosol microphysics simulations of the Mt. Pinatubo eruption with
the UM-UKCA composition-climate model S. S. Dhomse1, K. M. Emmerson2, G. W. Mann1,3, N. Bellouin4, K. S. Carslaw1, M. P. Chipperfield1, R. Hommel5,*,
N. L. Abraham3,5, P. Telford3,5, P. Braesicke3,5,**, M. Dalvi3,6, C. E. Johnson6, F. O’Connor6, O. Morgenstern7,
J. A. Pyle3,5, T. Deshler8, J. M. Zawodny9, and L. W. Thomason9 4Department of Meteorology, University of Reading, Reading, UK 5Department of Chemistry, University of Cambridge, Cambridge, UK
6 7National Institute of Water and Atmospheric Research (NIWA), Lauder, New Zealand
8University of Wyoming, Wyoming, USA 7National Institute of Water and Atmospheric Research (NIWA), Lauder, New Zealand
8University of Wyoming, Wyoming, USA 9NASA Langley Research Center, Hampton, Virginia, USA **now at: IMK-ASF Karlsruhe Institute of Technology, Karlsruhe, Germany **now at: IMK-ASF Karlsruhe Institute of Technology, Karlsruhe, Germany Correspondence to: S. S. Dhomse (s.s.dhomse@leeds.ac.uk) Received: 15 December 2013 – Published in Atmos. Chem. Phys. Discuss.: 28 January 2014
Revised: 16 September 2014 – Accepted: 16 September 2014 – Published: 24 October 2014 Received: 15 December 2013 – Published in Atmos. Chem. Phys. Discuss.: 28 January 2014
Revised: 16 September 2014 – Accepted: 16 September 2014 – Published: 24 October 2014 Abstract. We use a stratosphere–troposphere composition–
climate model with interactive sulfur chemistry and aerosol
microphysics, to investigate the effect of the 1991 Mount
Pinatubo eruption on stratospheric aerosol properties. Satel-
lite measurements indicate that shortly after the eruption,
between 14 and 23 Tg of SO2 (7 to 11.5 Tg of sulfur) was
present in the tropical stratosphere. Best estimates of the
peak global stratospheric aerosol burden are in the range 19
to 26 Tg, or 3.7 to 6.7 Tg of sulfur assuming a composition
of between 59 and 77 % H2SO4. In light of this large uncer-
tainty range, we performed two main simulations with 10 and
20 Tg of SO2 injected into the tropical lower stratosphere. Simulated stratospheric aerosol properties through the 1991
to 1995 period are compared against a range of available
satellite and in situ measurements. Stratospheric aerosol opti-
cal depth (sAOD) and effective radius from both simulations
show good qualitative agreement with the observations, with
the timing of peak sAOD and decay timescale matching well
with the observations in the tropics and mid-latitudes. Aerosol microphysics simulations of the Mt. Pinatubo eruption with
the UM-UKCA composition-climate model How-
ever, injecting 20 Tg gives a factor of 2 too high stratospheric
aerosol mass burden compared to the satellite data, with con- sequent strong high biases in simulated sAOD and surface
area density, with the 10 Tg injection in much better agree-
ment. Our model cannot explain the large fraction of the in-
jected sulfur that the satellite-derived SO2 and aerosol bur-
dens indicate was removed within the first few months after
the eruption. We suggest that either there is an additional al-
ternative loss pathway for the SO2 not included in our model
(e.g. via accommodation into ash or ice in the volcanic cloud)
or that a larger proportion of the injected sulfur was removed
via cross-tropopause transport than in our simulations. We also critically evaluate the simulated evolution of the
particle size distribution, comparing in detail to balloon-
borne optical particle counter (OPC) measurements from
Laramie, Wyoming, USA (41◦N). Overall, the model cap-
tures remarkably well the complex variations in particle con-
centration profiles across the different OPC size channels. However, for the 19 to 27 km injection height-range used
here, both runs have a modest high bias in the lowermost
stratosphere for the finest particles (radii less than 250 nm),
and the decay timescale is longer in the model for these parti-
cles, with a much later return to background conditions. Also, Atmos. Chem. Phys., 14, 11221–11246, 2014
www.atmos-chem-phys.net/14/11221/2014/
doi:10.5194/acp-14-11221-2014
© Author(s) 2014. CC Attribution 3.0 License.
Aerosol microphysics simulations of the Mt. Pinatubo eruption with
the UM-UKCA composition-climate model
S. S. Dhomse1, K. M. Emmerson2, G. W. Mann1,3, N. Bellouin4, K. S. Carslaw1, M. P. Chipperfield1, R. Hommel5,*,
N. L. Abraham3,5, P. Telford3,5, P. Braesicke3,5,**, M. Dalvi3,6, C. E. Johnson6, F. O’Connor6, O. Morgenstern7,
J. A. Pyle3,5, T. Deshler8, J. M. Zawodny9, and L. W. Thomason9
1School of Earth and Environment, University of Leeds LS2 9JT, UK
2CSIRO Marine and Atmospheric Research, Aspendale, Victoria 3195, Australia
3National Centre for Atmospheric Science (NCAS-Climate), UK
4Department of Meteorology, University of Reading, Reading, UK
5Department of Chemistry, University of Cambridge, Cambridge, UK
6Met Office, Exeter, UK
7National Institute of Water and Atmospheric Research (NIWA), Lauder, New Zealand
8University of Wyoming, Wyoming, USA
9NASA Langley Research Center, Hampton, Virginia, USA
*now at: IUP, University of Bremen, Bremen, Germany
**now at: IMK-ASF Karlsruhe Institute of Technology, Karlsruhe, Germany
Correspondence to: S. S. Dhomse (s.s.dhomse@leeds.ac.uk)
Received: 15 December 2013 – Published in Atmos. Chem. Phys. Discuss.: 28 January 2014
Revised: 16 September 2014 – Accepted: 16 September 2014 – Published: 24 October 2014 Atmos. Chem. Phys., 14, 11221–11246, 2014
www.atmos-chem-phys.net/14/11221/2014/
doi:10.5194/acp-14-11221-2014
© Author(s) 2014. CC Attribution 3.0 License. 1
Introduction Volcanic eruptions can have significant impacts on atmo-
spheric composition and climate (e.g. McCormick et al.,
1995; Robock, 2000). Powerful explosive eruptions can in-
ject large amounts of SO2, ash, water vapour and various
other chemical species directly into the stratosphere. Vol-
canic SO2 injected into the stratosphere is chemically con-
verted to sulfuric acid vapour over a timescale of days
to months, causing substantial new particle formation and
aerosol growth by condensation. Volcanic enhancements of
the stratospheric aerosol can be long lasting, with optically
active particle concentrations remaining substantially en-
hanced for several years in the case of tropical eruptions
(Deshler et al., 2003). The perturbed stratospheric aerosol al-
ters the Earth’s radiative balance with increased albedo via
enhanced back-scattering of solar radiation, cooling the sur-
face and increased absorption of terrestrial long-wave radi-
ation, warming the stratosphere (Labitzke and McCormick,
1992). The relative magnitude of these short-wave and long-
wave radiative effects are strongly influenced by the aerosol
particle size distribution (Lacis et al., 1992; Hansen et al.,
1992). We use the stratosphere–troposphere composition-climate
model UM-UKCA (Unified Model – UK Chemistry and
Aerosol) to simulate stratospheric aerosol interactively. The
model includes the GLOMAP-mode aerosol scheme and
calculates aerosol optical properties online and consistently
with the 3-D evolution of the particle size distribution, as
driven by the underlying microphysical processes. We use
the 1991 Mt Pinatubo eruption as a test case to examine
simulated aerosol properties comparing to a range of satel-
lite and in situ observations covering the background, vol-
canic perturbation and decay periods. Pinatubo erupted in
the Philippines (15.1◦N, 120.4◦E) on 15 June 1991 and
was the largest tropical eruption since Krakatoa 1883. Based
on column SO2 mass loadings derived from ultraviolet ra-
diation measurements from the Total Ozone Monitoring
Spectrometer (TOMS) instrument, Bluth et al. (1992) esti-
mated that the eruption injected approximately 20 Tg into
the tropical stratosphere. Guo et al. (2004a) re-evaluated the
post-Pinatubo TOMS data, and also analysed measurements
from the Television Infra-red Observation Satellite Vertical
Sounder (TOVS), finding total SO2 released to be in the
range 14 to 23 Tg (7 to 11.5 Tg of sulfur). The long-wave radiative heating induced by the thicker
aerosol layer also modifies the stratospheric circulation (e.g. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11222 whereas the 10 Tg run compared best to the satellite measure-
ments, a significant low bias is apparent in the coarser size
channels in the volcanically perturbed lower stratosphere. Overall, our results suggest that, with appropriate calibration,
aerosol microphysics models are capable of capturing the
observed variation in particle size distribution in the strato-
sphere across both volcanically perturbed and quiescent con-
ditions. Furthermore, additional sensitivity simulations sug-
gest that predictions with the models are robust to uncertain-
ties in sub-grid particle formation and nucleation rates in the
stratosphere. spheric aerosol, stratospheric aerosols are treated separately. Some models impose volcanic forcings and heating rates
(e.g. Stenchikov et al., 1998) or base these on prescribed
time-varying aerosol optical depth climatologies (such as
Sato et al., 1993). None of the CMIP5 climate models are
able to capture the complex dynamical changes associated
with large tropical eruptions (Driscoll et al., 2012). There
is now an established group of composition–climate models
(CCMs) which simulate stratospheric chemistry with inter-
active ozone radiative effects (e.g. SPARC, 2010), but few
include prognostic treatment of stratospheric aerosol. Even
relatively modest changes in stratospheric aerosol can exert
a significant radiative forcing (e.g. Solomon et al., 2011) and
expected future changes in stratospheric circulation further
motivate the need for interactive stratospheric aerosol in cli-
mate models. spheric aerosol, stratospheric aerosols are treated separately. Some models impose volcanic forcings and heating rates
(e.g. Stenchikov et al., 1998) or base these on prescribed
time-varying aerosol optical depth climatologies (such as
Sato et al., 1993). None of the CMIP5 climate models are
able to capture the complex dynamical changes associated
with large tropical eruptions (Driscoll et al., 2012). There
is now an established group of composition–climate models
(CCMs) which simulate stratospheric chemistry with inter-
active ozone radiative effects (e.g. SPARC, 2010), but few
include prognostic treatment of stratospheric aerosol. Even
relatively modest changes in stratospheric aerosol can exert
a significant radiative forcing (e.g. Solomon et al., 2011) and
expected future changes in stratospheric circulation further
motivate the need for interactive stratospheric aerosol in cli-
mate models. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11223 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM a sectional aerosol microphysics module with injection alti-
tude between 15.5 and 27 km, English et al. (2013) achieved
good agreement with SAGE- and AVHRR-observed sAOD
in NH mid-latitudes for the first 12 months after the eruption
but too rapid decay in sAOD through later months. They
also compared model aerosol effective radius (Reff) evolution
against observations from SAGE II and in situ measurements
(for e.g. Russell et al., 1996; Bauman et al., 2003), finding
that peak values in the model NH tropical stratosphere oc-
curred earlier than in the observations. Some of these model–
observation biases in earlier studies may be linked with the
transport-related issues (e.g. the lack of a quasi-biennial os-
cillation, QBO) in the underlying GCM, whereas some may
be linked to the simplified treatment of the particle size distri-
bution. Other causes such as interactions with ash, or missing
minor eruptions such as Mount Hudson in Chile (Septem-
ber 1991) have also been suggested. into a peak aerosol sulfur burden uncertainty range of 3.7
to 6.7 Tg of sulfur. Taken together, these findings suggest
that a large proportion of the sulfur was removed from the
stratosphere within the first few months after the eruption,
with potential loss pathways involving sedimentation, cross-
tropopause transport out of the stratosphere (Deshler, 2008)
or enhanced removal via interactions with ash or ice in the
Pinatubo cloud (Guo et al., 2004b). Monthly balloon sound-
ings of total and size-resolved particle concentrations carried
out at Laramie, Wyoming (e.g. Deshler, 1994) showed that
although substantially enhanced particle concentrations were
detected in the lowermost stratosphere by mid-July, the main
part of the volcanic plume was only transported to NH mid-
latitudes several months later. There have been many previous global modelling stud-
ies to investigate the evolution of the stratospheric aerosol
following the Pinatubo eruption. However, most have used
aerosol schemes that simulate only the evolution of aerosol
mass, prescribing a fixed particle size distribution for sedi-
mentation and radiative effects (e.g. Timmreck et al., 1999;
Oman et al., 2006; Aquila et al., 2012). However, size-
resolved stratospheric aerosol modules which include micro-
physical processes such as new particle formation, coagula-
tion and condensation have also been developed. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM The first
Pinatubo aerosol microphysics simulations were carried out
in 2-D models (Bekki and Pyle, 1994; Weisenstein et al.,
1997) with single-moment sectional schemes where mass in
numerous size bins is transported. More recently, several 3-
D general circulation models (GCMs) with aerosol micro-
physics schemes have also been developed, to predict sedi-
mentation and changes in radiative properties in conjunction
with an evolving stratospheric particle size distribution (e.g. Timmreck, 2001; Toohey et al., 2011; English et al., 2012,
2013). Although they have near-global spatial extent, satellite
measurements of sAOD and Reff constrain only integrated
stratospheric aerosol properties over the full particle size
range. Balloon-borne measurements (e.g. Deshler, 1994) en-
able a closer examination of the particle size distribution, but
are available at only a small number of sites. Here, we use both satellite and balloon-borne measure-
ments to evaluate the UM-UKCA simulated stratospheric
aerosol properties, and seek to provide a wider set of obser-
vational constraints for the models and to better understand
how the stratospheric aerosol layer was perturbed. In Sect. 2
we describe the model, including the experimental set-up
and the developments to the aerosol and chemistry schemes
which extend its applicability to both stratospheric and tro-
pospheric conditions. Section 3 describes the measurements
that are used to evaluate the model. Results and discussion
about potential causes of model–observation biases are pre-
sented in Sect. 4 and 5, respectively. Summary and major
conclusions are presented in Sect. 6. Although there have been this development of more so-
phisticated models, most of these studies evaluated their
simulations against a limited set of observational data sets,
primarily aerosol optical depths (AODs) derived using Ad-
vanced Very High Resolution Radiometer (AVHRR) and
SAGE II measurements. Using a mass-based prognostic
stratospheric aerosol module in a middle-atmosphere ver-
sion of the ECHAM4 climate model, Timmreck et al. (1999)
showed that the two distinct maxima in AOD apparent in
AVHRR- and SAGE-II-based stratospheric aerosol optical
depth (sAOD) could be simulated, but the model failed to
simulate the observed slow sAOD decay in the tropics af-
ter the peak. Similarly, Pitari and Mancini (2002) used a
GCM coupled to a global chemistry transport model with
interactive aerosol microphysics, and could simulate SH
sAOD reasonable well, but NH and tropical sAOD was bi-
ased low. Aquila et al. (2012) used a GCM with a mass-
based aerosol scheme radiatively coupled to the model dy-
namics. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM They found that simulated sAOD was higher than
both AVHRR and SAGE II during the first few months, but
showed very good agreement during the later phase. Using 1
Introduction Young et al., 1994), leading to indirect radiative effects via
dynamical changes in ozone and meridional transport, with
important implications for surface climate (Robock and Mao,
1992; Graf et al., 1993). Volcanically increased aerosol sur-
face area density (SAD) can also accelerate heterogeneous
chemistry perturbing stratospheric NOy species, halogens
and ozone (e.g. Solomon et al., 1996). Quantifying the net
impact from these direct and indirect radiative effects is very
important to better understand volcanic influences within the
historical climate records. g
g
g
Assuming a 50 % conversion of SO2 to sulfuric acid by
July and using an assumed size distribution and composition
to convert the Stratospheric Aerosol and Gas Experiment II
(SAGE II) satellite measurements of aerosol extinction, Mc-
Cormick and Veiga (1992) estimated that the total global
aerosol loading was increased by 20 to 30 Tg. Baran and Foot
(1994) used infrared satellite measurements from the High-
resolution Infrared Radiation Sounder (HIRS) instrument to
derive a time series of the global stratospheric aerosol mass
loading, finding a peak of 21 Tg in September 1991 with val-
ues in excess of 15 Tg persisting until November 1992 and
much earlier and steeper decay in the tropics than in Northern
Hemisphere (NH) mid-latitudes. Lambert et al. (1993) found
a peak aerosol loading of 19 to 26 Tg from the Improved
Stratospheric and Mesospheric Sounder (ISAMS) measure-
ments, which assuming the aerosol composition ranges from
59 to 77 % sulfuric acid (Grainger et al., 1993), translates There is an increasing recognition that having a good rep-
resentation of stratospheric processes is important for climate
projections (e.g. Scaife et al., 2012). However, whereas most
coupled atmosphere–ocean climate models (e.g. Jones et al.,
2011) that carried out historical integrations for CMIP5 (Tay-
lor et al., 2012) included a prognostic treatment of tropo- Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ 2.2
The aerosol microphysics module adapted
for the stratosphere In order to simulate stratospheric aerosol precursor gas
phase species, we have extended the existing UM-UKCA
stratospheric chemistry scheme to also include sulfur chem-
istry (see Sect. 2.1). The coupling to the GLOMAP-mode
aerosol microphysics module (Mann et al., 2010), and
its adaptation for stratospheric conditions, is described in
Sect. 2.2. Surface emissions of NOx, CO and HCHO are from
the RCP 4.5 scenario. Lower boundary conditions are ap-
plied for CH4, N2O, CFC-11 (CFCl3) and CFC-12 (CF2Cl2)
according to WMO (2011). Heterogeneous chemical reac-
tions use time-varying prescribed aerosol surface area den-
sity produced for the SPARC Assessment of the Strato-
spheric Aerosols Properties report (SPARC, 2006). We in-
clude surface and elevated emissions of anthropogenic SO2
from Lamarque et al. (2010) with also a 3-D source from
passively degassing volcanoes from Andres and Kasgnoc
(1998). DMS emissions are simulated interactively using
a seawater concentration climatology of Kettle and Andreae
(2000) with the sea–air exchange function of Liss and Mer-
livat (1986). For OCS, which has a tropospheric lifetime of
about 2 years (Montzka et al., 2007), we do not include an
emissions source, but instead apply a fixed lower boundary
condition of 550 pptm. The GLOMAP aerosol microphysics module was initially
developed as a component of the TOMCAT 3-D offline
Chemical Transport Model (Chipperfield, 2006) with both
2-moment sectional (Spracklen et al., 2005) and 2-moment
modal versions (Mann et al., 2010) available. The computa-
tionally faster modal scheme (GLOMAP-mode) was specif-
ically designed for longer integrations within UM-UKCA
and applies the same aerosol microphysics representations
as the sectional scheme but with the size distribution pa-
rameterised into seven log-normal modes, being similar in
framework to that used in ECHAM-HAM (e.g. Stier et al.,
2005). The GLOMAP-mode scheme produces aerosol prop-
erties in good agreement with the more sophisticated sec-
tional scheme under most tropospheric conditions (Mann
et al., 2012). Since this study investigates the evolution of the strato-
spheric aerosol layer after Pinatubo, we use only the four
soluble modes and treat only sulfate and sea salt components
– the latter included to give reasonable representation of tro-
pospheric aerosol optical properties. For this work, the model
approaches for water uptake, particle density, vapour conden-
sation and new particle formation have been adapted to be
applicable across stratospheric and tropospheric conditions. In the following sections, we briefly describe these updates. 2.2.1
Water uptake In the standard version of GLOMAP-mode described by
Mann et al. (2010), water uptake is calculated using ZSR
(Zdanovskii, 1948; Stokes and Robinson, 1966), which is not
applicable in stratosphere conditions. At pressures below 150
hPa we therefore instead use the expression of Carslaw et al. (1995) to provide the aerosol water content. At 225 K and
101 hPa, the composition of the solution is 74.5 % H2SO4
and 25.5 % water, approximating the 75 % weight fraction
assumed in some studies (e.g. Stenchikov et al., 1998; Oman
et al., 2006). 2.1
Stratospheric chemistry extended to include
the sulfur cycle The existing UM-UKCA stratospheric chemistry scheme
(Morgenstern et al., 2009) covers the oxidation of CH4 and
CO, with chlorine and bromine chemistry and their interac-
tion with HOx, NOx and Ox cycles including heterogeneous
reactions on polar stratospheric clouds (PSCs) and liquid sul-
fate aerosols (Chipperfield and Pyle, 1998). Here, we have
extended the scheme to also include a stratospheric aerosol
precursor chemistry scheme (Weisenstein et al., 1997) with
updates to reaction rates from Sander et al. (2006), see Ta-
ble 1. The added chemistry includes the steady background
source of SO2 from OCS, which principally maintains the
stratospheric aerosol during volcanically quiescent periods
(e.g. Carslaw and Kärcher, 2006). Also included are photol-
ysis reactions for H2SO4 and SO3, which occur above about
30 km and lead to a reservoir of SO2 building up during po-
lar winter, enabling new particle formation in the polar lower
stratosphere during spring (Mills et al., 2005). The chemistry
is integrated with the ASAD chemical integration package
(Carver et al., 1997) with the Newton–Raphson sparse matrix
solver from Wild et al. (2000). Photolysis rates are calculated
using the FAST-JX online photolysis (Neu et al., 2007) fol-
lowing the implementation described in Telford et al. (2013). The cross-section of H2SO4 is assumed analogous to the
cross-section of HCl (× 0.016) following the method of
Bekki and Pyle (1992). Aqueous sulfate production in (tropo-
spheric) liquid clouds is also passed to the GLOMAP module
for growth of accumulation and coarse soluble particles. 2.2.2
Particle density As composition of the aqueous sulfuric acid solution droplets
also affects their density, we modified GLOMAP-mode for
the stratosphere. For pressures lower than 150 hPa, density
values for each mode are replaced with values from a look-
up table based on the measurements of Martin et al. (2000)
as a function of the sulfuric acid weight-fraction. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM
2.2
The aerosol microphysics module adapted
for the stratosphere S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11224 2
Model description We use the stratospheric chemistry configuration of UM-
UKCA (Morgenstern et al., 2009) which here is an extended
configuration of the high-top version of the third-generation
Hadley Centre Global Environmental Model (HadGEM3)
(Hewitt et al., 2011) as used by Braesicke et al. (2013) and
Telford et al. (2013). Although UM-UKCA can be run with
fully coupled aerosol–chemistry dynamics with online ra-
diative effects from the simulated aerosol, O3, CH4, N2O
and other gases, here only the simulated O3 is radiatively
coupled. Sea-surface temperature and sea-ice fields are pre-
scribed from the AMIP time-varying data set (Hurrell et al.,
2008). The simulations are carried out at N48 horizontal res-
olution (2.5◦and 3.75◦in latitude and longitude) with 60 ver-
tical hybrid-height levels from the surface to 84 km. Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ 2.2.3
Condensation and vapour pressure of H2SO4 In all previous versions of the GLOMAP aerosol mod-
ule, gas-to-particle transfer of H2SO4 occurs assuming zero
vapour pressure, i.e. the transfer is represented as a conden-
sation process. Although this approach is entirely appropriate Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM
1
Table 1. Additional sulfur chemistry reactions and rates within UM-UKCA, W = Weisenstein et al. (1997), JPL = Sander et al. (
K03 = Kreidenweis et al. (2003). Rate
Reference
DMS + O(3P) →SO2 + 2CH3O2
1.3 × 10−11 exp(410/T )
W, JPL
DMS + OH →SO2 + CH3O2 + HCHO
1.2 × 10−11 exp(−260/T )
W
DMS + OH →SO2 + CH3O2
3.04 × 10−12 exp(350/T ) · (γ/1 + γ )
W
γ = 5.53 × 10−31 exp(7460/T ) × [O2]
DMS + NO3 →SO2 + HNO3 + CH3O2 + HCHO
1.9 × 10−13 exp(500/T )
W
OCS + O(3P) →CO + SO2
2.1 × 10−11 exp(−2200/T )
W, JPL
OCS + OH →CO2 + SO2
1.1 × 10−13 exp(−1200/T )
W, JPL
SO2 + OH + M →SO3 + HO2
k(T ) =
A
1+B × 0.6(1+(logB)2)−1
W
A = 3.0 × 10−31 × (300/T )3.3
B = A/1.5 × 10−12
SO2 + O3 →SO3
3.0 × 10−12 exp(−7000/T )
W, JPL
SO3 + H2O →H2SO4
8.5 × 10−41 exp(6540/T ) · [H2O]
JPL
SO2 + H2O2
aqueous
→
SO4
K03
OCS + hν →CO + SO2
Photolysis
W
H2SO4 + hν →SO3 + H2O
Photolysis
W
SO3 + hν →SO2 + O(3P)
Photolysis
W 11225 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Table 1. Additional sulfur chemistry reactions and rates within UM-UKCA, W = Weisenstein et al. (1997), JPL = Sander et al. (2006),
K03 = Kreidenweis et al. (2003). Table 1. Additional sulfur chemistry reactions and rates within UM-UKCA, W = Weisenstein et al. (1997), JPL = Sander et al. (2006),
K03 = Kreidenweis et al (2003) Table 1. Additional sulfur chemistry reactions and rates within UM-UKCA, W = Weisenstein et al. (1997), JPL = Sander et al. (2006),
K03 = Kreidenweis et al. (2003). nen and Kulmala (2002) to convert the cluster nucleation rate
from Vehkamäki et al. (2002) into an “apparent nucleation
rate” at 3 nm. The nucleation rate is set to zero in subsatu-
rated conditions. 2.2.5
Size distribution Balloon-borne optical particle counter and condensation nu-
cleus counter measurements in the mid-latitude stratosphere
in the 1990s (e.g. Deshler et al., 2003) suggest a bimodal
size distribution with the first mode at about 50–150 nm
radius with geometric standard deviation (σg) between 1.6
and 1.8 and a larger much narrower mode (σg ∼1.2) at
around 300–800 nm radius that is weak in volcanically qui-
escent conditions but much stronger (in number) following
the Pinatubo eruption (e.g. Carslaw and Kärcher, 2006). For
example Deshler et al. (2003) show that in March 1993
(21 months after the Pinatubo eruption), in the NH mid-
latitude lower stratosphere there was a 6 km layer (12–18 km)
with the number concentration of particles with radii larger
than 500 nm greater than 1 cm−3. Such coarse particles have
grown from their original size of around 1 nm due to coagu-
lation and gas-to-particle transfer of sulfuric acid. Modal mi-
crophysics schemes such as GLOMAP-mode represent this
condensational and coagulative growth, but must use a tech-
nique referred to as “mode-merging” (e.g. Binkowski and
Roselle, 2003) to transfer particles to adjacent larger modes
following strong growth. In the case of a large volcanic erup-
tion, the mode-merging may transfer particles first from the
nucleation mode to the soluble Aitken mode, and follow-
ing further growth up to the soluble accumulation mode and
then to the soluble coarse mode. In each case, when particles 2.2.3
Condensation and vapour pressure of H2SO4 in tropospheric conditions, above ∼25–30 km, the vapour
pressure of H2SO4 (pH2SO4) becomes significant as the tem-
perature increases in the stratosphere and above ∼35 km the
sulfuric acid droplets rapidly evaporate (Hamill et al., 1997;
Hommel et al., 2011). We therefore now calculate pH2SO4 online in the model
following Kulmala and Laaksonen (1990) and the conden-
sation rates are calculated consistently with the difference
between the vapour pressure and the gas phase partial pres-
sure. We also apply a simple approach to particle evaporation
whereby if the ambient gas phase H2SO4 partial pressure is
less than pH2SO4, the number concentration for all modes is
reduced at a fast decay rate of 50 % per condensation time
step, which corresponds to an e-folding timescale of around
17 min. 2.2.4
New particle formation Previous versions of GLOMAP (e.g. Mann et al., 2010)
formed new H2SO4–H2O particles based on the Kulmala
et al. (1998) parameterisation for binary homogeneous nu-
cleation. This is only applicable at temperatures in the range
233–298 K. Vehkamäki et al. (2002) suggested that condi-
tions for nucleation are also favourable at ∼200 K in the up-
per tropical troposphere and they updated the Kulmala et al. (1998) parameterisation to be applicable down to lower tem-
peratures and humidities. To allow GLOMAP-mode to be ap-
plied in both tropospheric and stratospheric conditions, we
have incorporated the Vehkamäki et al. (2002) parameteri-
sation, and used it within the recommended ranges of tem-
perature (190 to 305 K) and H2SO4 concentration (104 to
1011 cm−3). Note that we also use the expression of Kermi- Atmos. Chem. Phys., 14, 11221–11246, 2014 Atmos. Chem. Phys., 14, 11221–11246, 2014 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM are received from the adjacent smaller mode, the transferred
number and mass is added to that existing in the mode, with
the mean size reformulated reformulated consistently with
the σg value for the mode. The spin-up run started from zero aerosol and gas phase sul-
fur species, with other gases initialised from the UM-UKCA
REF-C1 integration from the SPARC Lifetimes of Strato-
spheric Ozone-Depleting Substances, Their Replacements,
and Related Species report (SPARC, 2013), representative of
1990 conditions. g
Kokkola et al. (2009) compared size distributions simu-
lated by a modal and three sectional schemes in a box model. While the four models agreed well in background strato-
spheric conditions, in volcanically perturbed conditions, the
size distributions were found to be better represented with
narrower mode widths. In particular, with the original coarse
mode σg of 2.0, they found the modal scheme overpre-
dicted the Reff compared to a reference sectional scheme
with a large number of bins. Niemeier et al. (2009) used an
improved version of the same modal microphysics scheme
whereby σg for the accumulation soluble mode was reduced
to 1.2 and the coarse mode was deactivated. To assess the robustness of the model to uncertainties in
particle formation processes, which are known to be consid-
erable uncertain in the stratosphere, we also carried out two
sensitivity simulations with are as run B_Control10, but we
switch off the sub-grid particle source (primary sulfate emis-
sion) from Pinatubo (D_noPrimary10) and reduce the nucle-
ation rate (E_ScaledStNuc10) in the stratosphere by a factor
of 2 (by multiplying it with 0.01). Here we are applying the modal GLOMAP scheme to vol-
canically perturbed stratospheric conditions, and also using
the same modes to represent tropospheric aerosol. In the tro-
posphere, the coarse soluble mode in GLOMAP-mode al-
most exclusively contains sea-salt, and the scheme follows
Wilson et al. (2001) and Vignati et al. (2004) in using a value
of 2.0 for σg in this mode, which are based on values given
in D’Almeida et al. (1991). 3
Measurements To evaluate the UM-UKCA simulations, we use measure-
ments from the SAGE II instrument (McCormick and Veiga,
1992), which was launched on the Earth Radiation Budget
Satellite (ERBS) in 1984. SAGE II was a seven-channel sun
photometer operated in solar occultation mode with a verti-
cal resolution of about 0.5 km. Spectral windows were cen-
tred at 385, 448, 453, 525, 600, 940 and 1020 nm. For eval-
uating the model stratospheric aerosol optical depth, we use
the gap-filled SAGE II (V6.2) product (Hamill et al., 2006)
produced for ASAP (Assessment of Stratospheric Aerosol
Properties SPARC, 2006). Simulated aerosol extinction is
compared against the recently updated version (V7.0) of
the SAGE II data (Damadeo et al., 2013). We also com-
pare to the SAGE-derived SAD product (Thomason et al.,
1997) that is obtained from http://www.sparc-climate.org/
data-center/data-access/asap/. Simulated SAD is also com-
pared against the recently available SAD data (Arfeuille
et al., 2013) which was created using SAGE II V7.0 data,
and is provided for the Chemistry Climate Model Initiative
(CCMI) simulations. Further evaluation of the post-Pinatubo
simulated sAOD evolution was carried out by comparing
to that measured by the Advanced Very High Resolution
Radiometer (AVHRR/2), which was onboard on the Na-
tional Oceanic and Atmospheric Administration (NOAA/11)
satellite. For details see http://www.nsof.class.noaa.gov/
release/data_available/avhrr/index.htm. The AVHRR instru-
ment measures the reflectance of the Earth in five spectral
bands centred around 0.6, 0.9, 3.5, 11 and 12 µm. To ensure the size distribution and vertical profile of the
simulated coarse sea-salt particles is retained as evaluated
in previous model versions (Mann et al., 2012), we retain
the σg value of 2.0 for the coarse soluble mode. However,
we now de-activate mode-merging between the accumulation
and coarse soluble modes, which allows the accumulation
soluble mode to continue to grow larger than 1 micron diam-
eter in strongly perturbed conditions. We also retain the σg
value of 1.4 for the soluble accumulation mode in GLOMAP-
mode, as reduced by Mann et al. (2012) from the value of
1.59 used in Mann et al. (2010), to better compare with size
distributions simulated by the sectional scheme and from ob-
servations. The σg=1.59 values for the nucleation and Aitken
modes are also retained. www.atmos-chem-phys.net/14/11221/2014/ 11226 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM
Table 2. Microphysical parameter settings used in model simulations. Run
SO2
Nucleation
Sub-grid primary
injection
sulphate emission
A_Control20
20 Tg (19–27 km)
Standard
Yes
B_Control10
10 Tg (19–27 km)
Standard
Yes
C_noPinatubo
No
Standard
Yes
D_noPrimary10
10 Tg (19–27 km)
Standard
No
E_ScaledStNuc10
10 Tg (19–27 km)
×0.01 in stratosphere
Yes coarse regions of the size spectrum. The OPC is a light
counter to derive integrated size distribution from measured
aerosol scattering in the forward direction. The standard
OPC design gives integral number concentrations larger than
150 nm and 250 nm radius, and has been used in balloon
sounding measurements of stratospheric aerosol since 1963
(Rosen, 1964), also giving important information about the
stratospheric aerosol changes induced by the 1963 Mt Agung
(Rosen, 1964), 1980 Mt St Helen’s (Hofmann and Rosen,
1982) and 1982 El Chichón (Hofmann and Rosen, 1984)
eruptions. Deshler et al. (1992) present the measurements
taken in July and August 1991, with most balloon flights
using this original two-channel OPC. An enhanced OPC, us-
ing an increased scattering angle, measured concentrations in
eight size channels for radii larger than 150 nm to around 10
microns. The eight-channel OPC had been developed shortly
before the eruption, and became the default measuring sys-
tem a few months after the eruption (Deshler et al., 1993). The measurement capabilities were later further enhanced to
measure up to 12 size ranges (see Deshler et al., 2003). ing the evolution of the background as well as volcanically
enhanced stratospheric aerosol layer. Stronger BD circula-
tion leads to more rapid transport of air masses (and chem-
ical species) from the tropics to high latitudes (e.g, We-
ber et al. 2003; Dhomse et al. 2006). This circulation also
affects aerosol removal from the stratosphere (e.g. Desh-
ler, 2008) via stratosphere–troposphere exchange (STE, e.g. Holton et al. 1995). However, the strength of the BD circu-
lation is also coupled with the phases of the QBO via the
Holton–Tan mechanism (Holton and Tan, 1980). Using satellite observations, Trepte and Hitchman (1992)
showed the importance of the QBO phase in determining the
initial dispersion of the Pinatubo plume. 4
Results Stratospheric aerosol sizes and concentrations are influenced
by dynamical, chemical and microphysical processes. For
example, background aerosol are formed by homogeneous
nucleation of H2SO4 and H2O, with H2SO4 concentrations
affected by oxidation of OCS and SO2. Microphysical pro-
cesses such as nucleation, condensational growth, coagu-
lation and sedimentation along with large-scale poleward
transport timescales affect stratospheric lifetimes of these
aerosol. To ensure the model is fully evaluated, it is neces-
sary to evaluate the model against a range of aerosol proper-
ties, but it is also important to assess stratospheric circulation
in the model and assess the evolution of key precursor gases
which influence the aerosol. A common metric used to assess stratospheric transport in
CCMs (e.g. Strahan et al., 2011) is the mean stratospheric
age-of-air. Figure 1c shows the latitude and altitude distri-
bution of the model zonal-mean age-of-air (for 1991–2000)
and Fig. 1d compares the model age of air at 50 hPa against
that derived from aircraft observations of the long-lived tro-
pospheric source gases SF6 and CO2 (Hall et al., 1999). The
values from other CCMs participating in the recent SPARC
Lifetime Assessment (e.g. Chipperfield et al., 2014) are also
shown for reference. In the tropics, the model age-of-air
agrees well with the observations, but at mid- and high lat-
itudes there is a low bias compared to the observations; up
to 1 yr too young air at high latitudes. The low bias in mid-
latitude age-of-air indicates that the model may have too
rapid meridional poleward transport and/or stronger STE. In S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM For the simulations
presented here, the model is initialised such that the lower
stratospheric winds are in the easterly phase of the QBO at
the time of the eruption, as observed. Figure 1a shows the
time evolution of the model monthly and zonal-mean zonal
wind in the tropics (15◦S–15◦N) against those from the ERA
interim re-analysis from 1990 until 1995 (Fig. 1b, Dee et al.,
2011). As in ERA interim, the model begins an easterly QBO
phase in mid-1991, although the model easterlies are weaker
than in ERA-interim in the lower stratosphere for the first
6 months after the eruption. Also, the model easterly QBO
phase begins slightly later than in ERA-interim, continu-
ing until around September 1993 (at 30 hPa), compared to
around January 1993 in the re-analysis. The semi-annual os-
cillation in the tropical middle and upper stratosphere is also
well represented in the model. 4.1
Stratospheric dynamics in the UM-UKCA One of the most important factors controlling stratospheric
aerosol is the stratospheric transport which is determined by
the strength of the stratospheric Brewer–Dobson (BD) cir-
culation. This circulation plays a crucial role in determin- Atmos. Chem. Phys., 14, 11221–11246, 2014 Atmos. Chem. Phys., 14, 11221–11246, 2014 2.3
Experimental setup For this study, we carried out several 5-year model integra-
tions, as summarised in Table 2. In addition to a background
run (C_noPinatubo) without any Pinatubo emission, two ref-
erence simulations were carried out with 20 (A_Control20)
and 10 Tg (B_Control10) of SO2 injected into the tropical
stratosphere on 15 June 1991 between 19 and 27 km. To en-
sure we closely match the initial spatial distribution of the
aerosol cloud, we inject the SO2 across the eight model grid
boxes between 0–20◦N along 120.5◦E. We emit 3 % of the
SO2 mass from Pinatubo directly as sulfuric acid particles
(assumed to form at the sub-grid scale) with half emitted
with assumed geometric mean radius of 15 and 40 nm as in
Spracklen et al. (2005). For all the simulations the entire set
of tracers were initialised from fields after 8 years spin-up. To examine the simulated particle size distribution that un-
derpins the model aerosol optical properties, we also com-
pare to profile measurements of size-resolved number con-
centration made at Laramie, USA (Deshler et al., 2003). The balloon-borne system includes a condensation nucleus
counter (CNC) to measure the concentrations of particles
larger than 10 nm and an optical particle counter (OPC,
Deshler et al., 1992) to measure size-resolved particle con-
centrations in several size ranges in the accumulation and www.atmos-chem-phys.net/14/11221/2014/ Atmos. Chem. Phys., 14, 11221–11246, 2014 11227 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Figure 1. (a) Model-simulated tropical (15◦S–15◦N) mean monthly mean zonal wind (m s−1, QBO propagation). (b) Same as (a) but from
ERA-interim reanalysis data (Dee et al., 2011). (c) Zonal mean age-of-air (years, mean 1991–2000), and (d) mean age-of-air (1991–2000)
comparison at 50 hPa. Triangles and filled circles show estimated age-of-air from CO2 and SF6 (Hall et al., 1999). Mean age-of-air from
various CCMs which participated in SPARC Lifetime Assessment are shown with yellow lines and the one from this study is shown with the
red line. Figure 1. (a) Model-simulated tropical (15◦S–15◦N) mean monthly mean zonal wind (m s−1, QBO propagation). (b) Same as (a) but from
ERA-interim reanalysis data (Dee et al., 2011). (c) Zonal mean age-of-air (years, mean 1991–2000), and (d) mean age-of-air (1991–2000)
comparison at 50 hPa. Triangles and filled circles show estimated age-of-air from CO2 and SF6 (Hall et al., 1999). Mean age-of-air from
various CCMs which participated in SPARC Lifetime Assessment are shown with yellow lines and the one from this study is shown with the
red line. Figure 2. Time series of the global burden (in Tg of sulfur) of SO2
(red), total sulfur (includes both SO2 and aerosol, black), aerosol
sulfur (dark blue) for runs A_Control20 (solid lines), B_Control10
(dashed lines), and C_noPinatubo (dotted lines). Integrated aerosol
sulfur burdens in the UTS and lower–middle troposphere (deter-
mined by above or below 400 hPa) are also shown with green and
aqua lines, respectively. The stratospheric aerosol burden derived
by Baran and Foot (1994) using HIRS measurements is shown by
the green line with filled circles, assuming 75% sulphuric acid by
weight. turn, such a mixing can cause too fast removal of aerosol
from the stratosphere into the mid- and high-latitude tropo-
sphere, and should be considered when drawing inference
from the evaluation of the model post-Pinatubo stratospheric
aerosol decay. www.atmos-chem-phys.net/14/11221/2014/ 11228 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM McCormick
and Veiga (1992) derived an approximate aerosol sulfur bur-
den assuming a 50 % conversion from SO2 to H2SO4 by the
end of July, which corresponds to an e-folding timescale of
43 days. Oman et al. (2006) found an SO2 e-folding conver-
sion timescale of 35 days in their model, which used fixed
OH concentrations. We note however that in the first month
of the eruption there is much slower conversion to aerosol of
the volcanic emitted SO2, compared to the timescale over 60
days. For example, at day 200 (34 days after the eruption)
there is ∼5.6 Tg of sulfur in the form of SO2, which gives an
e-folding timescale of 59 days. Bekki (1995) found that oxi-
dant concentrations can be strongly depleted after very large
volcanic eruptions, and in their Pinatubo simulation Bekki
and Pyle (1994), found a timescale of 40 days. volume mixing ratio (pptv) d) NH mid-lat Oct 91
10-2
100
102
104
106
volume mixing ratio (pptv)
10
20
30
40
50
Height [km] c) tropics Oct 91
10-2
100
102
104
106
volume mixing ratio (pptv)
10
20
30
40
50
Height [km] c) tropics Oct 91 d) NH mid-lat Oct 91 ht [km] ht [km] Figure 3. Volume mixing ratios of various sulfur containing species Figure 3. Volume mixing ratios of various sulfur containing species
(pptv) in the tropics (20◦S–20◦N, left) and NH mid-latitudes (35–
60◦N, right) during July 1991 (top) and October 1991 (bottom). Gas phase and particle phase H2SO4 ratios are shown with green
and black lines, respectively. OCS and SO2 are shown with blue and
purple lines, respectively. Runs A_Control20 and C_noPinatubo are
shown with solid and dashed lines, respectively. Also, A_Control20 has a peak stratospheric aerosol burden
of 9.3 Tg at about day 260, but the aerosol burden from
B_Control10 peaks around a month earlier, at around day
230, with 5.25 Tg of sulfur, which closely matches the HIRS
observations. For A_Control20 and B_Control10, we find
around 6.1 Tg and 3.5 Tg of sulfur by June 1992 (12 months
after the eruption, day 530, day 530, first vertical dashed
line in Fig. 2), suggesting e-folding timescales of 19 and
24 months, respectively. The shorter removal timescale for
the 20 Tg run is likely due to the particles growing to larger
sizes compared to the 10 Tg run (e.g. as seen in Fig. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11229 11229 a) tropics July 91
10-2
100
102
104
106
volume mixing ratio (pptv)
10
20
30
40
50
Height [km]
b) NH mid-lat July 91
10-2
100
102
104
106
volume mixing ratio (pptv)
10
20
30
40
50
Height [km]
SO2
OCS
H2SO4(g)
H2SO4(p)
c) tropics Oct 91
10-2
100
102
104
106
volume mixing ratio (pptv)
10
20
30
40
50
Height [km]
d) NH mid-lat Oct 91
10-2
100
102
104
106
volume mixing ratio (pptv)
10
20
30
40
50
Height [km]
Figure 3. Volume mixing ratios of various sulfur containing species
(pptv) in the tropics (20◦S–20◦N, left) and NH mid-latitudes (35–
60◦N, right) during July 1991 (top) and October 1991 (bottom). Gas phase and particle phase H2SO4 ratios are shown with green
and black lines, respectively. OCS and SO2 are shown with blue and
purple lines, respectively. Runs A_Control20 and C_noPinatubo are
shown with solid and dashed lines, respectively. a) tropics July 91
10-2
100
102
104
106
volume mixing ratio (pptv)
10
20
30
40
50
Height [km] (0.15 Tg S) found by Hommel et al. (2011). Tropospheric
aerosol burdens are also higher than other models (e.g. Tex-
tor et al., 2006) at around 1.25 Tg S on the annual mean. b) NH mid-lat July 91
10-2
100
102
104
106
volume mixing ratio (pptv)
10
20
30
40
50
Height [km]
SO2
OCS
H2SO4(g)
H2SO4(p) g
For run A_Control20, the global column SO2 burden
decays from an immediate post-eruption peak of 10.3 Tg
to around 2.0,Tg S SO2 burden on day 226 (60 days af-
ter the eruption). Subtracting the 0.3 Tg SO2 mass from
B_Control10 (which is all in the troposphere), gives 1.7 Tg S,
indicating that 8.3 of the emitted 10 Tg S emitted as SO2 has
been chemically converted to sulfuric acid over that period. We therefore estimate the e-folding timescale for conversion
of SO2 into sulfuric acid aerosol as 60 divided by ln(10/1.7)
which is 35 days, which agrees closely with most previous
studies. For example Bluth et al. (1992) derived an e-folding
timescale of 35 days from the TOMS satellite SO2 measure-
ments, but present this as a tentative estimate. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 8),
and therefore sedimenting faster, moving to altitudes closer
to the tropopause, where removal from the stratosphere is
more effective. We note that, despite the close agreement be-
tween the A_Control10 and the HIRS-derived burdens, the
timescale estimates are considerably longer than values cited
in the literature which range from around 12 to 14 months
(e.g. see Baran and Foot, 1994 and Bluth et al., 1997). In Fig. 2, we also show a time series of the stratospheric
aerosol sulfur burden derived from HIRS measurements by
Baran and Foot (1994), assuming a composition of 75% sul-
phuric acid by weight. For run A_Control20, we find the
peak in UTS aerosol sulfur burden occurs 3 months after the
eruption in September, in agreement with the timing derived
from HIRS. However, the stratospheric aerosol sulfur bur-
den from A_Control20 is much higher than the observations,
with a maximum of 9.3 Tg of sulfur (37 Tg aerosol mass as-
suming 75 % sulfuric acid composition), substantially higher
than the 5.4 Tg of sulfur (21.6 Tg of aerosol) from Baran
and Foot (1994). Based on ISAMS measurements, Lambert
et al. (1993) estimated the post-Pinatubo peak stratospheric
aerosol burden as between 19 and 26 Tg (4.75 to 6.5 Tg of
sulfur). Since A_Control20 gives much too much sulfur in
the stratospheric aerosol compared to both of these estimates,
we carried out a second control simulation – B_Control10
with 10 Tg of SO2 (dashed line in Fig. 2). 4.3
Perturbation in sulfur species Figure 3 shows vertical profiles of mixing ratios of the
three key gas phase sulfur species OCS, SO2, H2SO4,
and of sulfuric acid in the particle phase (P-H2SO4), from
runs A_Control20 and C_noPinatubo. The left and right
panels are for mean profiles in the tropics (20◦S–20◦N)
and NH mid-latitudes (35–60◦N), respectively with the top
and bottom rows indicating the means for July 1991 and The stratospheric aerosol sulfur burden from B_Control10
is in good agreement with the values derived from HIRS
through the second half of 1991 and the whole of 1992. How-
ever, the HIRS measurements suggest a return to approxi-
mately background stratospheric aerosol levels by the mid-
dle of 1993, while the model aerosol shows much slower de-
cay, even showing modest enhancement at the end of 1994. Atmos. Chem. Phys., 14, 11221–11246, 2014 4.2
Global burden and e-folding timescale Figure 2 shows the January 1991 to December 1994 time
evolution of the daily total global column mass burden of
sulfur in the gas phase (as SO2, red) and in the aerosol
particle phase (blue) from runs A_Control20 (solid line),
B_Control10 (dashed line) and C_noPinatubo (dotted line). Separate lines indicating the upper tropospheric and strato-
sphere (UTS) aerosol sulfur burden (above 400 hPa, green
lines) and that in the lower–middle troposphere (below 400
hPa, aqua lines) are also shown. From the no-Pinatubo run
C_noPinatubo, the global SO2 and aerosol sulfur burdens are
mostly in the troposphere, and their time series are dominated
by anthropogenic emission sources, which are mainly in NH
mid-latitudes. Photochemistry is strongest during summer,
with higher oxidants then causing efficient conversion of
SO2 to aerosol sulfate. Only 10 % of this background total
sulfur burden is in the form of SO2 during the NH sum-
mer, compared to around 50 % during winter. We find 30–
40 % of the total aerosol sulfur burden (around 0.5 Tg S) is in
the stratosphere, which is considerably higher than the 17 % Figure 2. Time series of the global burden (in Tg of sulfur) of SO2 Figure 2. Time series of the global burden (in Tg of sulfur) of SO2
(red), total sulfur (includes both SO2 and aerosol, black), aerosol
sulfur (dark blue) for runs A_Control20 (solid lines), B_Control10
(dashed lines), and C_noPinatubo (dotted lines). Integrated aerosol
sulfur burdens in the UTS and lower–middle troposphere (deter-
mined by above or below 400 hPa) are also shown with green and
aqua lines, respectively. The stratospheric aerosol burden derived
by Baran and Foot (1994) using HIRS measurements is shown by
the green line with filled circles, assuming 75% sulphuric acid by
weight. www.atmos-chem-phys.net/14/11221/2014/ Atmos. Chem. Phys., 14, 11221–11246, 2014 4.4
Stratospheric aerosol optical depth (sAOD)
comparison 3a) show large changes in concentrations of SO2 and P-
H2SO4 (between 20 and 30 km) relative to run C_noPinatubo
increasing by factors 103–104 and factor 102, respectively. The enhanced P-H2SO4 profile indicates that much of the
SO2 has already been oxidised and condensed into the par-
ticle phase. By contrast, the NH mid-latitude July profiles
show that the Pinatubo plume has not yet been transported,
with SO2 and aerosol H2SO4 still at quiescent concentrations
over almost the entire stratosphere, although some perturba-
tion can be observed in the lowermost stratosphere and up-
permost troposphere. It is notable that balloon-borne particle
concentration soundings at Laramie (41◦N) in July 1991 al-
ready show some enhanced layers between 15–18 km (Desh-
ler et al., 1992) which corresponds well with the altitude of
the SO2 and P-H2SO4 enhancement seen in the July-mean
NH mid-latitude profiles. g
However, consistent with Fig. 2 (more aerosol loading
than estimated by Baran and Foot, 1994) the simulated sAOD
in A_Control20 is much larger than both sets of observations. The sAOD distribution in B_Control10 is in better agree-
ment with the satellite measurements, comparing well to both
satellite measurements in mid- and high latitudes. Compar-
ing to the observed sAOD enhancements in the SH, both
model simulations are also in quite good qualitative agree-
ment. However, in the tropics the sAOD in B_Control10 is
still about 50 % larger than that derived from AVHRR, and
a factor of 2 larger than SAGE II. Possible causes for these
biases are discussed later in this section. The October mean SO2 profile is still strongly enhanced
(factor 100) in the tropics with the P-H2SO4 enhancement
only slightly higher than in July but over a much deeper
layer. This tropical enhancement in both SO2 and P-H2SO4
propagates up to about 40 km, and above that only the SO2
profiles show differences between runs A_Control20 and
C_noPinatubo. It is interesting that the October 1991 trop-
ical gas phase H2SO4 profile from run A_Control20 actually
shows lower values than in run C_noPinatubo in the main
part of the plume (15–30 km), due to the condensation sink to
aerosol being so much stronger. By contrast, above 30 km the
increase in vapour pressure shuts off the condensation sink
leading to the H2SO4 vapour concentrations being higher
than quiescent at those altitudes. 4.4
Stratospheric aerosol optical depth (sAOD)
comparison October 1991, selected to correspond to the 15–45 day post-
eruption period when the SO2 is oxidised to H2SO4 vapour,
and approximately when the peak global aerosol burden oc-
curs in the model. The profile of OCS shows the expected
shape, being constant in the troposphere and then reducing
with increasing altitude in the stratosphere as it is photol-
ysed. The SO2 profile from run C_noPinatubo shows a sharp
reduction with height across the tropopause but then reaches
a minimum and begins to increase with height to a local
maximum at 30 km corresponding to where the source from
OCS photolysis is largest. Below 30 km the sulfuric acid
vapour follows a similar shape as SO2 (but at lower concen-
trations) but above that altitude continues to increase up to
about 40 km. Below 35 km, the vertical profile of P-H2SO4
is approximately constant in the tropics in these quiescent
conditions, but has a slight decrease with altitude. In the
upper-middle stratosphere rapidly evaporating particles re-
lease their H2SO4 to the gas phase causing a sharp reduction
in P-H2SO4 about 40 km. Figures 4a and b show the time evolution of the model
mid-visible sAOD from runs A_Control20 and B_Control10
while Fig. 4c and d show the mid-visible sAOD measured
by SAGE II and derived from AVHRR. Since AVHRR is
a nadir viewing instrument, in Fig. 4d we have subtracted
monthly-mean total AODs for the year prior to the eruption,
matching the procedure recommended by Long and Stowe
(1994) and used by Aquila et al. (2012). Note that the SAGE-
II-derived sAOD is much lower than AVHRR in the trop-
ics during the very high loading period after Pinatubo due
to the measured extinction saturating at values above about
0.01 km−1 (Hamill et al., 2006). In both the A_Control20
and B_Control10 runs there is good qualitative agreement
with the satellite regarding spatial and temporal distribution. For example, there is high sAOD after the eruption centred
around the equator with peak sAOD in September 1991 in
both model simulations and in the two satellite data sets. However, the model feature is narrower, confined between
10◦N and 10◦S. Another well-captured feature in the model
is that there is no significant enhancement of sAOD in NH
mid- and high latitudes until October 1991. In the tropics, the July profiles from run A_Control20
(Fig. 4.4
Stratospheric aerosol optical depth (sAOD)
comparison The October 1991 NH mid-
latitude SO2 and P-H2SO4 profiles show only moderate en-
hancement, suggesting that the easterly phase of the QBO
has prevented transport of Pinatubo-enhanced air masses. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM
4.4
Stratospheric aerosol optical depth (sAOD)
comparison 11230 Atmos. Chem. Phys., 14, 11221–11246, 2014 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11231 Figure 4. Time series of model-simulated zonal mean sAOD at 525 nm (calculated by integrating the extinction above the tropopause) for
runs (a) A_Control20 and (b) B_Control10. (c) and (d) show the sAOD measured by SAGE II (525 nm) and derived from AVHRR (600 nm)
measurements. AVHRR sAOD is derived as the difference from the background total AOD from the 2 years before the eruption (Long and
Stowe, 1994). Figure 4. Time series of model-simulated zonal mean sAOD at 525 nm (calculated by integrating the extinction above the tropopause) for
runs (a) A_Control20 and (b) B_Control10. (c) and (d) show the sAOD measured by SAGE II (525 nm) and derived from AVHRR (600 nm)
measurements. AVHRR sAOD is derived as the difference from the background total AOD from the 2 years before the eruption (Long and
Stowe, 1994). average) particles sedimenting more slowly. Larger model
high bias is seen for simulated tropical extinctions at 32 km,
for both the runs (A_Control20 and B_Control10) that
may indicate that the upper altitude used for SO2 injection
was too high. At 32 km, the modelled extinction is slightly
larger than SAGE II and, although peaks and troughs are
mostly similar to the satellite measurements, the model
variability is less than in the observations. We note again
that the simulations presented here do not include the
dynamical effects of aerosol-induced radiative heating. Such
a radiative heating is known to cause increased tropical
upwelling, which would cause greater dilution, could alter
horizontal transport through the subtropical barrier and may
also alter microphysical processes such as evaporation and
coagulation. tropics (20◦S–20◦N). We choose these altitudes to allow
comparison with the evaluation presented in Weisenstein
et al. (2006, Fig. 6.20) for other stratospheric aerosol models. We compare extinction in the mid-visible (left panels) as well
as the near-infrared (right panels). Here we use the updated
v7.0 SAGE II data set and the profiles shown are averages
between 20◦S and 20◦N. Monthly mean observed values are
calculated based on both sunrise and sunset profiles. p
At
20
and
25 km,
both
runs
(A_Control20
and
B_Control10) capture the general evolution of the trop-
ical mid-visible extinction (Fig. 4.5
Extinction comparison Extinction profile measurements from SAGE II between July
and September show (e.g. McCormick et al., 1995) that trans-
port to the SH occurred mostly above about 24 km altitude. Aquila et al. (2012) highlighted the importance of resolving
the enhanced tropical upwelling which occurred due to the
long-wave absorption by the relatively larger stratospheric
aerosol after the Pinatubo eruption. As explained in Sect. 2,
in these simulations we do not radiatively couple the simu-
lated aerosol with the model dynamics, and yet we capture
quite well the SH post-Pinatubo sAOD evolution. We note
that Aquila et al. (2012) do not include evaporation of sulfu-
ric acid in their model, which could play an important role in
influencing transport to SH mid-latitudes. Figure 5 shows a time series of aerosol extinction from the
three model simulations (runs A_Control20, B_Control10,
C_noPinatubo) and SAGE II at 32, 25 and 20 km in the Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 5), with the magnitude and
timing of peak values, and the decay timescale, agreeing well
with SAGE II. However, before the eruption (background
conditions), modelled extinctions have a moderate low bias
of 20–50 % at these levels. For the tropical mid-visible ex-
tinction time series, run B_Control10 is in better agreement
with the observations than A_Control20, which tends to be
high biased (consistent with the sAOD and aerosol mass high
biases seen in Figs. 2 and 4 respectively). However, against
the tropical near-infrared extinction, run A_Control20 is in
better agreement, with run B_Control10 generally showing
modest low bias, although still in reasonable agreement. At 25 km, and for both wavelengths, the model tropical
extinction peaks in August 1991, whereas in the satellite
measurements, values plateau for 2–3 months before the
decay period begins. In the model, the decay is fastest in the
first 6–8 months after the peak value, with an approximately
constant e-folding timescale from mid-1992 onwards. The
faster decay in the early phase may be due to the shift in
size distribution which produced larger particles at this
time. Faster sedimentation would remove larger particles
during this initial period, with the remaining (smaller on Figure 6 shows a similar analysis to Fig. 5, but for NH
mid-latitudes (35–60◦N), again to compare against mod-
els shown in Weisenstein et al. (2006). At 20 km, there is
very good agreement between modelled and SAGE II ex-
tinctions at both mid-visible and near-infrared wavelengths. Similarly good agreement is also observed at 25 km. How-
ever, during 1993, SAGE II measurements show a signifi-
cant decrease at both wavelengths which is not captured by
the model. At 32 km the modelled extinction enhancement
is slightly larger than the observations. We also note that
whereas in the tropics the model and observations showed
a faster decay phase in the first 6–8 months after the peak
aerosol loading compared to the later phase, in NH mid-
latitudes, both model and observations have constant ex-
ponential decay timescale throughout the post-eruption pe-
riod. For this latitude band, the model shows much better
agreement with SAGE II measurements than seen in any of Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ Atmos. Chem. Phys., 14, 11221–11246, 2014 11232 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 10-6
10-5
10-4
10-3
10-2
10-1
ext [km-1]
SAGE II (525nm)
A_Control20
B_Control10
C_noPinatubo
Tropics (550nm)
32km
10-6
10-5
10-4
10-3
10-2
10-1
Tropics (1020nm)
32km
10-6
10-5
10-4
10-3
10-2
10-1
ext [km-1]
25km
10-6
10-5
10-4
10-3
10-2
10-1
25km
1990
1991
1992
1993
1994
1995
1996
10-6
10-5
10-4
10-3
10-2
10-1
ext [km-1]
20km
1990
1991
1992
1993
1994
1995
1996
10-6
10-5
10-4
10-3
10-2
10-1
20km
Figure 5. Comparison between modelled and SAGE II (V7.0) retrieved extinction at 525 nm (left) and 1020 nm (right) in the tropics (20◦S–
20◦N) for 20 km (bottom), 25 km (middle) and 32 km (top). Extinctions from runs A_Control20, B_Control10 and C_noPinatubo are shown
with red, orange and blue lines, respectively. The vertical black lines show the range of plus or minus one standard deviation over the
individual measurements used in the calculation of the monthly mean. 10-6
10-5
10-4
10-3
10-2
10-1
Tropics (1020nm)
32km
10-6
10-5
10-4
10-3
10-2
10-1
25km
1990
1991
1992
1993
1994
1995
1996
10-6
10-5
10-4
10-3
10-2
10-1
20km Tropics (1020nm) Figure 5. Comparison between modelled and SAGE II (V7.0) retrieved extinction at 525 nm (left) and 1020 nm (right) in the tropics (20◦S–
20◦N) for 20 km (bottom), 25 km (middle) and 32 km (top). Extinctions from runs A_Control20, B_Control10 and C_noPinatubo are shown
with red, orange and blue lines, respectively. The vertical black lines show the range of plus or minus one standard deviation over the
individual measurements used in the calculation of the monthly mean. 10-6
10-5
10-4
10-3
10-2
10-1
ext [km-1]
SAGE II (525nm)
A_Control20
B_Control10
C_noPinatubo
NH mid-lat (550nm)
32km
10-6
10-5
10-4
10-3
10-2
10-1
NH mid-lat (1020nm)
32km
10-6
10-5
10-4
10-3
10-2
10-1
ext [km-1]
25km
10-6
10-5
10-4
10-3
10-2
10-1
25km
1990
1991
1992
1993
1994
1995
1996
10-6
10-5
10-4
10-3
10-2
10-1
ext [km-1]
20km
1990
1991
1992
1993
1994
1995
1996
10-6
10-5
10-4
10-3
10-2
10-1
20km
Figure 6. Same as Fig. 5 but for NH mid-latitudes (35–65◦N). 10-6
10-5
10-4
10-3
10-2
10-1
NH mid-lat (1020nm)
32km
10-6
10-5
10-4
10-3
10-2
10-1
25km
1990
1991
1992
1993
1994
1995
1996
10-6
10-5
10-4
10-3
10-2
10-1
20km Figure 6. Same as Fig. www.atmos-chem-phys.net/14/11221/2014/ 5 but for NH mid-latitudes (35–65◦N). portion of aerosol is removed in the tropics in the 20 Tg run
than in 10 Tg run (likely related to stronger sedimentation). As we also saw in the tropics, in the NH mid-latitudes, at portion of aerosol is removed in the tropics in the 20 Tg run
than in 10 Tg run (likely related to stronger sedimentation). As we also saw in the tropics, in the NH mid-latitudes, at the simulations submitted for the Pinatubo intercomparison
in SPARC (2006). Interestingly, differences between runs
A_Control20 and B_Control10 are much smaller at this lati-
tude band than in the tropics (Fig. 5), suggesting a larger pro- Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11233 1
Figure 7. Comparison between zonal mean modelled (run
A_Control20) and satellite-derived V1 and V2 SAD (µm2 cm−3)
from SPARC (2006) and Arfeuille et al. (2013), respectively, for
various months before and after the eruption. one can see weak meridional transport to NH and SH sub-
tropics at about 20–22 km. Young et al. (1994) showed that
including the aerosol radiative effects on the model dynamics
broadens the latitudinal extent of the Pinatubo cloud which,
in our simulations would improve agreement with the satel-
lite observations. And as mentioned earlier, such a heating
can alter local circulation and may partially explain the SAD
high biases seen here. By January 1992, the model high bias
has reduced to a factor of 2, and the model shows meridional
transport to NH mid-latitudes in the lowermost stratosphere,
also seen in the observations. However, the satellite-derived
SAD suggests meridional transport also occurs to the SH,
but at slightly higher altitudes. By May 1992, high biases
in modelled SAD are much smaller and the general latitu-
dinal and altitudinal distribution is still in good qualitative
agreement with the observations, aside from the continued
low bias in the SH. Also, as observed in Fig. 2 (younger age-
of-air), in the lowermost stratosphere, the model seems to
have too much diffusion near the tropopause. Hence the dis-
tinct cross-tropopause gradients seen in satellite data are not
seen in our simulations. www.atmos-chem-phys.net/14/11221/2014/ While interpreting the model–observation SAD discrep-
ancies, one should consider how the satellite SAD product is
derived from the SAGE I, SAGE II, SAM II (Stratospheric
Aerosol Instrument II) and SME (Solar Mesosphere Ex-
plorer) measurements. As noted earlier, the extinction mea-
sured by the SAGE and SAM instruments has an upper limit
of 0.01 km−1, above which the atmosphere is effectively
opaque to the instruments (Hamill et al., 2006). During the
peak aerosol loading period, when the model SAD is a factor
of 2 high biased, it is apparent (for example in Fig. 5) that
the SAGE II 525 nm and 1020 nm extinctions in the tropi-
cal lower stratosphere are saturating at the upper limit value,
with actual extinction values likely to have been higher. The
late-1991 to 1992 period was flagged as missing data in the
original SAGE II extinction data set. The data gaps during
that period were addressed by Hamill et al. (2006), who
used lidar data from two tropical sites (Camaguey, Cuba and
Mauna Loa, Hawaii) and two mid-latitude sites (lidar from
NASA Langley, Virginia, USA and backscatter sonde from
Lauder, New Zealand), to fill the missing data. Figure 7. Comparison between zonal mean modelled (run
A_Control20) and satellite-derived V1 and V2 SAD (µm2 cm−3)
from SPARC (2006) and Arfeuille et al. (2013), respectively, for
various months before and after the eruption. 20 km, the 1020 nm extinction from run A_Control20 shows
better agreement with SAGE II than B_Control10. 4.6
Surface area density comparison Another important issue to consider with the SAGE-II-
derived SAD product is that, even outside the gap-filled part
of the data set, particles smaller than 50 nm are essentially
invisible to the satellite and there is little sensitivity to parti-
cles smaller than 100 nm. For example, Reeves et al. (2008)
derived extinction, SAD and volume concentration from air-
craft measurements of the aerosol particle size distribution
(in quiescent conditions) and compared to SAGE II prod-
ucts. They found that the aircraft measured SAD was a fac-
tor 1.5–3 higher than the SAGE-II-derived values, whereas
volume concentrations were only 35 % higher.The apparent
SAD high bias in the model may therefore partly be caused
by the contribution from smaller particles which are not ob-
servable from the SAGE II instrument Figure 7 compares the vertical and latitudinal distribution of
zonal mean SAD from run A_Control20 against two ver-
sions of the satellite-derived SAD data set, for 4 selected
months between May 1991 and May 1992. Before the erup-
tion (May 1991), the model captures the global SAD distri-
bution reasonably well compared to the SAGE-derived data
sets, although model values are higher in the upper tropo-
sphere and lower stratosphere (UTLS) region. For Septem-
ber 1991 (3 months after the eruption), although the simu-
lated SAD distribution broadly matches the observed shape,
it is up to a factor 2 to 3 high in the tropics. Also, the model
Pinatubo-enhanced SAD plume is strongly confined to the
tropical pipe, whereas in the satellite-derived SAD (Fig. 7d) 4.8
Particle size distribution However, above 25 km the model is not able
to simulate steeper decreases observed in vertical profiles
of N150 and N250. Intriguingly, in November 1991 (Fig. 9d)
run A_Control20 predicts only slightly higher N150 and N250
than run B_Control10, with particle concentrations at larger
sizes showing a much larger relative enhancement in the
20 Tg run than the 10 Tg run. The size distribution simulated
by the model is generally in good agreement with the ob-
servations, although in August and September 1991, for the
peak at about 18 km, the model N550 are low biased com-
pared to the observations, and there is a general overpredic-
tion of N5 in this initial post-eruption phase. The low bias
in the larger sizes could be related to the high bias in the
smallest sizes, with the condensation sink being shared out
across a larger number of particles leading to reduced parti-
cle growth. Another possibility is that there is faster STE as
observed in the age-of-air comparison (Fig. 1c and d). Obser-
vations also show approximately constant N5 between 20 and
30 km, but run A_Control20 (B_Control10) have elevated Figure 8 shows the evolution of the model zonal-mean Reff
at 20 and 25 km from runs A_Control20 and B_Control10
compared to that derived by Bauman et al. (2003) from
the SAGE II and CLAES satellite measurements. The gen-
eral spatial and temporal evolution of the model Reff is in
good qualitative agreement with the observations in both
runs, with values at 20 km larger than at 25 km, likely due
to sedimentation. In the tropics, at both altitudes, the ob-
servations suggest that, whereas sAOD and extinction are
decaying by November or December 1991 (Figs. 4 and 5),
the effective radius peaks several months later (early 1992)
with only a slow decay beginning later in 1992. By contrast,
in NH mid-latitudes, the observations suggest that the de-
cay in effective radius is slightly earlier and occurs faster. Both simulations capture the timing of these Reff peaks well,
although at 25 km, the model peak is later than observed,
matching the timing at 20 km. Effective radius values are
always higher in the tropics than at mid-latitudes, a feature
that is consistent between the model and observations. 4.8
Particle size distribution Reff =
Pm
i=1 Nir3
gi exp
9/2(lnσi)2
Pm
i=1 Nir2
gi exp
2(lnσi)2 . (1) To give a stronger observational constraint on the simulated
size distribution, we compare the model against balloon-
borne CNC and OPC measurements made at Laramie,
Wyoming, USA (41◦N, see Sect. 3). Figures 9 and 10 com-
pare model profiles of size-resolved number concentrations
(larger than a given particle diameter) against those mea-
sured by the CNC and OPC. In each case we are com-
paring a monthly-mean size-resolved particle concentration
from the model to a single balloon sounding. Note also
that whereas the number concentration profiles for particles
larger than 5 nm, 150 nm and 250 nm are exactly as measured
by the OPC, for the larger size channels we have interpolated
the observations (linearly in log N vs. log R space) onto reg-
ular Dp > 550 nm, 750 nm and 1000 nm size channels from
the irregular size thresholds given in the individual sounding
data files. (1) The two gap-filled SAGE/SAM extinction data products
provide 3-D time-varying volume concentration and SAD
which together give Reff throughout the Pinatubo period. This record therefore has the potential to provide informa-
tion on how the particle size distribution in the stratosphere
was perturbed by the eruption. However, again, when com-
paring the model to the satellite Reff, the limitations associ-
ated with the derived product need to be considered. In par-
ticular, because of the “blind spot” associated with particles
smaller than 50–100 nm, Hamill et al. (2006) state that since
the derived SAD may have an inherent low bias (whereas the
derived volume density will be less affected) the derived Reff
may overestimate the true value. Figure 9 shows the observed (plus signs) profile evolution
of the particle size distribution through August to Novem-
ber 1991, for the period after the Pinatubo plume was first
detected at Laramie on 16 July (Deshler et al., 1992). In
August and September 1991, both runs A_Control20 and
B_Control10 show elevated values of N150 and N250 be-
tween 14 and 20 km, whereas at higher altitudes (above 25
km) the profile remains close to background values (not
shown). The region with elevated N150 and N250 profiles
matches reasonably well with the observations, and indicates
efficient transport of air from the tropics in the lowermost
stratosphere. 4.7
Effective radius comparison tudes and before the eruption, which suggests that it is not
associated with sedimentation, since that would be expected
to occur mostly during the highest loading period. There ap-
pears to be a more persistent bias in simulated particle size
distribution, but it is unclear whether the model has too many
small particles, or too few large particles. Another product derived from the gap-filled satellite extinc-
tion record, that can be used to assess the evolution of the
stratospheric aerosol properties following the Pinatubo erup-
tion, is Reff, defined as the ratio of the third and second inte-
gral moments in radius, and which for multimodal distribu-
tion can be represented as (Russell et al., 1996, Eq. 6) www.atmos-chem-phys.net/14/11221/2014/ Atmos. Chem. Phys., 14, 11221–11246, 2014 11234 4.8
Particle size distribution How-
ever, although Reff from run A_Control20 is slightly larger
than from B_Control10, modelled values are up to 30–40 %
smaller than those derived from the satellite, with maximum
model Reff of around 0.4 and 0.35 µm, compared to around
0.6 µm from the satellites. At 20 km (Fig. 8b), despite combining the two sets of satel-
lite products, there is no observational constraint on the trop-
ical Reff between approximately June 1991 and August 1992,
but the overall shape suggests the Reff was likely even larger
than 0.6 µm during that period. The model low bias in Reff
is apparent at about the same extent at all latitudes and alti- Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11235 Figure 8. Satellite-derived (shaded, from Bauman et al., 2003) and modelled (contours) effective radii (Reff) in µm at 25 and 20 km from
runs A_Control20 (panels a and b) and B_Control10 (panels c and d). Figure 8. Satellite-derived (shaded, from Bauman et al., 2003) and modelled (contours) effective radii (Reff) in µm at 25 and 20 km from
runs A_Control20 (panels a and b) and B_Control10 (panels c and d). longer-timescale evolution of the size distribution through
the Pinatubo period, comparing soundings in March 1992,
1993 and 1994 (Fig. 10b, c and d). Before the eruption, the
observations show that N5 decreases with increasing altitude
between 12 and 23 km, whereas N150 and N250 show a much
slower decrease with height. This feature is well captured by
the model with N5 and N150, in excellent agreement with the
observations in this altitude range, although N250 has a slight
low bias. Between 25 and 30 km, the observed N5 profile
shows a layer of enhanced concentrations, by around a fac-
tor of 10 compared to a continuation of the decrease seen at
lower altitudes. This layer indicates a source of freshly nucle-
ated particles which have not grown to larger sizes. The simu-
lated N5 profile also shows this feature, but the enhancement
of particle concentrations is much stronger in the model, and
extends to lower altitudes, down to around 20km. Gas phase
sulfuric acid concentrations are known to increase rapidly
with height in this region from balloon-borne ion mass spec-
trometer measurements (e.g. Arnold et al., 1981). These ele-
vated concentrations of gas phase H2SO4 have been shown to
cause significant nucleation in the middle stratosphere (Hom-
mel et al., 2011) which is almost certainly the cause of this
feature. The high bias in the model N5 profile in this en-
hanced layer likely indicates that nucleation is too strong in
the model. The overpredicted nucleation rate in these vol-
canically quiescent conditions may be a result of gas phase
concentrations of H2SO4 being too high in the model. Pos-
sible explanations might be that the model does not include
the sink for gas phase H2SO4 provided by meteoric debris, N5 between 20 and 25 km. 5
Discussion In Sect. 4.2 we found that injecting 20 Tg SO2 into the tropi-
cal stratosphere substantially overestimates the stratospheric
aerosol sulfur burden, with a 10 Tg SO2 injection in much
better agreement with observations. Most previous modelling
studies of the Pinatubo eruption have also tended to inject
20 Tg of SO2, and we show here that the high bias in our
model is also found in other studies. Oman et al. (2006) and
English et al. (2013) found peak stratospheric sulfuric acid
aerosol burdens of 27 and 24 Tg respectively, translating to
36 and 32 Tg aerosol mass assuming 75 % weight sulfuric
acid, similar to our 37 Tg peak value. Niemeier et al. (2009)
injected 17 Tg of SO2, and their 30 Tg peak stratospheric
aerosol burden also agrees with our simulation, accounting
proportionally for the reduced sulfur source. We note also
that Niemeier et al. (2009) and English et al. (2013) have
presented the HIRS stratospheric aerosol burden time series
from Baran and Foot (1994) assuming the mass burden is
for sulfuric acid, without accounting for the fraction of water
content implicit in those values. In March 1993 (Fig. 10c), the observations show clear
separation between N5 and N150, although N150 and N250
are close together. This indicates the formation of a bimodal
size distribution consisting of an external mixture of particles
which have grown to larger sizes following condensation af-
ter oxidation of volcanic SO2 and a separate sub-population
of particles less influenced by the eruption. Observed pro-
files of N550, N750 and N1000 show peak values at around
12km at this time, much lower altitudes than at March 1992
(Fig. 10b). It is interesting that the March 1993 N550 and
N750 profiles are higher in the 10–15 km region than in
March 1992, likely indicating that, although slow at these
particle sizes, sedimentation is transporting the particles to
lower altitudes over these longer timescales. The model cap-
tures the observed size distribution fairly well, with N250 in
quite good agreement with the measurements. However, the
model N150 profile has a high bias of around a factor of 2 dur-
ing March 1993 and is still together with the N5 profile be-
tween 15 and 20 km. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11236 shown to be important by Saunders et al. (2012) and Brühl
et al. (2013) or underestimated H2SO4 photolysis rates in our
simulations. lated profiles not capturing the increase in particles larger
than N550 in the lowermost stratosphere. By March 1994
(Fig. 10d) the OPC measurements show that there has been
a general decay in all size channels towards background con-
ditions. The model N150 high bias seen in March 1993 has
worsened with the decay rate at these channels slower than
in the observations. In the N550, N750 and N1000 channels, the
model continues to have a low bias in both simulations. It is
notable that throughout the period, the model N150 and N250
profiles are remarkably similar between the A_Control20 and
B_Control10 simulations, with much larger differences in the
coarser sized particles. In March 1992 (Fig. 10b), 9 months after the eruption,
the observed particle concentration profiles show major en-
hancements throughout the upper troposphere and lower
stratosphere (10 to 25 km), for size channels 150 nm and
larger. By contrast, N5 shows a slight decrease compared
to March 1991, and is only marginally higher than N150
and N250 for this month, suggesting that a large propor-
tion of the particles have grown to sizes larger than 250 nm. The enhanced profiles of N550, N750 and N1000 are approx-
imately constant between 15 and 20 km with a fast decrease
at higher altitudes. Model run A_Control20 (solid line) cap-
tures this volcanically enhanced particle size distribution re-
markably well, with good qualitative and quantitative agree-
ment across all the size channels in the main part of the
plume. Run B_Control10 (dashed line) also captures well the
N5, N150 and N250 profiles, but is low biased in the larger size
channels. Despite generally very good agreement with the
Laramie OPC data at this time, in the lowermost stratosphere
and upper troposphere (between 10 and 15 km), both model
runs show a high bias in N150 and N250. We saw from the
previous comparisons that run A_Control20 has too high a
burden in the stratospheric aerosol compared to the HIRS and
ISAMS satellite measurements (Fig. 2) and that it is strongly
biased high in aerosol optical depth against the SAGE II and
AVHRR data (Fig. 4). S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM The comparisons to the OPC data sug-
gest that the high sAOD bias originates from the overpre-
dicted particle concentrations in the 150 to 550 nm radius
range in the lowermost stratosphere, with coarser particles
in that part of the atmosphere in reasonable agreement (run
A_Control20) or showing low bias (run B_Control10). S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM The observations show that con-
centrations of particles at 150 nm and larger reduce sharply
above 25 km, whereas the model profiles show only moder-
ate decline. This suggests that the simple approach to particle
evaporation may need improving. N5 between 20 and 25 km. The observations show that con-
centrations of particles at 150 nm and larger reduce sharply
above 25 km, whereas the model profiles show only moder-
ate decline. This suggests that the simple approach to particle
evaporation may need improving. p
y
p
g
For November 1991, the run A_Control20 shows enhance-
ment up to 25 km for all the particle size thresholds, with the
coarse mode higher than the run C_noPinatubo in the low-
ermost stratosphere (not shown) and the shape of the verti-
cal profile for each channel compares well with the obser-
vations. The model also shows an enhanced layer of N5,
N150 and N250 at about 35km, suggesting transport of the
Pinatubo plume to mid-latitudes throughout the lower and
middle stratosphere. In both the model and observations,
in these initial months, there is a layer where the N5 and
N150 lines come together, reflecting that few particles remain
smaller than 150 nm and indicating that particle growth at
these sizes is strongest in that part of the stratosphere. We
note however that in the observations this confluence occurs
at around 20 km, whereas in the model this occurs around
16–17 km. This discrepancy in altitude could be reflecting
the chosen injection height range, or be related to transport
deficiencies in the model, and the general good qualitative
agreement with the observations suggests that the modal ap-
proach to aerosol dynamics is capturing the evolution of the
size distribution rather well. In order to evaluate the model size distribution profile in
quiescent conditions, we compare to the Laramie balloon
measurements in March 1991 (Fig. 10a). We then probe the Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11237 Figure 9. August, September, October, November 1991 profiles of size-resolved number concentrations of particles (cm−3) with radii larger
than 5, 150, 250, 550, 750 and 1000 nm from Laramie (41.3◦N, 105.5◦W) are shown with plus (+) symbol. Solid and dashed lines show
aerosol profiles from the runs A_Control20 and B_Control10, respectively, highlighting the region where the model predicts perturbation
in the aerosol profiles. Horizontal coloured lines represent standard deviations (1σ) in number concentrations for a given month calculated
from daily values for run A_Control20. Figure 9. August, September, October, November 1991 profiles of size-resolved number concentrations of particles (cm−3) with radii larger
than 5, 150, 250, 550, 750 and 1000 nm from Laramie (41.3◦N, 105.5◦W) are shown with plus (+) symbol. Solid and dashed lines show
aerosol profiles from the runs A_Control20 and B_Control10, respectively, highlighting the region where the model predicts perturbation
in the aerosol profiles. Horizontal coloured lines represent standard deviations (1σ) in number concentrations for a given month calculated
from daily values for run A_Control20. Figure 10. Same as Fig. 9 but for March 1991, March 1992, March 1993 and March 1994. Figure 10. Same as Fig. 9 but for March 1991, March 1992, March 1993 and March 1994. the scattering efficiency of of incoming solar radiation. These
altered radiative effects illustrate the importance of resolv-
ing aerosol particle size changes and subsequent feedback on the scattering efficiency of of incoming solar radiation. These
altered radiative effects illustrate the importance of resolv-
ing aerosol particle size changes and subsequent feedback on a larger Reff will have caused important changes in the radia-
tive properties of the stratospheric aerosol, with significant
absorption of outgoing terrestrial radiation and a decrease in a larger Reff will have caused important changes in the radia-
tive properties of the stratospheric aerosol, with significant
absorption of outgoing terrestrial radiation and a decrease in Atmos. Chem. Phys., 14, 11221–11246, 2014 5
Discussion Also, simulated particle concentrations
in the larger size channels have a strong low bias of around
a factor of 10 (run A_Control20) or 20 (run B_Control10)
in the lowermost stratosphere at this time, with the simu- For our 10 Tg Pinatubo simulation, we found generally
good agreement with observed sAOD (section 4.4), extinc-
tion (section 4.5), and SAD (section 4.6). Our 20 Tg simu-
lation gives consistently too high sAOD in the tropics, mid-
latitudes and polar regions, whereas in most of the previous
studies mentioned above, reasonable agreement is found in
peak AOD, despite the high bias in stratospheric aerosol bur-
den. We note however that there is a considerable diversity
in the injection height-range, latitudinal spread and duration
of the volcanic source used in these different model experi-
ments. The comparisons against the balloon measurements
(Figs. 9 and 10) show that our model captures well the gen-
eral evolution of the particle size distribution in the strato-
sphere through the Pinatubo period. The observations indi-
cate how the huge injection of SO2 led after the eruption to
the growth of some particles to sizes larger than 1 µm at peak
loading (e.g. Fig. 10b), with a long-lasting perturbation to
concentrations larger than 150 nm causing a complex evo-
lution of Reff (Fig. 8). The shift in the size distribution to Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Also, our model has
too young age-of-air in mid-latitudes (see Fig. 1d) which may
also be affecting the simulated transport and particle size
evolution. Finally, we also note that nucleation rates at the
very low humidity and temperature conditions in the strato-
sphere are known to be highly uncertain. The Vehkamäki
et al. (2002) parameterisation used in this paper is the best
available for stratospheric conditions, but is essentially an
extrapolation from laboratory measurements at much higher
temperatures and humidities, based on classical nucleation
theory. Figure 11 suggests that, following Pinatubo, strong nu-
cleation occurred throughout the injection height range of
19–27 km for around 6 weeks after the eruption. Although
twice as much SO2 is injected in A_Control20 than in
B_Control10, the nucleation rates in the two runs are simi-
lar for July 1991. This could possibly be indicative of a de-
pletion of oxidants which is limiting SO2 oxidation at this
time, although an alternative explanation might be that there
is much more surface area in the 20 Tg injection run to act
as a condensation sink for sulfuric acid vapour. Nucleation
rates then reduce in magnitude through August and Septem-
ber as the emitted SO2 is completely converted to sulfuric
acid and there is a substantial surface area to provide a con-
densation sink of H2SO4. By October 1991, nucleation rates
in A_Control20 and B_Control10 have returned to similar
values to those found in the quiescent C_noPinatubo simu-
lation. Following the eruption of Mount Pinatubo, the bal-
loon observations at Laramie indicate that, by March 1992
(e.g. Fig. 10b), N150 is increased by a factor of 8, whereas N5
has already returned to pre-eruption values, consistent with
the reduced nucleation rate seen here. As a consequence, N5
and N150 profiles are separated by only a few tens of percent,
indicating that the majority of particles in the lower strato-
sphere have grown larger than 150 nm at that time. This fea-
ture was well captured by the model in runs A_Control20
and B_Control10 with the N5, N150 and N250 profiles being
remarkably similar between the two runs. Our study is the first to fully examine the variation in sim-
ulated particle size distribution through the Pinatubo post-
eruption period, and we therefore choose to document the nu-
cleation rate occurring in our simulations. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11238 dynamics in stratospheric CCMs, and we aim to include and
assess the impact of these feedbacks in a future study (Mann
et al., in prep., 2014). cleated particles grow to sizes larger than 150 nm, with most
remaining at smaller sizes. We note however that nucleation
can be seen in the middle stratosphere at SH mid-latitudes
in the volcanically quiescent C_noPinatubo September 1991
monthly-mean, indicating the occurrence of nucleation in
springtime, as seen in the McMurdo OPC record (Campbell
and Deshler, 2014). Note that the mechanism here is that par-
ticle evaporation and subsequent photolysis of sulfuric acid
leads to a reservoir of SO2 building up during polar winter,
which leads to new particle formation in polar spring (Mills
et al., 2005). This is the same mechanism that is leading to
the layer of elevated N5 at 25–30 km in the March Laramie
profiles (see Fig. 10). p p
Our simulations here indicate that the model is capable
of capturing the main features of the observed evolution of
the particle size distribution very well, with particularly good
agreement with the measurements in the most perturbed post-
eruption period through to mid-1992. However, Fig. 10c and
d suggest that the decay phase is not well captured, with N150
reducing much more slowly than the measurements and the
return to a background size distribution occurs much later
in the model. We have seen that simulated particle concen-
trations in the 5–250 nm size range, whilst agreeing well in
background conditions, have moderate high bias in the first
year after the eruption, with the bias worsening as the model
decays too slowly in the subsequent period. There are sev-
eral possible causes for this model size distribution bias. It
could be that the simplified modal representation of aerosol
dynamics may be only partly capturing the different parti-
cle growth and removal rates across the particle size range. However, it is also worth noting that the largest biases oc-
curred in the lowermost stratosphere and upper troposphere
where STE-related processes may not be well captured in our
low-resolution GCM. Another related issue is that we again
note that these simulations do not include the coupling to dy-
namics which would increase the altitude of the aerosol layer
and reduce concentrations in the lower part of the plume,
where the high bias is mostly evident. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11239 Figure 11. Modelled nucleation rates (cm3 s−1) from runs A_Control20 (left), B_Control10 (middle), and C_noPinatubo (right) for (top to
bottom) July, August, September and October 1991. Figure 11. Modelled nucleation rates (cm3 s−1) from runs A_Control20 (left), B_Control10 (middle), and C_noPinatubo (right) for (top to
bottom) July, August, September and October 1991. cesses are highly uncertain in the stratosphere and the two
additional simulations essentially test how robust the model
is to changes in the model physics. In the first 12 months
after the eruption the tropical and global sAOD is around
80 % higher in run A_Control20 than B_Control10, but in
the second half of 1992 the difference in sAOD between
the two control runs reduces to only around 10 %. In run
D_noPrimary10, sAOD is only very slightly lower than in
B_Control10, suggesting that including the source of pri-
mary particles has only a minor impact on the aerosol evo-
lution post-Pinatubo. The factor-100 reduced nucleation run
E_ScaledStNuc10 causes a prolonged peak in tropical mid-
visible AOD, with values around 10 % higher during Septem-
ber 1991, with E_ScaledStNuc10 continuing to have AOD
around 5 % higher than B_Control10 through the remainder
of the simulation. Intriguingly, the impact of the nucleation
rate reduction on N150 is, in the first 6 months after the erup-
tion, to reduce N150, which is opposite to the slight increase
in mid-visible AOD. This likely is due to a reduced num-
ber of smaller particles growing to radii larger than 150 nm,
with the sAOD increase caused by larger particles which will part of the plume (28 to 30 km). However, the accumulation
mode SAD fraction (Fig. 12c) shows that even during the
early phase of eruption total SAD is primarily determined
by these larger particles in the lower–middle stratosphere. At
a later stage (December 1991, not shown), the contribution
from nucleation and Aitken mode is insignificant and, as ex-
pected, the accumulation mode then contributes the vast ma-
jority of the SAD. We note that in Fig. 7 the model shows
highest biases in simulated SAD against the observations
during the first few months after the eruption. Figure 13 compares tropical (panel a) and global (panel b)
mid-visible sAOD and N150 evolution from the three main
simulations A_Control20, B_Control10 and C_noPinatubo
against the satellite observations from AVHRR. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Figure 11 shows,
for runs A_Control20, B_Control10 and C_noPinatubo, the
zonal-mean nucleation rate against latitude and altitude for
monthly means through August to October 1991. In volcani-
cally quiescent conditions (C_noPinatubo), the model has
nucleation occurring mainly in the tropical upper troposphere
with negligible new particle formation in the stratosphere. Note that the observed and simulated lower stratospheric N5
and N150 profiles at Laramie in March 1991 (Fig. 10a) are in
very good agreement, and Fig. 11 indicates that these strato-
spheric particles were actually formed in the tropical upper
troposphere, consistent with the stratospheric aerosol lifecy-
cle described by Hamill et al. (1997). The observations at
Laramie indicate that only a small proportion of these nu- Since uptake of reactive gases is dependent on particle
size, accounting for the shift in size distribution may also
be important for better quantification of the influence that
volcanically enhanced aerosol has on stratospheric ozone
through accelerated heterogeneous chemistry. We therefore
investigate the evolution of the SAD distribution across the
three stratospheric aerosol modes (Fig. 12) from July 1991,
15–45 days after the eruption (panels a to d) and in October
1991, when aerosol loading was close to its peak (panels e to
h). Nucleation mode particles are always smaller than 10 nm,
so even during July 1991, when substantial nucleation is oc-
curring (Fig. 11), their contribution to total SAD is at most
only around 10 %. However, although the Aitken mode par-
ticles are smaller than 100 nm, during the early part of the
eruption they contribute significantly to SAD in the upper Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM We also
compare time series of simulated N150 at 18 and 22 km al-
titude against the long time series OPC measurements from
Laramie. Also presented in Fig. 13 are results from two
additional 10 Tg simulations, designed to test the sensitiv-
ity of the model predictions to sub-grid particle formation
(run D_noPrimary10) and with much reduced new particle
formation rate (run E_ScaledStNuc10). Both of these pro- Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11240 S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM
a) AOD - 20oS-20oN
1991
1992
1993
1994
1995
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
stratospheric AOD
b) AOD - 60oS-60oN
1991
1992
1993
1994
1995
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
stratospheric AOD
OBS. (AVHRR)
A_Control20
B_Control10
C_noPinatubo
D_noPrimary10
E_ScaledStNuc10
c) N150 at 18km
1991
1992
1993
1994
1995
0
5
10
15
20
N150 conc. (cm-3)
d) N150 at 22km
1991
1992
1993
1994
1995
0
5
10
15
20
N150 conc. (cm-3)
Figure 13. (a) Mean tropical (20◦S, 20◦N) and (b) near-global
(60◦S, 60◦N) sAOD from AVHRR (550 nm) and various model
simulations (525 nm). (c) and (d) show comparison between mod-
elled and observed N150 time series from Laramie (shown with +
sign), at 18 km and 22 km, respectively. a) AOD - 20oS-20oN
1991
1992
1993
1994
1995
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
stratospheric AOD Figure 12. Percentage zonal mean surface area densities in nucle-
ation, Aitken and accumulation modes from run A_Control20 for
July 1991 (panels a, b, c) and December 1991 (panels e, f, g). Total
SAD for July 1991 and December 1991 (µm2 cm−3) are shown in
panels (d) and (h), respectively. b) AOD - 60oS-60oN
1991
1992
1993
1994
1995
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
stratospheric AOD
OBS. (AVHRR)
A_Control20
B_Control10
C_noPinatubo
D_noPrimary10
E_ScaledStNuc10
d) N150 at 22km c) N150 at 18km
1991
1992
1993
1994
1995
0
5
10
15
20
N150 conc. (cm-3) d) N150 at 22km
1991
1992
1993
1994
1995
0
5
10
15
20
N150 conc. (cm-3) Figure 13. (a) Mean tropical (20◦S, 20◦N) and (b) near-global
(60◦S, 60◦N) sAOD from AVHRR (550 nm) and various model
simulations (525 nm). (c) and (d) show comparison between mod-
elled and observed N150 time series from Laramie (shown with +
sign), at 18 km and 22 km, respectively. 6
Summary and conclusions We have extended the UM-UKCA composition-climate
model to incorporate stratospheric sulfur chemistry and up-
dated the process descriptions in the GLOMAP aerosol mi-
crophysics module to be applicable for both tropospheric and
stratospheric conditions. Using stratospheric aerosol changes
after the Mt Pinatubo eruption as a test case, we have evalu-
ated simulated aerosol properties against a wide range of ob-
servations in both quiescent and volcanically perturbed con-
ditions. The improvements to the model enable a prognos-
tic treatment of stratospheric aerosol with dynamically vary-
ing particle size distribution alongside stratospheric transport
and chemistry up to a model top of 80 km. Figure 12. Percentage zonal mean surface area densities in nucle-
ation, Aitken and accumulation modes from run A_Control20 for
July 1991 (panels a, b, c) and December 1991 (panels e, f, g). Total
SAD for July 1991 and December 1991 (µm2 cm−3) are shown in
panels (d) and (h), respectively. In general, the model captures the observed distribution
and evolution of stratospheric aerosol properties well, in
both quiescent and volcanically perturbed conditions. For the
Pinatubo test case, the timing of the peak in global aerosol
mass and decay timescale are captured well compared to val-
ues derived from HIRS satellite measurements (Baran et al.,
1993). However, our control simulation, with a 20 Tg emis-
sion of SO2 produces much too high a burden of aerosol sul-
fur compared to the HIRS measurements, whereas a 10 Tg
injection is in good agreement. The 10 Tg run also com-
pares better to the magnitude of the enhanced sAOD distri-
bution seen in SAGE II and AVHRR, and captures well the
transport to the Southern Hemisphere. Modelled extinction
in the tropical and NH mid-latitude lower stratosphere shows
good agreement with SAGE V7 data in both the mid-visible
and near-infrared. However, for the first 6 months after the have received more gas-to-particle transfer of sulfuric acid,
enhancing condensational growth and increasing their scat-
tering efficiency. In summary, however, although these mi-
crophysical sensitivities are interesting, the results suggest
a low sensitivity to uncertainties in the nucleation rate, and
to model treatment of sub-grid particle formation. The low
sensitivity gives additional credibility to the aerosol micro-
physics models, suggesting the models are robust to known
uncertainties in some processes in stratospheric conditions. have received more gas-to-particle transfer of sulfuric acid,
enhancing condensational growth and increasing their scat-
tering efficiency. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Comparing the evolution and altitude of the high biases
in extinction to those seen in the OPC profile measurements
of the size distribution suggest that the main source of the
biases is in particles in the 150 to 550 nm size range in the
lowermost stratosphere. The discrepancy could be related to
the modal aerosol dynamics failing to capture the differential
growth across the particle size range. However, an alternative
explanation could be that too young age-of-air and too rapid
STE is affecting the simulated stratospheric aerosol evolu-
tion. We also note that enhanced upwelling from radiative
heating of the enhanced aerosol layer, not included in these
uncoupled simulations, will also change the vertical distri-
bution and transport of the aerosol. It is worth noting that
in radiatively coupled simulations, we expect that increased
tropical upwelling would dilute the lower part of the plume,
decreasing particle concentrations in the lowermost strato-
sphere. eruption simulated sAOD and SAD are larger than the satel-
lite measurements, and the model enhancement in Reff is too
low (e.g. compared to Bauman et al., 2003). Lack of radia-
tive coupling in these simulations is likely to be the dominant
contribution to the high AOD, since radiative heating of the
Pinatubo cloud in the tropical lower stratosphere is known to
have enhanced upwelling and reduces aerosol optical depth
in model simulations (e.g. Young et al., 1994). To better understand how the particle size distribution was
perturbed during the Pinatubo eruption, we have compared
against mid-latitude balloon-borne measurements during that
time period, allowing a strong observational constraint on
concentrations of particles larger than 5, 150, 250, 550, 750
and 1000 nm. Although there have been many model studies
covering the Pinatubo period over the 22 yr since the erup-
tion, to our knowledge, this is the first time the full profile
of a simulated size distribution in a global model has been
compared to these measurements in volcanically perturbed
conditions. Overall, the general good agreement with the size dis-
tribution measurements from Laramie and the global effec-
tive radius evolution from satellite suggest that the modal
aerosol microphysics module used in our CCM is capable of
representing the variation in particle size distribution in the
strongly volcanically perturbed post-Pinatubo period. The
sensitivity simulations also suggest that such simulated per-
turbations to stratospheric aerosol properties are robust to
known uncertainties in nucleation rate and sub-grid parti-
cle formation. 6
Summary and conclusions In summary, however, although these mi-
crophysical sensitivities are interesting, the results suggest
a low sensitivity to uncertainties in the nucleation rate, and
to model treatment of sub-grid particle formation. The low
sensitivity gives additional credibility to the aerosol micro-
physics models, suggesting the models are robust to known
uncertainties in some processes in stratospheric conditions. Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ 11241 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Our results underline the importance of bet-
ter constraining transport and growth of Aitken-mode-sized
particles in the first few months after the eruption to improve
prediction of volcanic impacts on climate with stratospheric
aerosol microphysics models. The findings highlight the need
for a coordinated set of experiments to inter-compare and
evaluate current global stratospheric aerosol models against
the wide set of observations available through the Pinatubo-
perturbed period. The model finds that in the volcanically quiescent strato-
sphere, nucleation occurs only during polar spring, with
stratospheric particles at Laramie mostly originating from the
tropical upper troposphere. In such background conditions
the model agrees very well with size distribution observed
at Laramie, with only a small proportion of these nucleated
particles have grown to 150 nm by coagulation and conden-
sation, with N5 larger than N150 by around a factor of 10. We have investigated the impact on the size distribution of
20 and 10 Tg tropical injections of SO2 from Pinatubo. g
p
j
2
In the first 2 months after the eruption, nucleation is found
to occur throughout the volcanic plume and the large in-
jection of SO2 leads to strong growth of these particles to-
gether with growth of older particles formed in the tropi-
cal upper troposphere. Comparing the 10 and 20 Tg control
simulations, we find much larger relative difference between
concentrations of particles larger than 550 nm between runs
A_Control20 and B_Control10 than in the smaller particles
between 150 and 550 nm, which may be indicative of two
types of volcanically enhanced particles. Overall the simu-
lated profile of the particle size distribution agrees remark-
ably well with the observations, capturing most of the com-
plex shape of the concentration profiles in the different size
channels. However, the decay timescale for N150 is slower in
the model than the observations, which leads to an initially
modest high bias increasing to around a factor of two by mid-
1993 (e.g. Fig. 13), with the return to a background size dis-
tribution occurring much later in the model. The spatial and
temporal evolution of the Reff in the lower stratosphere seen
by satellite (Bauman et al., 2003) is also well captured by the
model, albeit with a low bias in size compared to the mea-
surements. www.atmos-chem-phys.net/14/11221/2014/ Atmos. Chem. Phys., 14, 11221–11246, 2014 References Carslaw, K. and Kärcher, B.: Stratospheric aerosol processes. In:
Thomason L. and Peter Th. (Eds.) SPARC Assessment of Strato-
spheric Aerosol Properties (ASAP), pp. 1–28, SPARC Report
No. 4, World Climate Research Programme, 2006. Andres, R. and Kasgnoc, A.: A time-averaged inventory of subaerial
volcanic sulfur emissions, J. Geophys. Res.-Atmos., 103, 25251–
25261, 1998. Aquila, V., Oman, L. D., Stolarski, R. S., Colarco, P. R., and New-
man, P. A.: Dispersion of the volcanic sulfate cloud from a Mount
Pinatubo-like eruption, J. Geophys. Res.-Atmos., 117, D06216,
doi:10.1029/2011JD016968, 2012. Carslaw, K. S., Luo, B., and Peter, T.: An analytic expression for the
composition of aqueous HNO3–H2SO4 stratospheric aerosols
including gas phase removal of HNO3, Geophys. Res. Lett., 22,
1877–1880, 1995. Arfeuille, F., Luo, B. P., Heckendorn, P., Weisenstein, D., Sheng,
J. X., Rozanov, E., Schraner, M., Brönnimann, S., Thomason,
L. W., and Peter, T.: Modeling the stratospheric warming follow-
ing the Mt. Pinatubo eruption: uncertainties in aerosol extinc-
tions, Atmos. Chem. Phys., 13, 11221–11234, doi:10.5194/acp-
13-11221-2013, 2013. Carver, G. D., Brown, P. D., and Wild, O.: The ASAD atmospheric
chemistry integration package and chemical reaction database,
Comput. Phys. Commun., 105, 197–215, 1997. Chipperfield, M.: New version of the TOMCAT/SLIMCAT of-
fline chemical transport model: Intercomparison of stratospheric
tracer experiments, Q. J. Roy. Meteor. Soc., 132, 1179–1203,
2006. Arnold, F., Fabian, R., and Joos, W.: Measurements of the height
variation of sulfuric acid vapor concentrations in the strato-
sphere, Geophys. Res. Lett., 8, 293–296, 1981. Chipperfield, M. and Pyle, J.: Model sensitivity studies of Arctic
ozone depletion, J. Geophys. Res.-Atmos., 103, 28389–28403,
1998. Baran, A. J. and Foot, J. S.: New application of the operational
sounder HIRS in determining a climatology of sulphuric acid
aerosol from the Pinatubo eruption, J. Geophys. Res.-Atmos., 99,
25673–25679, 1994. Chipperfield, M., Liang, Q., Strahan, S., Morgenstern, O., Dhomse,
S., Abraham, N., Archibald, A., Bekki, S., Braesicke, P., Di Gen-
ova, G., Fleming, E. L., Hardiman, S. C., Iachetti, D., Jack-
man, C. H., Kinnison, D. E., Marchand, M., Pitari, G., Pyle,
J. A., Rozanov, E., Stenke, A., and Tummon, F.: Multimodel es-
timates of atmospheric lifetimes of long-lived ozone-depleting
substances: Present and future, J. Geophys. Res.-Atmos., 119,
2555–2573, 2014. Baran, A. J., Foot, J. S., and Dibben, P. C.: Satellite detection of
volcanic sulfuric-acid aerosol, Geophys. Res. Lett., 20, 1799–
1801, 1993. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM We acknowledge
use of the MONSooN system, a collaborative facility supplied
under the Joint Weather and Climate Research Programme, which
is a strategic partnership between the UK Met Office and the
Natural Environment Research Council. This work has been
also supported by NIWA as part of its Government-funded, core
research programme. The in situ measurements were supported by
the US National Science Foundation. Current measurements are
supported under grant number ATM-0437406. Bekki, S.: Oxidation of volcanic SO2: a sink for stratospheric OH
and H2O, Geophys. Res. Lett., 22, 913–916, 1995. Bekki, S. and Pyle, J.: Two dimensional assessment of the impact
of aircraft sulphur emissions on the stratospheric sulphate aerosol
layer, J. Geophys. Res.-Atmos., 97, 15839–15847, 1992. Bekki, S. and Pyle, J.: A two-dimensional modeling study of the
volcanic eruption of Mount Pinatubo, J. Geophys. Res.: Atmo-
spheres (1984–2012), 99, 18861–18869, 1994. Binkowski, F. S. and Roselle, S. J.: Models-3 Community Mul-
tiscale Air Quality (CMAQ) model aerosol component 1. Model description, J. Geophys. Res.-Atmos., 108, D64183,
doi:10.1029/2001JD001409, 2003. Bluth, G. J., Doiron, S. D., Schnetzler, C. C., Krueger, A. J., and
Walter, L. S.: Global tracking of the SO2 clouds from the June,
1991 Mount Pinatubo eruptions, Geophys. Res. Lett., 19, 151–
154, 1992. Bluth, G. J. S., Rose, W. I., Sprod, I. E., and Krueger, A. J.: Strato-
spheric loading of sulfur from explosive volcanic eruptions, Jour-
nal of Geology, 105, 671–683, 1997. Braesicke, P., Keeble, J., Yang, X., Stiller, G., Kellmann, S., Abra-
ham, N. L., Archibald, A., Telford, P., and Pyle, J. A.: Circula-
tion anomalies in the Southern Hemisphere and ozone changes,
Atmos. Chem. Phys., 13, 10677–10688, 2013,
http://www.atmos-chem-phys.net/13/10677/2013/. Brühl, C., Lelieveld, J., Höpfner, M., and Tost, H.: Stratospheric
SO2 and sulphate aerosol, model simulations and satellite ob-
servations, Atmos. Chem. Phys. Discussions, 13, 11 395–11 425,
doi:10.5194/acpd-13-11395-2013, 2013. Edited by: T. Röckmann Edited by: T. Röckmann Campbell, P. and Deshler, T.: Condensation nuclei measurements in
the midlatitude (1982–2012) and Antarctic (1986–2010) strato-
sphere between 20 and 35 km, J. Geophys. Res.-Atmos., 119,
137–152, 2014. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11242 Acknowledgements. We would like to thank P. B. Russell for
effective radius data. This work was supported by the UK Natural
Environment Research Council (NERC grants NE/E005659/1
and NE/E017150/1). GWM and KSC received funding from
the National Centre for Atmospheric Science, one of the NERC
research centres. GWM received EU funding from the European
Research Council (ERC) under Seventh Framework Programme
(FP7) consortium projects MACC and MACC-II (grant agreements
218793 and 283576 respectively). NB, MD, CEJ, FOC were
supported as part the UK Integrated Climate Programme funded by
the Department for Energy and Climate Change (DECC) and De-
partment for Environment Food and Rural Affairs – DECC/Defra
(GA01101). RH was partly funded by German Federal Ministry of
Education and Research (BMBF) project ROSA (reference code
01LG1212A) within the ROMIC (Role Of the Middle Atmosphere
in Climate) research programme. We would also like to thank James
Keeble for his help during model development. We acknowledge
use of the MONSooN system, a collaborative facility supplied
under the Joint Weather and Climate Research Programme, which
is a strategic partnership between the UK Met Office and the
Natural Environment Research Council. This work has been
also supported by NIWA as part of its Government-funded, core
research programme. The in situ measurements were supported by
the US National Science Foundation. Current measurements are
supported under grant number ATM-0437406. Acknowledgements. We would like to thank P. B. Russell for
effective radius data. This work was supported by the UK Natural
Environment Research Council (NERC grants NE/E005659/1
and NE/E017150/1). GWM and KSC received funding from
the National Centre for Atmospheric Science, one of the NERC
research centres. GWM received EU funding from the European
Research Council (ERC) under Seventh Framework Programme
(FP7) consortium projects MACC and MACC-II (grant agreements
218793 and 283576 respectively). NB, MD, CEJ, FOC were
supported as part the UK Integrated Climate Programme funded by
the Department for Energy and Climate Change (DECC) and De-
partment for Environment Food and Rural Affairs – DECC/Defra
(GA01101). RH was partly funded by German Federal Ministry of
Education and Research (BMBF) project ROSA (reference code
01LG1212A) within the ROMIC (Role Of the Middle Atmosphere
in Climate) research programme. We would also like to thank James
Keeble for his help during model development. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Tavolato, C., Thapaut, J.-N., and Vitart, F.: The ERA-Interim re-
analysis: Configuration and performance of the data assimilation Deshler, T.: In situ measurements of Pinatubo aerosol over Kiruna
on four days between 18 January and 13 February 1992, Geo-
phys. Res. Lett., 21, 1323–1326, 1994. Deshler, T.: A review of global stratospheric aerosol: Measure-
ments, importance, life cycle, and local stratospheric aerosol, At-
mospheric Research, 90, 223–232, 2008. Hansen, J., Lacis, A., Ruedy, R., and Sato, M.: Potential climate im-
pact of Mount Pinatubo eruption, Geophys. Res. Lett., 19, 215–
218, 1992. Deshler, T., Hofmann, D., Johnson, B., and Rozier, W.: Balloon-
borne measurements of the Pinatubo aerosol size distribution and
volatility at Laramie, Wyoming during the summer of 1991, Geo-
phys. Res. Lett., 19, 199–202, 1992. Hewitt, H. T., Copsey, D., Culverwell, I. D., Harris, C. M., Hill, R. S. R., Keen, A. B., McLaren, A. J., and Hunke, E. C.: Design
and implementation of the infrastructure of HadGEM3: the next-
generation Met Office climate modelling system, Geosci. Model
Dev., 4, 223–253, doi:10.5194/gmd-4-223-2011, 2011. Deshler, T., Johnson, B. J., and Rozier, W. R.: Balloonborne mea-
surements of Pinatubo aerosol during 1991 and 1992 at 41◦N:
Vertical profiles, size distribution, and volatility, Geophys. Res. Lett., 20, 1435–1438, doi:10.1029/93GL01337, 1993. Hofmann, D. and Rosen, J.: Balloo-sorne observations of strato-
spheric aerosol and condensation nuclei during the year follow-
ing the Mt. St. Helens eruption, J. Geophys. Res.-Ocean, 87,
11039–11061, 1982. Deshler, T., Hervig, M. E., Hofmann, D. J., Rosen, J. M., and Liley,
J. B.: Thirty years of in situ stratospheric aerosol size distri-
bution measurements from Laramie, Wyoming (41 degrees N),
using balloon-borne instruments, J. Geophys. Res.-Atmos., 108,
D54167, doi:10.1029/2002JD002514, 2003. Hofmann, D. and Rosen, J.: On the temporal variation of strato-
spheric aerosol size and mass during the first 18 months follow-
ing the 1982 eruptions of El Chichón, J. Geophys. Res.-Atmos,
89, 4883–4890, 1984. Dhomse, S., Weber, M., Wohltmann, I., Rex, M., and Burrows,
J. P.: On the possible causes of recent increases in northern
hemispheric total ozone from a statistical analysis of satellite
data from 1979 to 2003, Atmos. Chem. Phys., 6, 1165–1180,
doi:10.5194/acp-6-1165-2006, 2006. Holton, J. R. and Tan, H.-C.: The influence of the equatorial quasi-
biennial oscillation on the global circulation at 50 mb, J. Atmos. Sci., 37, 2200–2208, 1980. Holton, J. R., Haynes, P. H., McIntyre, M. E., Douglass, A. R.,
Rood, R. References Bauman, J., Russell, P., Geller, M., and Hamill, P.: A stratospheric
aerosol climatology from SAGE II and CLAES measurements:
2. Results and comparisons, 1984–1999, J. Geophys. Res., 108,
D134383, doi:10.1029/2002JD002993, 2003. Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11243 Guo, S., Bluth, G. J., Rose, W. I., Watson, I. M., and Prata, A.: Re-
evaluation of SO2 release of the 15 June 1991 Pinatubo eruption
using ultraviolet and infrared satellite sensors, Geochem. Geo-
phy. Geosys., 5, Q04001, doi:10.1029/2003GC000654, 2004a. D’Almeida, G. A., Koepke, P., and Shettle, E. P.: Atmospheric
aerosols: global climatology and radiative characteristics, A. Deepak Pub. Hampton, Virginia, USA, 1991. Damadeo, R. P., Zawodny, J. M., Thomason, L. W., and Iyer, N.:
SAGE version 7.0 algorithm: application to SAGE II, Atmos. Meas. Tech. Discuss., 6, 5101–5171, doi:10.5194/amtd-6-5101-
2013, 2013. Guo, S., Rose, W. I., Bluth, G. J., and Watson, I. M.: Par-
ticles in the great Pinatubo volcanic cloud of June 1991:
The role of ice, Geochem. Geophy. Geosys., 5, Q05003,
doi:10.1029/2003GC000655, 2004b. Dee, D. P., Uppala, S. M., Simmons, A. J., Berrisford, P., Poli,
P., Kobayashi, S., Andrae, U., Balmaseda, M. A., Balsamo, G.,
Bauer, P., Bechtold, P., Beljaars, A. C. M., van de Berg, L., Bid-
lot, J., Bormann, N., Delsol, C., Dragani, R., Fuentes, M., Geer,
A. J., Haimberger, L., Healy, S., Hersbach, H., Holm, E. V., Isak-
sen, L., Kallberg, P., Koehler, M., Matricardi, M., McNally, A. P.,
Monge-Sanz, B. M., Morcrette, J.-J., Peubey, C., de Rosnay, P.,
Tavolato, C., Thapaut, J.-N., and Vitart, F.: The ERA-Interim re-
analysis: Configuration and performance of the data assimilation
system, Q. J. R. Meteor. Soc., 133, 1972–1990, 2011. Hall, T. M., Waugh, D. W., Boering, K. A., and Plumb, R. A.: Eval-
uation of transport in stratospheric models, J. Geophys. Res.-
Atmos., 104, 18815–18839, 1999. lot, J., Bormann, N., Delsol, C., Dragani, R., Fuentes, M., Geer, Hamill, P., Jensen, E. J., Russell, P., and Bauman, J. J.: The life
cycle of stratospheric aerosol particles, B. Am. Meteorol. Soc.,
78, 1395–1410, 1997. A. J., Haimberger, L., Healy, S., Hersbach, H., Holm, E. V., Isak- sen, L., Kallberg, P., Koehler, M., Matricardi, M., McNally, A. P., Monge-Sanz, B. M., Morcrette, J.-J., Peubey, C., de Rosnay, P., Hamill, P., Brogniez, C., Thomason, L., Deshler, T., Antuña, J.,
Baumgardner, D., Bevilacqua, R., Brock, C., David, C., Fussen,
D., Hervig, M., Hostettler, C. A., Lee, S.-H., Mergenthaler, J.,
Osborn, M. T., Raga, G., Reeves, J. M., Rosen, J., and Wilson,
J. C.: Instrument Descriptions, edited by: Thomason, L. and Pe-
ter, Th., SPARC Assessment of Stratospheric Aerosol Properties
(ASAP), 77–106, SPARC Report No. 4, World Climate Research
Programme, 2006. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11244 ber concentration for atmospheric nucleation events, J. Aerosol
Sci., 33, 609–622, 2002. Martin, E., George, C., and Mirabel, P.: Densities and surface ten-
sions of H2SO4/HNO3/H2O solutions, Geophys. Res. Lett., 27,
197–200, 2000. Kettle, A. and Andreae, M.: Flux of dimethylsulfide from the
oceans: A comparison of updated data sets and flux models, J. Geophys. Res.-Atmos., 105, 26793–26808, 2000. McCormick, M. and Veiga, R.: SAGE II measurements of early
Pinatubo aerosols, Geophys. Res. Lett., 19, 155–158, 1992. McCormick, M. P., Thomason, L. W., and Trepte, C. R.: Atmo-
spheric effects of the Mt Pinatubo eruption, Nature, 373, 399–
404, 1995. Kokkola, H., Hommel, R., Kazil, J., Niemeier, U., Partanen, A.-I.,
Feichter, J., and Timmreck, C.: Aerosol microphysics modules in
the framework of the ECHAM5 climate model – intercomparison
under stratospheric conditions, Geosci. Model Dev., 2, 97–112,
doi:10.5194/gmd-2-97-2009, 2009. Mills, M. J., Toon, O. B., Vaida, V., Hintze, P. E., Kjaergaard,
H. G., Schofield, D. P., and Robinson, T. W.: Photolysis of
sulfuric acid vapor by visible light as a source of the polar
stratospheric CN layer, J. Geophys. Res.-Atmos., 110, D08201,
doi:10.1029/2004JD005519D8, 2005. Kreidenweis, S. M., Walcek, C. J., Feingold, G., Gong, W., Ja-
cobson, M. Z., Kim, C.-H., Liu, X., Penner, J. E., Nenes, A.,
and Seinfeld, J. H.: Modification of aerosol mass and size dis-
tribution due to aqueous-phase SO2 oxidation in clouds: Com-
parisons of several models, J. Geophys. Res., 108, D74213,
doi:10.1029/2002JD002697, 2003. Montzka, S., Calvert, P., Hall, B., Elkins, J., Conway, T., Tans,
P., and Sweeney, C.: On the global distribution, seasonality,
and budget of atmospheric carbonyl sulfide (COS) and some
similarities to CO2, J. Geophys. Res.-Atmos., 112, D09302,
doi:10.1029/2006JD007665, 2007. Kulmala, M. and Laaksonen, A.: Binary nucleation of water-
sulfuric acid system: Comparison of classical theories with dif-
ferent H2SO4 saturation vapor pressures, J. Chem. Phys., 93,
696, 1990. Morgenstern, O., Braesicke, P., O’Connor, F., Bushell, A., John-
son, C., Osprey, S., and Pyle, J.: Evaluation of the new UKCA
climate-composition model Part 1: The stratosphere, Geosci. Model Dev., 2, 43–57, 2009. Kulmala, M., Laaksonen, A., and Pirjola, L.: Parameterizations for
sulfuric acid/water nucleation rates, J. Geophys. Res.-Atmos.,
103, 8301–8307, 1998. Neu, J. L., Prather, M. J., and Penner, J. E.: Global atmospheric
chemistry: Integrating over fractional cloud cover, J. Geophys. Res.-Atmos., 112, D11306, doi:10.1029/2006JD008007, 2007. Labitzke, K. and McCormick, M.: Stratospheric temperature in-
creases due to Pinatubo aerosols, Geophys. Res. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Lett., 19, 207–
210, 1992. Niemeier, U., Timmreck, C., Graf, H.-F., Kinne, S., Rast, S., and
Self, S.: Initial fate of fine ash and sulfur from large volcanic
eruptions, Atmos. Chem. Phys., 9, 9043–9057, doi:10.5194/acp-
9-9043-2009, 2009. Lacis, A., Hansen, J., and Sato, M.: Climate forcing by stratospheric
aerosols, Geophys. Res. Lett., 19, 1607–1610, 1992. Lamarque, J.-F., Bond, T. C., Eyring, V., Granier, C., Heil, A.,
Klimont, Z., Lee, D., Liousse, C., Mieville, A., Owen, B.,
Schultz, M. G., Shindell, D., Smith, S. J., Stehfest, E., Van Aar-
denne, J., Cooper, O. R., Kainuma, M., Mahowald, N., Mc-
Connell, J. R., Naik, V., Riahi, K., and van Vuuren, D. P.: His-
torical (1850–2000) gridded anthropogenic and biomass burning
emissions of reactive gases and aerosols: methodology and ap-
plication, Atmos. Chem. Phys., 10, 7017–7039, doi:10.5194/acp-
10-7017-2010, 2010. Oman, L., Robock, A., Stenchikov, G. L., Thordarson, T.,
Koch, D., Shindell, D. T., and Gao, C. C.: Modeling the
distribution of the volcanic aerosol cloud from the 1783–
1784 Laki eruption, J. Geophys. Res.-Atmos., 111, D12209,
doi:10.1029/2005JD006899, 2006. Pitari, G. and Mancini, E.: Short-term climatic impact of the 1991
volcanic eruption of Mt. Pinatubo and effects on atmospheric
tracers, Natural Hazards and Earth System Science, 2, 91–108,
doi:10.5194/nhess-2-91-2002, 2002. Lambert, A., Grainger, R., Remedios, J., Rodgers, C., Corney,
M., and Taylor, F.: Measurements of the evolution of the Mt. Pinatubo aerosol cloud by ISAMS, Geophys. Res. Lett., 20,
1287–1290, 1993. Reeves, J., Wilson, J., Brock, C., and Bui, T.: Comparison of aerosol
extinction coefficients, surface area density, and volume density
from SAGE II and in situ aircraft measurements, J. Geophys. Res.-Atmos., 113, D10202, doi:10.1029/2007JD009357113,
2008. Liss, P. S. and Merlivat, L.: Air-sea gas exchange rates: Introduction
and synthesis, 113–127, Springer, Netherlands, 1986. Robock, A.: Volcanic eruptions and climate, Rev. Geophys., 38,
191–219, 2000. Long, C. S. and Stowe, L. L.: Using the NOAA/AVHRR to
study stratospheric aerosol optical thicknesses following the Mt. Pinatubo eruption, Geophys. Res. Lett., 21, 2215–2218, 1994. Robock, A. and Mao, J.: Winter warming from large volcanic erup-
tions, Geophys. Res. Lett., 19, 2405–2408, 1992. Mann, G., Carslaw, K., Spracklen, D., Ridley, D., Manktelow, P.,
Chipperfield, M., Pickering, S., and Johnson, C.: Description
and evaluation of GLOMAP-mode: a modal global aerosol mi-
crophysics model for the UKCA composition-climate model,
Geosci. Model Dev., 3, 519–551, 2010. Rosen, J. M.: The vertical distribution of dust to 30 kilometers, J. Geophys. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM B., and Pfister, L.: Stratosphere-troposphere exchange,
Rev. Geophys., 33, 403–439, 1995. Driscoll, S., Bozzo, A., Gray, L. J., Robock, A., and Stenchikov,
G.: Coupled Model Intercomparison Project 5 (CMIP5) simula-
tions of climate following volcanic eruptions, J. Geophys. Res.-
Atmos., 117, D17105, doi:10.1029/2012JD017607, 2012. Hommel, R., Timmreck, C., and Graf, H.: The global middle-
atmosphere aerosol model MAECHAM5-SAM2: comparison
with satellite and in-situ observations, Geosci. Model Dev., 4,
809–834, doi:10.5194/gmd-4-809-2011, 2011. English, J. M., Toon, O. B., and Mills, M. J.: Microphysical simula-
tions of sulfur burdens from stratospheric sulfur geoengineering,
Atmos. Chem. Phys., 12, 4775–4793, doi:10.5194/acp-12-4775-
2012, 2012. Hurrell, J. W., Hack, J. J., Shea, D., Caron, J. M., and Rosinski, J.:
A new sea surface temperature and sea ice boundary dataset for
the Community Atmosphere Model, J. Climate, 21, 5145–5153,
2008. English, J. M., Toon, O. B., and Mills, M. J.: Microphysical simula-
tions of large volcanic eruptions: Pinatubo and Toba, J. Geophys. Res.-Atmos., 118, 1880–1895, doi:10.1002/jgrd.50196, 2013. Jones, C., Hughes, J., Bellouin, N., Hardiman, S., Jones, G., Knight,
J., Liddicoat, S., O’Connor, F., Andres, R. J., and Bell, C.:
The HadGEM2-ES implementation of CMIP5 centennial sim-
ulations, Geosci. Model Dev., 4, 543–570, 2011. Graf, H. F., Kirchner, I., Robock, A., and Schult, I.: Pinatubo erup-
tion winter climate effects: Model versus observations, Clim. Dy-
nam., 9, 81–93, 1993. Grainger, R., Lambert, A., Taylor, F., Remedios, J., Rodgers, C.,
Corney, M., and Kerridge, B.: Infrared absorption by volcanic
stratospheric aerosols observed by ISAMS, Geophys. Res. Lett.,
20, 1283–1286, 1993. Kerminen, V.-M. and Kulmala, M.: Analytical formulae connecting
the "real" and the “apparent” nucleation rate and the nuclei num- www.atmos-chem-phys.net/14/11221/2014/ Atmos. Chem. Phys., 14, 11221–11246, 2014 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Thomason, L., Poole, L., and Deshler, T.: A global climatology of
stratospheric aerosol surface area density deduced from Strato-
spheric Aerosol and Gas Experiment II measurements 1984–
1994, J. Geophys. Res.-Atmos., 102, 8967–8976, 1997. SPARC: SPARC Assessment of stratospheric aerosol properties
(ASAP), SPARC Report No. 4, World Clim. Res. Programme,
WCRP-124, WMO/TD-No.1295, 2006. Timmreck, C.: Three-dimensional simulation of stratospheric back-
ground aerosol: First results of a multiannual general circulation
model simulation, J. Geophys. Res.-Atmos., 106, 28313–28332,
2001. SPARC: SPARC Report on the Evaluation of Chemistry-Climate
Models, World Clim. Res. Programme, WCRP-132, WMO/TD-
No.1526, 2010. SPARC: SPARC Report on Lifetimes of Stratospheric Ozone-
Depleting Substances, Their Replacements, and Related Species,
SPARC Report No. 6, World Clim. Res. Programme, WCRP-
15/2013, 2013. Timmreck, C., Graf, H.-F., and Feichter, J.: Simulation of Mt. Pinatubo volcanic aerosol with the Hamburg climate model
ECHAM4, Theor. Appl. Climatol., 62, 85–108, 1999. Toohey, M., Krüger, K., Niemeier, U., and Timmreck, C.: The influ-
ence of eruption season on the global aerosol evolution and radia-
tive impact of tropical volcanic eruptions, Atmos. Chem. Phys.,
11, 12351–12367, doi:10.5194/acp-11-12351-2011, 2011. Spracklen, D. V., Pringle, K. J., Carslaw, K. S., Chipperfield, M. P.,
and Mann, G. W.: A global off-line model of size-resolved
aerosol microphysics: I. Model development and prediction
of aerosol properties, Atmos. Chem. Phys., 5, 2227–2252,
doi:10.5194/acp-5-2227-2005, 2005. Trepte, C. R. and Hitchman, M. H.: Tropical stratospheric circula-
tion deduced from satellite aerosol data, Nature, 355, 626–628,
1992. Stenchikov, G. L., Kirchner, I., Robock, A., Graf, H., Antuna, J. C.,
Grainger, R., Lambert, A., and Thomason, L.: Radiative forcing
from the 1991 Mount Pinatubo volcanic eruption, J. Geophys. Res.-Atmos., 103, 13837–13857, 1998. Vehkamäki, H., Kulmala, M., Napari, I., Lehtinen, K. E., Timmreck,
C., Noppel, M., and Laaksonen, A.: An improved parameteri-
zation for sulfuric acid–water nucleation rates for tropospheric
and stratospheric conditions, J. Geophys. Res., 107, D224622,
doi:10.1029/2002JD002184, 2002. Stier, P., Feichter, J., Kinne, S., Kloster, S., Vignati, E., Wilson, J.,
Ganzeveld, L., Tegen, I., Werner, M., Balkanski, Y., Schulz, M.,
Boucher, O., Minikin, A., and Petzold, A.: The aerosol-climate
model ECHAM5-HAM, Atmos. Chem. Phys., 5, 1125–1156,
doi:10.5194/acp-5-1125-2005, 2005. Vignati, E., Wilson, J., and Stier, P.: M7: An efficient size Re-
solved aerosol microphysics module for large scale aerosol
transport models, J. Geophys. Res.-Atmos., 109, D22202,
doi:10.1029/2003JD004485, 2004. Stokes, R. and Robinson, R.: Interactions in aqueous nonelectrolyte
solutions. I. Solute-solvent equilibria, J. Phys. Chem., 70, 2126–
2131, 1966. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11245 Taylor, K. E., Stouffer, R. J., and Meehl, G. A.: An overview of
CMIP5 and the experiment design, B. Am. Meteorol. Soc., 93,
485–498, 2012. JPL Publication 06-2, Vol. 4915, Jet Propulsion Laboratory, Cal-
ifornia Insitute of Technology, Pasadena, California, 2006. Sato, M., Hansen, J. E., McCormick, M. P., and Pollack, J. B.:
Stratospheric aerosol optical depths, 1850–1990, J. Geophys. Res.-Atmos., 98, 22987–22994, 1993. Telford, P., Abraham, N., Archibald, A., Braesicke, P., Dalvi, M.,
Morgenstern, O., O’Connor, F., Richards, N., and Pyle, J.: Im-
plementation of the Fast-JX Photolysis scheme (v6. 4) into the
UKCA component of the MetUM chemistry-climate model (v7. 3), Geosci. Model Dev., 6, 161–177, 2013. Saunders, R. W., Dhomse, S., Tian, W. S., Chipperfield, M. P., and
Plane, J. M. C.: Interactions of meteoric smoke particles with
sulphuric acid in the Earth’s stratosphere, Atmos. Chem. Phys.,
12, 4387–4398, doi:10.5194/acp-12-4387-2012, 2012. Textor, C., Schulz, M., Guibert, S., Kinne, S., Balkanski, Y.,
Bauer, S., Berntsen, T., Berglen, T., Boucher, O., Chin, M.,
Dentener, F., Diehl, T., Easter, R., Feichter, H., Fillmore, D.,
Ghan, S., Ginoux, P., Gong, S., Grini, A., Hendricks, J.,
Horowitz, L., Huang, P., Isaksen, I., Iversen, I., Kloster, S.,
Koch, D., Kirkevåg, A., Kristjansson, J. E., Krol, M., Lauer, A.,
Lamarque, J. F., Liu, X., Montanaro, V., Myhre, G., Penner, J.,
Pitari, G., Reddy, S., Seland, Ø., Stier, P., Takemura, T., and
Tie, X.: Analysis and quantification of the diversities of aerosol
life cycles within AeroCom, Atmos. Chem. Phys., 6, 1777–1813,
doi:10.5194/acp-6-1777-2006, 2006. Scaife, A. A., Spangehl, T., Fereday, D. R., Cubasch, U., Lange-
matz, U., Akiyoshi, H., Bekki, S., Braesicke, P., Butchart, N., and
Chipperfield, M. P.: Climate change projections and stratosphere-
troposphere interaction, Clim. Dynam., 38, 2089–2097, 2012. Ghan, S., Ginoux, P., Gong, S., Grini, A., Hendricks, J., Horowitz, L., Huang, P., Isaksen, I., Iversen, I., Kloster, S., Solomon, S., Portmann, R. W., Garcia, R. R., Thomason, L. W.,
Poole, L. R., and McCormick, M. P.: The role of aerosol varia-
tions in anthropogenic ozone depletion at northern midlatitudes,
J. Geophys. Res.-Atmos., 101, 6713–6727, 1996. Koch, D., Kirkevåg, A., Kristjansson, J. E., Krol, M., Lauer, A., Solomon, S., Daniel, J., Neely, R., Vernier, J.-P., Dutton, E.,
and Thomason, L.: The persistently variable background strato-
spheric aerosol layer and global climate change, Science, 333,
866–870, 2011. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Res., 69, 4673–4676, 1964. Russell, P., Livingston, J., Pueschel, R., Bauman, J., Pollack, J.,
Brooks, S., Hamill, P., Thomason, L., Stowe, L., and Deshler, T.:
Global to microscale evolution of the Pinatubo volcanic aerosol
derived from diverse measurements and analyses, J. Geophys. Res., 101, 18745–18763, 1996. Mann, G. W., Carslaw, K. S., Ridley, D. A., Spracklen, D. V.,
Pringle, K. J., Merikanto, J., Korhonen, H., Schwarz, J. P.,
Lee, L. A., Manktelow, P. T., Woodhouse, M. T., Schmidt, A.,
Breider, T. J., Emmerson, K. M., Reddington, C. L., Chipper-
field, M. P., and Pickering, S. J.: Intercomparison of modal and
sectional aerosol microphysics representations within the same
3-D global chemical transport model, Atmos. Chem. Phys., 12,
4449–4476, doi:10.5194/acp-12-4449-2012, 2012. Sander, S., Friedl, R., Golden, D., Kurylo, M., Huie, R., Orkin, V.,
Moortgat, G., Wine, P., Ravishankara, A., Kolb, C., Molina, M.,
Finlayson-Pitts, B., Huie, R., Orkin, V. L., and Keller-Rudek, H.:
Chemical kinetics and photochemical data for use in atmospheric
studies, NASA Panel for Data Evaluation, Evaluation number 15, Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Weber, M., Dhomse, S., Wittrock, F., Richter, A., Sinnhuber, B. M.,
and Burrows, J. P.: Dynamical control of NH and SH win-
ter/spring total ozone from GOME observations in 1995-2002,
Geophys. Res. Lett., 30, 2003. Strahan, S. E., Douglass, A. R., Stolarski, R. S., Akiyoshi, H.,
Bekki, S., Braesicke, P., Butchart, N., Chipperfield, M. P.,
Cugnet, D., Dhomse, S., Frith, S. M., Gettelman, A., Hardi-
man, S. C., Kinnison, D. E., Lamarque, J. F., Mancini, E., Marc-
hand, M., Michou, M., Morgenstern, O., Nakamura, T., Olivie,
D., Pawson, S., Pitari, G., Plummer, D. A., Pyle, J. A., Scinocca,
J. F., Shepherd, T. G., Shibata, K., Smale, D., Teyssedre, H., Tian,
W., and Yamashita, Y.: Using transport diagnostics to understand
chemistry climate model ozone simulations, J. Geophys. Res.-
Atmos., 116, D17302, doi:doi:10.1029/2010JD015360, 2011. Weisenstein, D., Bekki, S., Mills, M., Pitari, G., and Timmreck, C.:
Modeling of stratospheric aerosols, edited by: Thomason, L. and
Peter, Th., SPARC Assessment of Stratospheric Aerosol Prop-
erties (ASAP), 219–272, SPARC Report No. 4, World Climate
Research Programme, 2006. Weisenstein, D. K., Yue, G. K., Ko, M. K. W., Sze, N. D., Ro-
driguez, J. M., and Scott, C. J.: A two-dimensional model of sul-
fur species and aerosols, J. Geophys. Res.-Atmos., 102, 13019–
13035, 1997. Atmos. Chem. Phys., 14, 11221–11246, 2014 Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11246 Wild, O., Zhu, X., and Prather, M. J.: Fast-J: Accurate simulation
of in-and below-cloud photolysis in tropospheric chemical mod-
els, J. Atmos. Chem., 37, 245–282, 2000. Young, R. E., Houben, H., and Toon, O. B.: Radiatively forced dis-
persion of the Mt. Pinatubo volcanic cloud and induced tempera-
ture perturbations in the stratosphere during the first few months
following the eruption, Geophys. Res. Lett., 21, 369–372, 1994. Wilson, J., Cuvelier, C., and Raes, F.: A modeling study of global
mixed aerosol fields, J. Geophys. Res.-Atmos., 106, 34081–
34108, 2001. Zdanovskii, A.: New methods for calculating solubilities of elec-
trolytes in multicomponent systems, Zh. Fiz. Khim, 22, 1475–
1485, 1948. WMO: Scientific Assessment of Ozone Depletion: 2010, Global
Ozone Research and Monitoring Project Report 52, World Mete-
orological Organization, Geneva, 2011. Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/
|
https://openalex.org/W3204775518
|
https://comum.rcaap.pt/bitstream/10400.26/40992/1/Microbiological%20evaluation%20in%20oral%20health%20units.pdf
|
English
| null |
Microbiological evaluation in oral health units: detection of antibiotic resistant bacteria
|
Annals of medicine (Helsinki)/Annals of medicine
| 2,021
|
cc-by
| 1,520
|
Annals of Medicine ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/iann20 Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=iann20 Annals of Medicine Annals of Medicine Acknowledgements The authors acknowledge funding from FCT projects – PTDC/BIM-MEC/6631/2014 and Egas Moniz, Cooperativa de Ensino
Superior CRL. DOI: 10.1080/07853890.2021.1895585 Inês Gama & Helena Barroso Inês Gama & Helena Barroso To cite this article: Inês Gama & Helena Barroso (2021) Microbiological evaluation in oral health
units: detection of antibiotic resistant bacteria, Annals of Medicine, 53:sup1, S95-S96, DOI:
10.1080/07853890.2021.1895586 To link to this article: https://doi.org/10.1080/07853890.2021.1895586 © 2021 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group
Published online: 28 Sep 2021. Submit your article to this journal
Article views: 41
View related articles
View Crossmark data © 2021 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group
Published online: 28 Sep 2021. Submit your article to this journal
Article views: 41
View related articles
View Crossmark data S95
ANNALS OF MEDICINE S9
ANNALS OF MEDICINE Figure 1. Viability of cells treated with docetaxel for 48 h. Untreated cells are considered to have 100% of viability. Values are pre-
sent has average ± SEM of three independent MTT assays. *p-value <.05 in relation to untreated cells within the same cell line. cells treated with docetaxel for 48 h Untreated cells are considered to have 100% of viability Values are pre- Figure 1. Viability of cells treated with docetaxel for 48 h. Untreated cells are considered to have 100% of viability. Values are pre-
sent has average ± SEM of three independent MTT assays. *p-value <.05 in relation to untreated cells within the same cell line. ABSTRACT Introduction: The environment in oral health care units can represent an important source of transmission of infections,
which can be acquired through aerosols, bleeding, saliva and respiratory secretions [1]. It is increasingly important to pre-
vent cross-infection in dental clinics [2]. Resistance to antibiotics is a serious public health problem. Presence of resistant
microorganisms in health care units is a worrying reality, but little is known about oral health care units [3]. The aim of
this study was the detection of microorganisms resistant to antibiotics at the Clınica Dentaria Egas Moniz, in the dentist’s
chair, trays and lamp handles. y
p
Materials and methods: Environmental samples were collected at the dental clinic with a swab. Sampling was made at
trays, chairs and lamp handles, at the end of the appointments. All samples were inoculated in Trypticase soy agar (TSA),
Mannitol salt agar (MSA) and MacConkey agar. All the bacteria that grew in MSA and were mannitol positive were inocu-
lated in chromogenic agar, because we wanted to detect Methicillin resistant Staphylococcus aureus (MRSA). g
g ,
p y
(
)
Results: Of the 123 samples obtained in 41 working stations, only two (1.6%) lactose negative bacteria were found. One
was isolated from a tray and the other from a lamp handle, in two different working stations. We found 51 mannitol positive Staphylococcus samples (41.5%), were isolated from 36 different working stations, being
14 samples identified as MRSA (11.4%). These MRSA were isolated from 13 different working stations. In our study, we
cannot identify if there was a preferential location for the presence of MRSA, but we found it mainly at trays and dentist’s
chairs. We found 51 mannitol positive Staphylococcus samples (41.5%), were isolated from 36 different working stations, being
14 samples identified as MRSA (11.4%). These MRSA were isolated from 13 different working stations. In our study, we
cannot identify if there was a preferential location for the presence of MRSA, but we found it mainly at trays and dentist’s
chairs. Discussion and conclusions: There was a low contamination by Enterobacteriaceae. However, a percentage of MRSA iso-
lation of 11.4% was obtained. There are few similar studies. Although, in a study where 95 surfaces from 7 different uni-
versity dental clinics were evaluated, 8 MRSA were found, which corresponds to 8.4%. Comparing to our study, we
obtained a slightly higher percentage. In^es Gamaa and Helena Barrosoa,b aInstituto Universitario Egas Moniz (IUEM), Caparica, Portugal; bCentro de Investigac¸~ao Interdisciplinar Egas Moniz (CiiEM), IUEM;
Cooperativa de Ensino Superior, C.R.L., Caparica, Portugal ABSTRACT Discussion and conclusions: There was a low contamination by Enterobacteriaceae. However, a percentage of MRSA iso-
lation of 11.4% was obtained. There are few similar studies. Although, in a study where 95 surfaces from 7 different uni-
versity dental clinics were evaluated, 8 MRSA were found, which corresponds to 8.4%. Comparing to our study, we
obtained a slightly higher percentage. g
y
g
p
g
These results demonstrate that oral health care units are also sites of contamination, where bacteria with resistance to g
y
g
p
g
These results demonstrate that oral health care units are also sites of contamination, where bacteria with resistance to S96 ABSTRACTS antibiotics can circulate. They also reinforce the need for good hygiene and disinfection of the site between
appointments. Acknowledgements The authors are grateful to ESSEM, for the support given on the Pedagogical Simulacrum. No funding to declare. Acknowledgements The authors acknowledge funding from Egas Moniz, CRL. [1]
Pozzoli SML, Cecılio LCO. Sobre o cuidar e o ser cuidado na atenc¸~ao domiciliar. Saude Debate. 2017;41(115):1116–1129.
[2]
Dias C. Oxigenoterapia de longa durac¸~ao no domicılio. Med Interna. 1999;6(4):270–275. ABSTRACT ABSTRACT
Introduction: Oxygen therapy is applied in a variety of clinical situations where patients have low levels of oxygen in the
blood and is part of one of the procedures in nursing care. Knowing that home care is more beneficial for the patient, because
it promotes a faster recovery and guarantees comfort and safety, preserving its autonomy [1], it is pertinent to train (enable)
both patients and informal caregivers (e.g. family, friends), with the correct knowledge (empowerment) and procedures to fol-
low. Nurses have a fundamental role in the training of patients and/or informal caregivers, knowing their difficulties and real-
ities, thus enabling caregivers to be autonomous in-home care [2–4]. This work presents an original educational tool that uses
pictograms to teach and empower patients and/or caregivers to use oxygen therapy materials correctly at home. Materials and methods: An Educational Tool for Health purposes was originally designed using pictograms, which illus-
trates/anticipates the therapeutic procedures, destined for patients who need oxygen therapy with regularity. The instru-
ment is made up of a set of pictograms that complement the clinical nursing procedure on oxygen therapy (teaching
step by step the therapeutic) and a good practice manual (helps to understand oxygen therapy and serves as a record
and monitoring of the difficulties experienced). Results: The use of pedagogic tools for health using pictograms can be a strategy to improve interpersonal communica-
tion between nurse and the patient and/or informal caregiver [5]. They can also be used as complementary learning
tools, because of their universal character, the pictograms constitute an effective way to overcome individual constraints,
such as literacy level, familiarity with the images, interpretation, and perception of what is being pictorially represented
[6]. The suggested good practice manual is interactive and dynamic, enabling both patient and informal caregiver to
focus and actively participate. y p
p
Discussion and conclusions: The Strategies for Health Education are increasingly relevant on what concerns the change
in health behaviour. Pedagogical materials of this nature facilitate the understanding and use of therapeutic procedures
used daily, allowing to reduce the excessive dependence of professionals and health services and are fundamental contri-
butions for a greater autonomy and adherence to the health behaviours of the patient and/or informal caregiver. CONTACT Ana Cristina Neves
anaccseven@gmail.com The authors are grateful to ESSEM, for the support given on the Pedagogical Simulacrum. No funding to declare. A teaching tool for nursing procedure with oxygen therapy Maria Rodrigueza, Catia Balsinhaa, Nataliya Garciaa, Valeria Vicentea and Ana Cristina Nev aEscola Superior de Saúde Egas Moniz (ESSEM), Egas Moniz Cooperativa de Ensino Superior, Caparica, Portugal;
Investigação Interdisciplinar Egas Moniz (CiiEM), Egas Moniz Cooperativa de Ensino Superior, Caparica, Portugal CONTACT Ana Cristina Neves
anaccseven@gmail.com References [1]
Al-Marzooq F, Bayat SA, Sayyar F, et al. Can probiotic cleaning solutions replace chemical disinfectants in dental clinics? Eur J Dentistry. 2018;12:532–539. [2]
Silva CR Jorge AOCJ Evaluation of surface disinfectants utilized in dentistry Pesqui Odontol Bras 2002;16(2):107–114 [1]
Al-Marzooq F, Bayat SA, Sayyar F, et al. Can probiotic cleaning solutions replace chemical disinfe
Eur J Dentistry. 2018;12:532–539. [2]
Silva CR, Jorge AOCJ. Evaluation of surface disinfectants utilized in dentistry. Pesqui Odontol Bras. 2002;16(2):107–114. [3]
Roberts MC, Soge OO, Horst JA, et al. Methicillin-resistant Staphylococcus aureus from dental school clinic surfaces and
students. Am J Infect Control. 2011;39(8):628–632. [ ]
,
g
y
q
;
( )
[3]
Roberts MC, Soge OO, Horst JA, et al. Methicillin-resistant Staphylococcus aureus from dental school clinic surfaces and
students. Am J Infect Control. 2011;39(8):628–632. [3]
Roberts MC, Soge OO, Horst JA, et al. Methicillin-resistant Staphylococcus aureus from dental sc
students. Am J Infect Control. 2011;39(8):628–632. DOI: 10.1080/07853890.2021.1895586 References [1]
Pozzoli SML, Cecılio LCO. Sobre o cuidar e o ser cuidado na atenc¸ao domiciliar. Saude Debate. 2017;41(115):1116 1129. [2]
Dias C. Oxigenoterapia de longa durac¸~ao no domicılio. Med Interna. 1999;6(4):270–275.
|
https://openalex.org/W4235135380
|
https://journals.vgtu.lt/index.php/JEELM/article/download/7929/6870
|
English
| null |
APPLICATION OF BACKWARD AIR MASS TRAJECTORY ANALYSIS IN EVALUATING AIRBORNE POLLEN DISPERSION
|
Journal of environmental engineering and landscape management
| 2,006
|
cc-by
| 6,013
|
1. Introduction identify the beginning of bloom of the plant that is chang-
ing year after year and is partially determined by mete-
orological factors in the period of blossom formation [5]. Investigaions into the dynamics of airborne birch
(Betula L.) pollen are performed in many European states
[1–4]. The attention of aerobiologists, botanists, planters,
clinicists to this biological kind of air pollution has ap-
peared because of large amounts of pollen dispersed during
bloom period and decreasing life quality of the part of
human population. It has been estimated that most often in
spring the allergy caused by birch pollen is diagnosed [5]. Using special methods, it has been shown that people are
sensitive even to small amounts of pollen: people experi-
ence unpleasant sensations when the number of birch pol-
len in 24 hours reaches 35 grains/m³, for people ill in
asthma the process of illness becomes more complicated
when 68–81 grains/m³ is recorded, 81–252 grains/m³
causes conjunctivitis and/or rhinitis [6]. The periods of
birch pollination at different aerobiology stations in
Lithuania were evaluated [7], but the influence of long-
range transport on pollen dynamics was not shown. Performing the forecast, the necessary evaluation of
interaction between airborne pollen concentration and
environmental factors is conducted [9]. It is shown that
warm and wet weather influences bigger airborne birch
pollen concentrations [2], and comparing pollen dynam-
ics with temperature (minimum and/or maximum) strong
positive correlation is observed, while comparing it with
rainfall negative correlation occurs [4]. It has been esti-
mated that airborne birch pollen concentration signifi-
cantly increases when anticyclone is dominating, relative
humidity falls below 70 %, and the temperature reaches
6–11 °C [6]. Thus, meteorological conditions and
weather types have a vital influence not only on pollen
formation, but also on the release of pollen from anthers
and airborne concentration. To investigate the movement of airborne pollen
aerobiologists use backward air mass trajectories [9]. Using this method, long-range transport and the move-
ment of airborne pollen is investigated. Airborne pollen dispersion may be forecast having
evaluated biological features of a plant, climate factors
and circulation of the atmosphere. The situation analysis
becomes more complicated because of the lack of unani-
mous opinion on birch reproduction cycle. In some au-
thors’ opinion, the triennial rhythm is characteristic of
birch [8] maintaining synchrony above large territories; in
other authors’ opinion, the rhythm is biennial (Spieksma
et al., 2003). APPLICATION OF BACKWARD AIR MASS TRAJECTORY ANALYSIS IN
EVALUATING AIRBORNE POLLEN DISPERSION Ingrida Šaulienė, Laura Veriankaitė
Dept of Environmental Sciences, Šiauliai University,
Vilniaus g. 88, LT-76285 Šiauliai, Lithuania. E-mail: oikos@fm.su.lt
Submitted 30 Mar 2006; accepted 15 Apr 2006 Ingrida Šaulienė, Laura Veriankaitė Abstract. To investigate airborne pollen movement aerobiologists use backward air mass trajectories. In the present paper
the peculiarities of airborne birch pollen dispersion are analysed. In 2005 at Šiauliai Aerobiology Station pollen was re-
corded using Hirst-type spore trap. The situation of birch bloom in neighbouring European countries was evaluated ac-
cording to the European Aeroallergen Network database. It was generalized and used to prove long-range pollen transport. Air mass trajectories were calculated according to the HYSPLIT 4 model the trajectory drawing principle of which is
based on the integration of the position of air mass with regard to time. The present study has shown that backward air
mass trajectory analysis may be applied in investigating relationship among pollen dispersion, meteorological conditions
and air mass transport. It is estimated that at Šiauliai Aerobiology Station the dynamics of pollen concentration is determi-
nated by local flora and airborne pollen long-range transport. The evaluation of pollen dispersion by the backward air
mass trajectory method presented in the paper established precondition to look for possibilities of application of the
HYSPLIT 4 model to prepare the forecasts of pollen dispersion. Keywords: pollen, meteorological conditions, long-range transport, backward air mass trajectories, birch pollination, air
pollution. 113 113 ISSN 1648–6897 print
ISSN 1822–4199 online
JOURNAL OF ENVIRONMENTAL ENGINEERING
AND LANDSCAPE MANAGEMENT 2006, Vol XIV, No 3, 113–120 2006, Vol XIV, No 3, 113–120 2.3. Backward trajectory analysis Air mass trajectories were calculated according to
HYSPLIT 4 model [12] the trajectory drawing principle
of which is based on the integration of the position of air
mass with regard to time. Since air mass is transported
with the help of the wind, its passive transport may be
calculated evaluating three-dimensional means of speed
vector when the particles are in the primary position
( )t
P
and the first approximate position
(
)
dt
t
P
+
′
. Speed
vectors are interpolated with regard both to space and
time. Using the HYSPLIT 4 model the first approximate
position is calculated: The study of airborne pollen dynamics was con-
ducted choosing 3 days the peaks of which were the most
distinct during the entire period of birch pollination: on 3,
13 and 21 May (Fig 2). On 3 and 21 May at Šiauliai Aerobiological Station
peaks of pollen concentration atypical of morning hours
were recorded. On May 13 it was the middle of pollina-
tion season, but abundant rainfall produced atypical con-
ditions for pollen dispersion. Having established
presumption about the dispersion of pollen of a nonlocal
origin, backward air mass trajectory analysis to perform
the case evaluation was used. (
)
dt
t
P
+
′
= ( )
(
) dt
t,
P
V
t
P
+
. (1) (1) While the last position of air particles: While the last position of air particles: While the last position of air particles: p
p
(
)
dt
t
P
+
= ( )
(
)
(
)
[
] dt
dt
t
P
V
t
P
V
t
P
+
′
+
+
,
,
5,0
. (2) p
p
(
)
dt
t
P
+
= ( )
(
)
(
)
[
] dt
dt
t
P
V
t
P
V
t
P
+
′
+
+
,
,
5,0
. (2) p
p
(
)
dt
t
P
+
= ( )
(
)
(
)
[
] dt
dt
t
P
V
t
P
V
t
P
+
′
+
+
,
,
5,0
. (2) Thus, backward air mass trajectories may be integra-
ted and presented graphically. Backward air mass trajectories were calculated for
every day analysed (at midnight, at 6 a.m., at noon, at
6 p.m. according to local time) at three different heights:
1500 m, 1000 m, 500 m. 2.3. Backward trajectory analysis The following heights were
chosen because to identify air mass trajectories it is rec-
ommended to choose heights with the pressure interval of
950–10 mb. It is suggested that the use of pressures less
than 950 mb often results in parcels going into the
ground [9]. 2.1. Object Birch forests in the whole Šiauliai region cover
about 29 % (61 300 ha) of the forest area and make up
15 % of the Lithuanian territory [10]. In Šiauliai city
surroundings birch is in the second place according to
number and makes up about 32 % of the forest area. 3.1. Blooming season of birch genus (Betula L.) in
Šiauliai city in 2005 For the case study, data about the amount of air-
borne birch pollen are obtained from Šiauliai Aerobi-
ological Station. Pollen was recorded using the Hirst-type
spore trap raised 18 m above the ground and localized in
the western part of the city (55°56'96 N, 23°16'95 E). Samples were prepared, and the data were processed
according to a standard method [11]. Birch pollen counts
were recorded every 2 hours and expressed as the number
of grains counted per transect (field of view of 0,55 mm
diameter). Each transect represented 0,001 % of the total
volume of sampled air. The blooming season of the plants of birch genus in
2005 lasted for 27 calendar days. During these days 5
concentration peaks dangerous to human health were
recorded: May 3(397 grains/m³), May 6 (212 grains/m³),
May 8 (205 grains/m³), May 13–15 (560–286 grains/m³)
and May 21 (347 grains/m³) (Fig 1). Fig 1. Birch pollen season in Šiauliai in 2005 The situation of birch bloom in May in neighbouring
European countries was evaluated referring to the Euro-
pean Aeroallergen Network (EAN) database. It was gen-
eralized and used to prove long-range pollen transport
with the help of air mass. 2. Materials and methods With the help of the HYSPLIT 4 model the rainfall
in the way of air masses was evaluated, the data were
taken from the National Weather Service's NCEP (the
National Weather Service's National Centers for Envi-
ronmental Prediction) database. 2.2. Meteorological data The meteorological data for Šiauliai city were pro-
vided by Šiauliai Meteorological Station of Hydrometeo-
rological Service of Lithuania at the Ministry of
Environment. The station is in the eastern part of the city
about 3 km from Šiauliai Aerobiological Station. Fig 1. Birch pollen season in Šiauliai in 2005 Having evaluated the meteorological situation dur-
ing the period of birch pollination it was identified that
conditions to release pollen from anthers were sufficient:
relative humidity during the season fluctuated from 53–
88 %, average weather temperature in 24 hours was from
7,5–20,5 °C, average wind speed in 24 hours reached
1,4–4,8 m/s, the sunniness was changing at an interval of
1,3–15,2 hours, the rainfall was 0,6–11,6 mm. 1. Introduction For the forecasts, it is not less important to The aim of the present study is to establish relation-
ship of pollen dispersion, meteorological conditions and
air mass transport using a selected case for modeling. Such a study helps to forecast probability for pollen of a
nonlocal origin to get to the territory of Lithuania and the
dynamics of biological pollution in the atmosphere. 3.4. Pollen dispersion and backward air mass trajectory
analysis on 21 May 2005 On 21 May 2005 the peak of birch pollen concentra-
tion (Fig 1), recorded at Šiauliai Aerobiological Station
was evaluated as noncharacteristic of the period because
at the end of pollination season such an emission from
local flora was impossible. The analysis of the aerobiolo-
gical preparations of the 24 hours analysed (Fig 2) high-
lighted the pollen peak of 6 a.m.–8 a.m. showing probabi-
lity of pollen of a nonlocal origin. Analysis of backward 3.2. Pollen dispersion and backward air mass trajectory
analysis on 3 May 2005 The HYSPLIT 4 model
showed that on May 13 air mass came from the northern
part of Europe: 500 m above the ground air mass retained
similar direction during 24 hours, while at a height of
1000 m and 1500 m it went from the North East to the
North where exceptionally large amounts of birch pollen
were recorded. A part of pollen could have settled toge-
ther with rainfall (especially in morning hours), and the
evening pollen peak occurred due to dried and airborne
spread local pollen (Fig 2). The situation analysis allows
to establish presumption that at Šiauliai Aerobiological
Station pollen concentration in the morning of May 13
increased due to long-range transport of particles, and in
the evening – due to the emission from the local flora. Using the HYSPLIT 4 model and the EAN database
it was revealed that on 2 May very small amounts of
birch pollen were recorded in Latvia, and air mass cros-
sed this region during night hours, therefore, it could not
bring pollen from this European region. Having evaluated
that maximum pollen concentrations were recorded from
midnight to 6 a.m. and on a bigger part of the day on May
2 local air mass was dominating, therefore, increased
amounts of pollen were related to blooming of local
plants in the central and southern parts of Lithuania. Du-
ring later hours of May 3 little amounts of airborne pollen
recorded may be related to an unfavourable meteorologi-
cal situation in Šiauliai city (a high relative humidity,
rainfall, a low pressure) preventing to spread pollen and
an unfavourable air mass circulation because at the after-
noon hours air mass came from the central and northern
parts of Europe. Although, according to the EAN data, in
Denmark and Poland much pollen was recorded these
days, however, having evaluated meteorological condi-
tions with the help of the model, it is possible to establish
presumption that pollen may have been washed by rain-
fall that occurred in the way of air mass. 3.2. Pollen dispersion and backward air mass trajectory
analysis on 3 May 2005 During 24 hours of analysis the pollen peak was re-
corded at night hours (Fig 2). Having evaluated pollen
recorded during 24 hours it was estimated that 67 % of
the total amount was recorded from midnight to 6 a.m.). I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 115 0
20
40
60
80
100
120
140
160
180
200
00–02 02–04 04–06 06–08 08–10 10–12 12–14 14–16 16–18 18–20 20–22 22–24
Pollen concentration (grains/m3)
3 May
13 May
21 May
hours
Fig 2. Birch pollination on 3, 13 and 21 May Fig 2. Birch pollination on 3, 13 and 21 May The biggest pollen concentration that day was from
4 a.m. to 6 a.m. (184,11 grains/m³) and significantly dec-
reased in the afternoon hours. The meteorological situa-
tion of 24 hours was unfavourable for pollen dispersion
(Table 1). An average temperature reached 8,8 °C, the
rainfall (from 5:28 to 11:06) was to 1,1 mm, the day was
cloudy, the variable wind speed reached 2,8 m/s. Knowing that for the release of pollen from anthers a dry
and sunny weather is essential, the problem of the situa-
tion analysis occurs. To evaluate the changes of pollen
concentration of that day, analysis of air mass transport
was conducted (Figs 3–6). decreasing towards the end of the day. The total rainfall
amount reached 11,6 mm, its biggest part fell until
11 a.m. Other meteorological conditions (Table 2) were
unfavourable for the release of pollen, but airborne pollen
concentration under such conditions reached a maximum
of the entire season (560 grains/m³). Local pollen (of
plants from Šiauliai surroundings) was wet and heavy and
could not spread, so it was possible to establish presump-
tion that the part of pollen could be brought together with
air mass (Figs 7–10) from neighbouring regions. Having
evaluated plant pollination in Europe (according to the
EAN database) it was estimated that in Latvia and in the
southern part of Finland big amounts of birch pollen were
recorded, while in the southern and central parts of Fin-
land birch bloom was over. I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120
116 I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120
116 116 Table 1. Meteorological data recorded on May 3, 2005
Time
Surface level
pressure
mb
Average tem-
perature,
°C
Minimal tem-
perature,
°C
Wind speed,
m/s
Wind direc-
tion,
degrees
Relative
humidity,
%
24
1006,6
8,5
8,4
2
20
62
6
1004,0
9
8,2
3
180
71
12
999,6
7,9
7,7
3
160
82
18
997,7
8,7
8,6
3
180
92
NOAA HYSPLIT MODEL
Backward trajectories ending at 21 UTC 2 May 2005
FNL Meteoroogical Data
Fig 3. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 00:00, May 3, 2005
NOAA HYSPLIT MODEL
Backward trajectories ending at 3 UTC 3 May 2005
FNL Meteoroogical Data
NOAA HYSPLIT MODEL
Backward trajectories ending at 9 UTC 3 May 2005
FNL Meteoroogical Data
Fig 5. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 12:00, May 3, 2005
NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 3 May 2005
FNL Meteoroogical Data Table 1. Meteorological data recorded on May 3, 2005
Time
Surface level
pressure
mb
Average tem-
perature,
°C
Minimal tem-
perature,
°C
Wind speed,
m/s
Wind direc-
tion,
degrees
Relative
humidity,
%
24
1006,6
8,5
8,4
2
20
62
6
1004,0
9
8,2
3
180
71
12
999,6
7,9
7,7
3
160
82
18
997,7
8,7
8,6
3
180
92 Table 1. Meteorological data recorded on May 3, 2005
Time
Surface level
pressure
mb
Average tem-
perature,
°C
Minimal tem-
perature,
°C
Wind speed,
m/s
Wind direc-
tion,
degrees
Relative
humidity,
%
24
1006,6
8,5
8,4
2
20
62
6
1004,0
9
8,2
3
180
71
12
999,6
7,9
7,7
3
160
82
18
997,7
8,7
8,6
3
180
92 Table 1. Meteorological data recorded on May 3, 2005 18
997,7
8,7
8,6
3
180
92
NOAA HYSPLIT MODEL
Backward trajectories ending at 21 UTC 2 May 2005
FNL Meteoroogical Data
Fig 3. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 00:00, May 3, 2005
NOAA HYSPLIT MODEL
Backward trajectories ending at 3 UTC 3 May 2005
FNL Meteoroogical Data
Fig 4. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 6:00, May 3, 2005
NOAA HYSPLIT MODEL
Backward trajectories ending at 9 UTC 3 May 2005
FNL Meteoroogical Data
Fig 5. 3.3. Pollen dispersion and backward air mass trajectory
analysis on 13 May 2005 The 24 hours analysed were distinguished by an
abundant rainfall that began at night and was gradually I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120
116 Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 117 Table 2. Meteorological data recorded on May 13, 2005
Time
Surface level pressure
mb
Average
temperature,
°C
Minimal
temperature,
°C
Wind speed,
m/s
Wind direc-
tion,
degrees
Relative
humidity,
%
24
1002,4
6,4
5,9
5
360
91
6
1000,0
6,8
6,1
5
360
93
12
1000,5
10,5
7,9
6
340
64
18
1001,6
12,9
11,4
5
340
55
NOAA HYSPLIT MODEL
Backward trajectories ending at 21 UTC 12 May 2005
FNL Meteoroogical Data
Fig 7. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 00:00, May 13, 2005
NOAA HYSPLIT MODEL
Backward trajectories ending at 3 UTC 13 May 2005
FNL Meteoroogical Data
NOAA HYSPLIT MODEL
Backward trajectories ending at 9 UTC 13 May 2005
FNL Meteoroogical Data
Fig 9. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 12:00, May 13, 2005
NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 13 May 2005
FNL Meteoroogical Data Table 2. Meteorological data recorded on May 13, 2005
Time
Surface level pressure
mb
Average
temperature,
°C
Minimal
temperature,
°C
Wind speed,
m/s
Wind direc-
tion,
degrees
Relative
humidity,
%
24
1002,4
6,4
5,9
5
360
91
6
1000,0
6,8
6,1
5
360
93
12
1000,5
10,5
7,9
6
340
64
18
1001,6
12,9
11,4
5
340
55 Table 2. Meteorological data recorded on May 13, 2005
Time
Surface level pressure
mb
Average
temperature,
°C
Minimal
temperature,
°C
Wind speed,
m/s
Wind direc-
tion,
degrees
Relative
humidity,
%
24
1002,4
6,4
5,9
5
360
91
6
1000,0
6,8
6,1
5
360
93
12
1000,5
10,5
7,9
6
340
64
18
1001,6
12,9
11,4
5
340
55 Table 2. Meteorological data recorded on May 13, 2005 ,
,
,
NOAA HYSPLIT MODEL
Backward trajectories ending at 21 UTC 12 May 2005
FNL Meteoroogical Data
Fig 7. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 00:00, May 13, 2005
NOAA HYSPLIT MODEL
Backward trajectories ending at 3 UTC 13 May 2005
FNL Meteoroogical Data
Fig 8. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 6:00, May 13, 2005
NOAA HYSPLIT MODEL
Backward trajectories ending at 9 UTC 13 May 2005
FNL Meteoroogical Data
Fig 9. I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120
116 Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 12:00, May 3, 2005
NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 3 May 2005
FNL Meteoroogical Data
Fig 6. Backward trajectory analysis (24 hours) at three al-
titudes, start of run 18:00 hours, May 3, 2005 NOAA HYSPLIT MODEL
Backward trajectories ending at 21 UTC 2 May 2005
FNL Meteoroogical Data NOAA HYSPLIT MODEL
Backward trajectories ending at 21 UTC 2 May 2005
FNL Meteoroogical Data NOAA HYSPLIT MODEL
Backward trajectories ending at 9 UTC 3 May 2005
FNL Meteoroogical Data NOAA HYSPLIT MODEL
Backward trajectories ending at 9 UTC 3 May 2005
FNL Meteoroogical Data NOAA HYSPLIT MODEL
Backward trajectories ending at 9 UTC 3 May 2005
FNL Meteoroogical Data
Fig 5. Backward trajectory analysis (24 hours) at three
titudes, start of run at 12:00, May 3, 2005 NOAA HYSPLIT MODEL
Backward trajectories ending at 21 UTC 2 May 2005
FNL Meteoroogical Data
Fig 3. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 00:00, May 3, 2005 Fig 5. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 12:00, May 3, 2005 Fig 3. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 00:00, May 3, 2005 NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 3 May 2005
FNL Meteoroogical Data NOAA HYSPLIT MODEL
Backward trajectories ending at 3 UTC 3 May 2005
FNL Meteoroogical Data
Fig 4. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 6:00, May 3, 2005 NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 3 May 2005
FNL Meteoroogical Data
Fig 6. Backward trajectory analysis (24 hours) at three a
titudes, start of run 18:00 hours, May 3, 2005 NOAA HYSPLIT MODEL
Backward trajectories ending at 3 UTC 3 May 2005
FNL Meteoroogical Data Fig 6. Backward trajectory analysis (24 hours) at three al-
titudes, start of run 18:00 hours, May 3, 2005 Fig 6. Backward trajectory analysis (24 hours) at three al-
titudes, start of run 18:00 hours, May 3, 2005 Fig 4. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 6:00, May 3, 2005 Fig 4. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 6:00, May 3, 2005 I. Šaulienė, L. I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120
116 Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 12:00, May 13, 2005
NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 13 May 2005
FNL Meteoroogical Data
Fig 10. Backward trajectory analysis (24 hours) at three
altitudes, start of run at 18:00, May 13, 2005 NOAA HYSPLIT MODEL
Backward trajectories ending at 21 UTC 12 May 2005
FNL Meteoroogical Data NOAA HYSPLIT MODEL
Backward trajectories ending at 9 UTC 13 May 2005
FNL Meteoroogical Data Backward trajectories ending at 21 UTC 12 May 2005
FNL Meteoroogical Data
Fig 7. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 00:00, May 13, 2005 Backward trajectories ending at 9 UTC 13 May 2005
FNL Meteoroogical Data
Fig 9. Backward trajectory analysis (24 hours) at three al
titudes, start of run at 12:00, May 13, 2005 g
FNL Meteoroogical Data Fig 7. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 00:00, May 13, 2005 Fig 9. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 12:00, May 13, 2005 NOAA HYSPLIT MODEL
Backward trajectories ending at 3 UTC 13 May 2005
FNL Meteoroogical Data NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 13 May 2005
FNL Meteoroogical Data NOAA HYSPLIT MODEL
Backward trajectories ending at 3 UTC 13 May 2005
FNL Meteoroogical Data
Fig 8. Backward trajectory analysis (24 hours) at three al
titudes, start of run at 6:00, May 13, 2005 NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 13 May 2005
FNL Meteoroogical Data
Fig 10. Backward trajectory analysis (24 hours) at three
altitudes, start of run at 18:00, May 13, 2005 Fig 8. Backward trajectory analysis (24 hours) at three al-
titudes, start of run at 6:00, May 13, 2005 Fig 10. Backward trajectory analysis (24 hours) at three
altitudes, start of run at 18:00, May 13, 2005 Fig 10. Backward trajectory analysis (24 hours) at three
altitudes, start of run at 18:00, May 13, 2005 I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120
118 118 Table 3. I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120
116 Backward trajectory analysis (24 hours) at three
altitudes, start of run at 18:00, May 21, 2005 O
S
O
Backward trajectories ending at 15 UTC 21 May 2005
FNL M t
i
l D t j
g
FNL Meteoroogical Data The meteorological data of 24 hours (Table 3) were
favourable for pollination: the day was sunny, a sudden
change of relative humidity from 95 % to 33 %, quite a
high pressure. Evaluating the air mass that reached Šiau-
liai Aerobiological Station at a different time, the air
mass movement of the same direction was stated: air
mass moved from the southern part of Estonia through
Russia, Latvia and northern Lithuania (Figs 11–12). On
the way of the air mass there was no rainfall. According
to the EAN data, in Latvia there was a large amount of
birch pollen and it could get into the air mass moving
towards Lithuania. A low morning temperature (Table 3)
unfavourable for the pollination of local plants allows
consideration of the probability of long-range transport,
so it is possible to establish presumption that the birch
pollen from southern Latvia could have been recorded at
Šiauliai Aerobiological Station. I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120
116 Meteorological data recorded on May 21, 2005
Time
Surface level
pressure
mb
Average
temperature,
°C
Minimal
temperature,
°C
Wind speed,
m/s
Wind
direction,
degrees
Relative
humidity, %
24
1012,3
7
6,9
0
Tyla
73
6
1011,1
3,5
3,2
0
Tyla
95
12
1009,7
16,3
12,4
3
135
38
18
1006,1
19,3
18,1
3
90
33 Wind speed,
m/s
Wind
direction,
degrees
Relative
humidity, %
0
Tyla
73
0
Tyla
95
3
135
38
3
90
33
NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 21 May 2005
FNL Meteoroogical Data
Fig 12. Backward trajectory analysis (24 hours) at three
altitudes, start of run at 18:00, May 21, 2005 corded on May 21, 2005
Average
temperature,
°C
Minimal
temperature,
°C
Wind speed,
m/s
Wind
direction,
degrees
Relative
humidity, %
7
6,9
0
Tyla
73
3,5
3,2
0
Tyla
95
16,3
12,4
3
135
38
19,3
18,1
3
90
33
erent height helped to explain
concentration occurred at the
a of 24 hours (Table 3) were
the day was sunny, a sudden
from 95 % to 33 %, quite a
e air mass that reached Šiau-
at a different time, the air
me direction was stated: air
hern part of Estonia through
n Lithuania (Figs 11–12). On
re was no rainfall. According
there was a large amount of
et into the air mass moving
morning temperature (Table 3)
ation of local plants allows
ility of long-range transport,
h presumption that the birch
could have been recorded at
on. that backward trajectory ana-
ate specific pollen episodes. PLIT MODEL
t 21 UTC 20 M
2005
NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 21 May 2005
FNL Meteoroogical Data
Fig 12. Backward trajectory analysis (24 hours) at three
altitudes, start of run at 18:00, May 21, 2005 Table 3. Meteorological data recorded on May 21, 2005 NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 21 May 2005
FNL Meteoroogical Data air mass trajectories at a different height helped to explain
why the increase of pollen concentration occurred at the
end of pollination season. NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 21 May 2005
FNL Meteoroogical Data
Fig 12. Backward trajectory analysis (24 hours) at three
altitudes, start of run at 18:00, May 21, 2005 NOAA HYSPLIT MODEL
Backward trajectories ending at 15 UTC 21 May 2005
FNL Meteoroogical Data
Fig 12. References 1. Filon, F. L.;
Bosco, A.;
Berbina, P.;
Sauli, M. L.;
Longo, L. R. Betulaceae and Corylaceae in Trieste (NE-
Italy): Aerobiological and clinical data. Aerobiologia,
Vol 16, 2000, p. 87–91. 10. Kuliešis, A.; Kasperavičius, A.;
Kulbokas, G.;
Kval-
kauskienė, M. Lietuvos nacionalinė miškų inventorizacija
1998–2002. Atrankos
schema,
metodai,
rezultatai. Kaunas: Naujasis lankas, 2003. 256 p. 2. Corden, J.; Millington, W; Bailey, J.; Brookes, M.; Caul-
ton, E.; Emberlin, J.; Mullins, J.; Simpson, C.; Wood, A. UK regional variations in Betula pollen (1993–1997). Aerobiologia, Vol 16, 2000, p. 227–232. 11. Mandrioli, P.; Comtois, P.; Levizzani, V. Methods in
Aerobiology. Bologna: Pitagora Editrice, 1998. 262 p. 12. Draxler, R. R.; Rolph, G. D. HYSPLIT (HYbrid Single-
Particle Lagrangian Integrated Trajectory) Model access
via
NOAA
ARL
READY
Website
(http://www.arl.noaa.gov/ready/hysplit4.html). NOAA
Air Resources Laboratory, Silver Spring, MD, 2003. 3. Radišić, P.; Šikoparija, B. Betula spp. Pollen in the at-
mosphere of Novi Sad (2000–2002). Aerobiologia,
Vol 21, 2005, p. 63–67. 4. Mendez, J.; Comtois, P.; Iglesias, I. Betula pollen: One of
the most important aeroallergens in Ourense, Spain. Aerobiological studies from 1993 to 2000. Aerobiologia,
Vol 21, 2005, p. 115–123. 4. Discussion This study has shown that backward trajectory ana-
lysis can be used to investigate specific pollen episodes. Fig 12. Backward trajectory analysis (24 hours) at three
altitudes, start of run at 18:00, May 21, 2005 NOAA HYSPLIT MODEL
Backward trajectories ending at 21 UTC 20 May 2005
FNL Meteoroogical Data
Fig 11. Backward trajectory analysis (24 hours) at three
altitudes, start of run at 00:00, May 21, 2005 NOAA HYSPLIT MODEL
Backward trajectories ending at 21 UTC 20 May 2005
FNL Meteoroogical Data On May 3, 13, 21 the evaluation of the aerobiologi-
cal situation was successful having used backward air
mass trajectory analysis. Using the HYSPLIT 4 model, it
is revealed why uncommonly large pollen concentrations
were recorded. Possibility to use the EAN data allowed to
establish presumption that under atypical conditions at
Šiauliai Aerobiological Station the increase of pollen
concentration may have been recorded not only because
of the dynamics of the pollination of local flora, but also
because of airborne pollen long-range transport. Evaluating the importance of air mass movement for
long-range pollen transport, it is necessary to choose a
correct model. Application of the HYSPLIT 4 model
revealed the regularities of transport and may be succes-
sfully used. Evaluation of pollen dispersion by the
backward air mass trajectory method presented in the
paper established the precondition to look for possibilities
of application of the HYSPLIT 4 model to prepare the
forecasts of pollen dispersion. Such a permanent control
of pollen spread (together with evaluating the processes
that determine long-range transport) is important for pa-
tients with allergic illnesses, patient associations, doctors,
pharmaceutical companies, organizers of international
sport and leisure events, tourism business. Precise pollen Fig 11. Backward trajectory analysis (24 hours) at three
altitudes, start of run at 00:00, May 21, 2005 I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2 identification may decrease losses experienced because of
mistakes in choosing medicine against a specific allergen
that is usually of a nonlocal origin. 5. Latałowa, M.; Miętus, M.; Uruska, A. Seasonal variations
in the atmospheric Betula pollen count in Gdańsk (south-
ern Baltic coast) in relation to meteorological parameters. Aerobiologia, Vol 18, 2002, p. 33–43. 6. Laaidi, K. Predicting days of high allergenic risk during
Betula pollination using weather types. International
Journal of Biometeorology, Vol 45, 2001, p. 124–132. 5. Conclusions 1. It is shown that backward air mass analysis may
be applied in investigating relationship among pollen
dispersion, meteorological conditions and air mass trans-
port. 1. It is shown that backward air mass analysis may
be applied in investigating relationship among pollen
dispersion, meteorological conditions and air mass trans-
port. 7. Šaulienė, I.;
Motiekaitytė, V. Pecularities
of
Aero-
palinological Monitoring in Northern Lithuania. In: The
6th International Conference on Environmental Engineer-
ing. Selected Papers, Vol 1 (May 26–27 2005, Vilnius,
Lithuania). Vilnius: Technika, 2005, p. 252–256. p
2. It is shown that the HYSPLIT 4 model may be
used to investigate long-range pollen transport. Š p
2. It is shown that the HYSPLIT 4 model may be
used to investigate long-range pollen transport. 8. Jager, S.; Spieksma, F. Th. M.; Nolard, N. Fluctuations
and trends in airborne concentrations of some abundant
pollen types, monitored at Vienna, Leiden, and Brussels. Grana, Vol 30, 1991, p. 3009–312. g
g
g p
p
3. It is revealed that at Šiauliai Aerobiological Sta-
tion the dynamics of pollen concentration is conditioned
by the local flora and airborne pollen long-range trans-
port. 3. It is revealed that at Šiauliai Aerobiological Sta-
tion the dynamics of pollen concentration is conditioned
by the local flora and airborne pollen long-range trans-
port. 9. Smith, M.; Emberlin, J.; Kress, A. Examing high magni-
tude grass pollen episodes at Worcester, United Kingdom,
using back-trajectory analysis. Aerobiologia, Vol 21,
2005, p 85–94. ОЦЕНКА ДИСПЕРСИИ ПЫЛЬЦЫ В АТМОСФЕРЕ ПО АНАЛИЗУ ТРАЕКТОРИЙ ОБРАТНЫХ
ВОЗДУШНЫХ МАСС И. Шаулене, Л. Верянкайте S a n t r a u k a Žiedadulkių judėjimą atmosferoje aerobiologai tiria analizuodami atgalinių oro masių trajektorijas. Šiame straipsnyje gili-
namasi į beržo žiedadulkių sklaidos atmosferoje ypatumus. Šiaulių aerobiologijos stotyje 2005 m. žiedadulkės buvo fik-
suojamos Hirst tipo sporų gaudykle. Žiedadulkių judėjimas žydint beržams aplinkinėse Europos šalyse buvo įvertintas
remiantis Europos aeroalergenų tinklo duomenų baze. Apibendrintais duomenimis pasinaudota, įrodant tolimąją
žiedadulkių pernašą. Oro masių trajektorijos apskaičiuotos pagal HYSPLIT 4 modelį, trajektorijų brėžimo principą
grindžiant oro masės padėties integravimu laiko atžvilgiu. Tyrimas parodė, kad atgalinių oro masių trajektorijų analizė
gali būti taikoma tiriant sąsają tarp žiedadulkių dispersijos, meteorologinių sąlygų ir oro masių pernašos. Nustatyta, kad
Šiaulių aerobiologijos stotyje žiedadulkių koncentracijos dinamiką lemia vietinė augalija ir žiedadulkių atmosferoje to-
limosios pernašos. Straipsnyje pateiktas žiedadulkių dispersijos vertinimas atgalinių oro masių trajektorijų analizės me-
todu suformavo prielaidas siekti modelio HYSPLIT 4 taikymo galimybių žiedadulkių sklaidai prognozuoti. Prasminiai žodžiai: žiedadulkės, meteorologinės sąlygos, tolimosios pernašos, atgalinės oro masių trajektorijos, beržo
dulkėjimas, oro tarša. r of Ecology and Environmental Research, Šiauliai University. Research interests: meteorology, environmental re-
erobiology. Р е з ю м е Р е з ю м е Для исследования пыльцы в атмосфере аэробиологи используют траектории обратных атмосферных масс. В
статье анализируются особенности рассеянности берёзовой пыльцы в атмосфере в 2005 г. Пыльца фиксировалась
с использованием ловушки спор типа Хирст. Ситуация цветения берёзы в соседних странах Европы была оценена
согласно данным базы Европейской сети по аэроаллергенам. Данные обобщены, использованы, доказан дальний I. Šaulienė, L. Verinkaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 120 перенос пыльцы. Траектории обратных воздушных масс установлены согласно модели HYSPLIT 4. Исследования
показали, что анализ обратных воздушных масс может быть использован для изучения связи между дисперсией
пыльцы, метеоусловиями и переносом атмосферных масс. Установлено, что на аэробиологической станции
города Шяуляй динамика пыльцы предопределена местной флорой и дальними переносами. Представленная
оценка дисперсии пыльцы может служить основой при изучении возможности использования HYSPLIT 4 для
прогнозов. Ключевые слова: пыльца, метеоусловия, дальний перенос, обратные траектории атмосферных масс, пыление
берёзы, загрязнение воздуха. Ingrida ŠAULIENĖ. Dr, Assoc Prof and head of Dept of Environmental Research, Šiauliai University (ŠU),
Vilniaus g. 88, LT-76285, Šiauliai, Lithuania. Ingrida ŠAULIENĖ. Dr, Assoc Prof and head of Dept of Environmental Research, Šiauliai University (ŠU),
Vilniaus g. 88, LT-76285, Šiauliai, Lithuania. Doctor of Natural Sciences (botany), 2002. Probation: Poznan University (Poland), Certificate of Aerobiologist, 2003; In-
stitute of Zoology and Botany, Estonian Agricultural University, 2003; Faculty of Geography and Earth Sciences, Univer-
sity of Latvia, 2005. Membership: Lithuanian Association of Plant Physiology, Phenology Community of Lithuanian
Association of Nature, International Association for Aerobiology, Nordic Aerobiology Federation. Publications: author of
11 research papers. Research interests: plant physiology (influence of environmental factors on plant regeneration proc-
esses), aerobiology, environment quality. Laura VERIANKAITĖ. Master student, Dept of Hydrology and Climatology, Vilnius University (VU),
M. K. Čiurlionio g. 21/27, LT-03101, Vilnius, Lithuania. Bachelor of Ecology and Environmental Research, Šiauliai University. Research interests: meteorology, environmental re-
search, aerobiology.
|
https://openalex.org/W1584571495
|
https://pure.eur.nl/files/47611818/REPUB_87999_OA.pdf
|
English
| null |
Dynamic prediction of outcome for patients with severe aortic stenosis: application of joint models for longitudinal and time-to-event data
|
BMC cardiovascular disorders
| 2,015
|
cc-by
| 4,815
|
© 2015 Andrinopoulou et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated. Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28
DOI 10.1186/s12872-015-0035-z Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28
DOI 10.1186/s12872-015-0035-z Dynamic prediction of outcome for patients with
severe aortic stenosis: application of joint models
for longitudinal and time-to-event data Eleni-Rosalina Andrinopoulou1,4*, Dimitris Rizopoulos1, Marcel L. Geleijnse2, Emmanuel Lesaffre1,3, Ad J. J. C. Bogers4
and Johanna J. M. Takkenberg4 Abstract Background: Physicians utilize different types of information to predict patient prognosis. For example: confronted
with a new patient suffering from severe aortic stenosis (AS), the cardiologist considers not only the severity of
the AS but also patient characteristics, medical history, and markers such as BNP. Intuitively, doctors adjust their
prediction of prognosis over time, with the change in clinical status, aortic valve area and BNP at each outpatient
clinic visit. With the help of novel statistical approaches to model outcomes, it is now possible to construct
dynamic event prediction models, employing longitudinal data such as AVA and BNP, and mimicking the dynamic
adjustment of prognosis as employed intuitively by cardiologists. We illustrate dynamic prediction of patient
survival and freedom from intervention, using baseline patient characteristics and longitudinal BNP data that are
becoming available over time, from a cohort of patients with severe aortic stenosis. Methods: A 3-step approach was employed: (1) construction of a mixed-effects model to describe temporal BNP
progression, (2) jointly modeling the mixed-effects model with time-to-event data (death and freedom from
intervention), and (3) using the joint model to build subject-specific prediction risk models. The dataset used for
this purpose includes 191 patients with severe aortic stenosis who were followed over a 3-year time period. Results: In the mixed-effects model BNP was significantly influenced by time, baseline patient age, gender, LV
fractional ejection fraction and creatinine. Additionally, the joint model showed that an increasing BNP trend over
time was found to be a significant predictor of death. Conclusions: By jointly modeling longitudinal data with time-to-event outcomes it is possible to construct individualized
dynamic event prediction models that renew over time with accumulating evidence. It provides a potentially valuable
evidence-based tool for everyday use in medical practice. ar disease, Brain natriuretic peptide, Aortic valve disease, Survival, Individualized prediction Keywords: Valvular disease, Brain natriuretic peptide, Aortic valve disease, Survival, Individualized p RESEARCH ARTICLE Open Access * Correspondence: e.andrinopoulou@erasmusmc.nl
1Department of Biostatistics, Erasmus MC, Rotterdam, The Netherlands
4Department of Cardiothoracic Surgery, Erasmus MC, Rotterdam, The
Netherlands
Full list of author information is available at the end of the article Background age and comorbidities, New York Heart Association
(NYHA) functional class and patient history, in order to
make an assessment of patient prognosis. Additionally,
biomarkers such as brain natriuretic peptide (BNP) can
be used to further assess AS severity and prognosis. A
small AVA and a high BNP are both associated with a
more severe disease and a worse outcome [1-3]. In clinical practice, physicians utilize different sources of
information to predict patient prognosis. For example, in
diagnosing a new patient with severe aortic stenosis
(AS), a cardiologist considers not only the severity of the
AS (for example through aortic valve area AVA mea-
surement) but also patient characteristics such as patient Empirically, cardiologists adjust their prognosis over
time at each outpatient clinic visit, with the change in
functional class, AVA and BNP. Based on emerging
evidence on determinants of the outcome in AS, and
with the help of novel statistical approaches to model * Correspondence: e.andrinopoulou@erasmusmc.nl
1Department of Biostatistics, Erasmus MC, Rotterdam, The Netherlands
4Department of Cardiothoracic Surgery, Erasmus MC, Rotterdam, The
Netherlands
Full list of author information is available at the end of the article Page 2 of 8 Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 outcomes, it is now possible to construct dynamic predic-
tion models for patient outcome, employing repeatedly
collected (longitudinal) data such as BNP, mimicking the
dynamic adjustment of prognosis as employed intuitively
by cardiologists at each outpatient clinic visit. and longitudinal BNP measurement, requires that we first
describe the evolution of BNP over time, correcting for
baseline variables. Second, we use this information in a
time-to-event model. Finally, using the combined model,
we perform dynamic event predictions. In the next para-
graphs we describe in detail the statistical methods that
were employed in this 3-step process, and the rationale be-
hind these methods. This paper aims to illustrate the use of joint models of
longitudinal and time-to-event data to dynamically pre-
dict individualized event occurrence severe AS. For this
purpose, data from a prospective cohort study of 191 pa-
tients with severe AS is modeled to dynamically predict
prognosis of two patients: Mr. Jones and Mr. Smith; who
were recently diagnosed with severe AS. First, we fitted a mixed-effects model to describe the
evolution of BNP over time. Patient dataset We used the patient dataset of a previously reported pro-
spective cohort study of 191 adult patients, who were diag-
nosed with severe aortic valve disease in seven cardiology
clinics in the wider Rotterdam area between 2006 and
2009, and who were followed for 2 years [4]. Inclusion cri-
teria were AVA ≤1 cm2, peak transaortic jet velocity
(Vmax) ≥4 m/s, or aortic valve/left ventricular outflow tract
velocity time integral ratio ≥4. The patients were followed
clinically, including BNP measurements, and echocardio-
graphically at baseline and then after 6, 12 and 24 months. Baseline patient characteristics are displayed in Table 1. In
total 561 BNP measurements were collected over a 2-year
period (mean 0.9 years; range 0–2.5 years). During the
follow-up period, 15 % of the patients (N = 28) died and
48 % (N = 91) received an aortic valve replacement of trans-
catheter aortic valve implantation. Second, to investigate the effect of the repeated BNP
measurements on death and intervention probabilities,
separate joint models of longitudinal and survival out-
comes were constructed [5,6]. AVA, age, symptoms, gen-
der, LV fraction and creatinine (all at baseline) were
included as additional confounders. More details about
the joint models are presented in the Appendix. The study protocol was approved by the medical ethics
committee of Erasmus University Medical Center (MEC
2006–066); all patients provided written informed consent. Background Particularly, the model in-
cluded time (years) and the baseline covariates: AVA
(cm2), patient age (years), symptoms (yes/no), gender,
transformed LV ejection fraction (%) and transformed
creatinine (micromol/L). Transformation was done by
dividing the values with the standard deviations of the
specific covariates. Moreover, due to heterogeneity in
the residuals plot the logarithmic scale of BNP was used. An advantage of the mixed-effects models is that they
account for the positive correlation between the mea-
surements that are observed within the same patient. For example, the values of BNP that are observed over
time from the same patient are expected to be more cor-
related than between patients. Moreover, these models
account for the biological variability in the longitudinal
outcome. Specifically, if we measure BNP twice a day,
we may not obtain the same result. By taking this into
account using the mixed-effects model, more reliable re-
sults will be observed. Statistical methods Third, we considered the joint modeling framework
and focused on the assessment of the predictive ability
of our survival outcomes. Specifically, it was of interest
to predict patient survival and aortic valve intervention-
free for a new patient that has provided us with a set of
BNP measurements and baseline characteristics, using
the fitted joint model for all patients. Due to the fact
that BNP is time-dependent and not constant between
the visits and therefore providing longitudinal measure-
ment up to a specific time, assumes survival up to this
time, it was more relevant to calculate the probability of
surviving a future time point, given that the patient was
alive until his last follow-up visit [7,8]. Using this approach,
we applied the resulting joint modeling framework to two
hypothetical patients: Mr. Jones and Mr. Smith and pre-
dicted their future survival and aortic valve intervention-
free probabilities. Specifically, Mr. Jones is a 72 year old
male, with creatinine value at baseline 92 micromol/L,
AVA of 0.96 cm2, LV ejection fraction 61 % and BNP The development of a dynamic event prediction model
that takes into account both baseline patient characteristics The development of a dynamic event prediction model
that takes into account both baseline patient characteristics Table 1 Baseline patient characteristics
All patients (Number = 191)
Male gender (n, %)
118, 62 %
Age in years (mean, sd)
72.6, 11.4
Symptomatic at study entry (n, %)
132, 69 %
Smoking (n, %)
115, 60 %
Hypertension (n, %)
100, 52 %
Diabetes (n, %)
39, 20 %
Dyslipidemia (n, %)
93, 49 %
AVA in cm2 (mean, sd)
0.74, 0.27
LV ejection fraction in % (mean, sd)
61, 6.7
Creatinine in micromol/L (mean, sd)
89, 125
AVA = aortic valve area; LV = left ventricular Table 1 Baseline patient characteristics
All patients (Number = 191) Table 1 Baseline patient characteristics Page 3 of 8 Page 3 of 8 Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 Table 2 Coefficients, standard error of coefficients and
p-values for the mixed-effects model describing the
evolution of BNP over time
Coef
Se(coef)
p-value
(Intercept)
2.92
0.95
0.0025
Time (years)
0.23
0.04
<0.0001
AVA (cm2)
−1.48
0.3
<0.0001
Age (years)
0.05
0.007
<0.0001
Symptoms
0.43
0.18
0.0188
Male gender
−0.34
0.18
0.0607
*LV ejection fraction (%)
−0.16
0.08
0.0486
*Creatinine (micromol/L)
0.4
0.09
<0.0001
AVA = aortic valve area; LV = left ventricular. Statistical methods * Trasnformed LV ejection fraction
and Creatinine in the models Table 2 Coefficients, standard error of coefficients and
p-values for the mixed-effects model describing the
evolution of BNP over time 287, 1068 and 1070 pg/ml measured at 0, 0.9, 1.2 and
2 years. Furthermore, we performed internal validation using a
bootstrapping procedure (size of 1000). Specifically, we
focused on discrimination, that is, how well can the
model discriminate between patients who are about to
experience the event within a time frame after the last
measurement, from patients that are going to surpass
this time frame. Since the patients were visiting their
physician approximately every half year, we set this time
frame. In particularly, we relied on the receiver operating
characteristic (ROC) approach to assess the predictive
ability of the longitudinal biomarker BNP [7]. All analyses have been implemented in R-3.2.0, which
can be downloaded as freeware at http://www.r-project.org,
using the JM package [9]. values over time 64, 70, 72 and 78 pg/ml measured at 0.5,
0.9, 1.5 and 1.5 years. Moreover, he is asymptomatic at
baseline. Additionally, Mr. Smith is a 79 year old male that
has creatinine equal to 92 micromol/L, AVA equal to
0.61 cm2, LV ejection fraction equal to 61 % and he is
symptomatic at baseline. Finally, his BNP values are 381, values over time 64, 70, 72 and 78 pg/ml measured at 0.5,
0.9, 1.5 and 1.5 years. Moreover, he is asymptomatic at
baseline. Additionally, Mr. Smith is a 79 year old male that
has creatinine equal to 92 micromol/L, AVA equal to
0.61 cm2, LV ejection fraction equal to 61 % and he is
symptomatic at baseline. Finally, his BNP values are 381, ptide; AVA = aortic valve area; LV = left ventricular; HR = hazard ratio. * Trasnformed LV ejection fraction and Creatinine in the models Results As illustrated in Table 2 in the mixed-effects model de-
scribing the evolution of BNP over time, all covariates
have a strong association with the levels of BNP, except
baseline gender. Specifically, a longer follow-up, lower
AVA at baseline, older patient baseline age, symptomatic Fig. 1 Effect plot of AVA described from the joint model for Mr. Jones and another patient with the same age, with impaired LV ejection fraction
of 61, creatinine level equal to 92 and both patients with no symptoms at baseline Fig. 1 Effect plot of AVA described from the joint model for Mr. Jones and another patient with the same age, with impaired LV ejection fraction
of 61, creatinine level equal to 92 and both patients with no symptoms at baseline Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 Page 4 of 8 Page 4 of 8 Table 3 Coefficients, standard error of coefficients and p-values for the joint model predicting death and aortic valve intervention
Coef
Exp(coef):HR
Se(coef)
p-value
Death
BNP at specific time point (pg/ml)
0.5
1.65
0.3
0.0962
AVA (cm2)
−2.61
0.07
1.5
0.0815
Age (years)
0.02
1.02
0.04
0.5674
Male gender
1.12
3.06
0.6
0.0623
Symptoms
1.87
6.49
1.05
0.0753
*LV ejection fraction (%)
0.01
1.01
0.25
0.9539
*Creatinine (micromol/L)
0.18
1.2
0.15
0.2162
Aortic valve intervention
BNP at specific time point (pg/ml)
0.18
1.2
0.25
0.4787
AVA (cm2)
−1.12
0.33
1.04
0.2804
Age (years)
−0.04
0.96
0.02
0.0077
Male gender
0.39
1.48
0.49
0.4287
Symptoms
1.08
2.94
0.46
0.0183
*LV ejection fraction (%)
0.24
1.27
0.21
0.2388
*Creatinine (micromol/L)
−1.43
0.24
1.31
0.2761
BNP = brain natriuretic peptide; AVA = aortic valve area; LV = left ventricular; HR = hazard ratio. * Trasnformed LV ejection fraction and Creatinine in the models Table 3 Coefficients, standard error of coefficients and p-values for the joint model predicting death and aortic valve intervention
Coef
Exp(coef):HR
Se(coef)
p-value Fig. 2 Dynamic prediction for the survival probability for Mr. Jones for the survival probability for Mr Jones Fig. 2 Dynamic prediction for the survival probability for Mr. Jones Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 Page 5 of 8 same can be seen in Fig. 3, where the intervention-free
probabilities are presented. This can be explained by the
fact that Mr. Jones’ BNP measurements are relatively low
and stable. In contrast, Mr. Results Smith has more steep curves
for both expected survival and aortic valve intervention-
free probabilities indicating that the patient should be
monitored frequently. Specifically, one year after his first
follow-up visit Mr. Smith has a survival probability of
70 %, while one year after his last visit his survival probabi-
lity is less than 50 %. The reason could be that Mr. Smith
has a high BNP value at baseline and his progression is fas-
ter within the 2 year period compared to Mr. Jones. Thus,
Mr. Smith has a much lower survival probability one year
after his last follow-up. patient at baseline, lower baseline LV ejection fraction
and a higher baseline serum creatinine are highly associ-
ated with an increased BNP. Moreover, Fig. 1 shows the
evolutions
of
BNP
over
time
of
two
hypothetical
patients, Mr. Jones and another patient that has the
same characteristics as Mr. Jones except for the AVA level
which is 0.61. It is obvious in Fig. 1 that a smaller AVA is
associated with a higher BNP at baseline. Furthermore,
there is no difference in the progression of BNP between
the two patients. From the joint model with the death as
outcome, in Table 3, we observe that smaller AVA at base-
line, male patient, symptoms at baseline and higher BNP
at a specific time point (since we used all repeated mea-
surements in the model for the specific covariate) tend to
be associated with death. The joint model with the aortic
valve intervention as outcome shows that a younger
patient and symptoms at baseline are strongly associated
with aortic valve intervention probabilities. Finally, from the bootstrapping we observe the area
under the ROC curve for death and aortic valve inter-
vention to be 0.88 and 0.59, respectively. This indicates a
good discriminative capability of the BNP for death, and
little added value for the prediction of aortic valve
intervention. Figures 2, 3, 4 and 5 represent the dynamic prediction
of survival and aortic valve intervention-free respectively
for Mr. Jones and Mr. Smith, employing the joint mode-
ling framework. It can be seen in Fig. 2 that as more BNP
measurements accumulated over time for Mr. Jones, the
survival curve does not show big changes. Moreover, the Discussion In this paper we illustrated the use of joint models of
longitudinal and time-to-event data for individualized In this paper we illustrated the use of joint models of
longitudinal and time-to-event data for individualized Fig. 3 Dynamic prediction for the aortic valve intervention-free probability for Mr. Jones ig. 3 Dynamic prediction for the aortic valve intervention-free probability for Mr. Jones Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 Page 6 of 8 Fig. 4 Dynamic prediction for the survival probability for Mr. Smith Fig. 4 Dynamic prediction for the survival probability for Mr. Smith Fig. 4 Dynamic prediction for the survival probability for Mr. Smith dynamic event prediction using serial BNP measurements
in patients with severe AS. Patient prognostication may be
improved by the use of such models that take into account
all available medical information that accumulates over
time. In the case of Mr. Jones and Mr. Smith, their prob-
abilities of survival and aortic valve intervention-free were
calculated accounting for all BNP values that accumulated
over time and were updated when new BNP mea-
surements became available. This approach provides the
cardiologist with a useful evidence-based tool to assess
the impact of BNP on patient prognosis. Importantly,
the calculated probabilities for survival and aortic valve
intervention-free can be used as an early warning system,
allowing the necessary time for the physicians to plan an
intervention. Given the impaired quality of life (QOL) of
symptomatic patients with AS [10] and the considerable
improvement in QOL after the aortic valve replacement,
dynamic event predictions may be of great value in opti-
mizing the timing of the intervention [11]. realistic for biomarkers due to the fact that such cardio
data as BNP values cannot be assumed to be constant
between visits. Of course, the proposed methodology has several
(potential) limitations, both from a clinical and a statis-
tical point of view. From a clinical point of view, every
patient is unique, and analysis based on group data may
not account for the special characteristics of an indi-
vidual patient. Moreover, there are factors that are not
included in the statistical models that may play an im-
portant role and thus influence the decision making. In
this respect we acknowledge that the proposed metho-
dology may be supportive in clinical decision making,
but can never replace clinical expertise. Discussion Also, for clini-
cians with limited understanding of advanced statistical
models, the proposed methodology may be difficult to
comprehend, and tutorials aimed at clinicians are needed
to further educate clinical professionals [6]. From the stat-
istical point of view, the analysis of more than one longitu-
dinal outcomes such as BNP, AVA and symptoms over
time together with survival outcomes requires advanced
computational work and standard statistical packages do
not yet provide these options. Moreover, there is not yet a
package performing dynamic event prediction accounting
for the competing risk problem: specifically, patients could The joint model of longitudinal and survival data [5]
represents a powerful statistical tool capable of capturing
the association between longitudinal and survival data. An alternative approach is to utilize the time-dependent
Cox model. However, this model assumes a step func-
tion between the repeated measurements, which is not Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 Page 7 of 8 Fig. 5 Dynamic prediction for the aortic valve intervention-free probability for Mr. Smith ig. 5 Dynamic prediction for the aortic valve intervention-free probability for Mr. Smith to produce. Particularly, an easy web application could
give the opportunity to every physician to derive up-
dated predictions for new patients when more longitu-
dinal outcomes are available. die or require an intervention, in this case is aortic valve
intervention. The analysis, then, becomes more compli-
cated by the fact that the two censored outcomes are not
completely independent, thus it is clear that analyzing the
two outcomes separately is not appropriate and may lead
to bias. However, in order to keep the analysis simple and
thus to use only available packages, in the paper we did
not accounted for the competing risk problem. Further-
more, a topic that was not addressed in this paper, con-
cerns the validation of the derived predictions in terms of
calibration. Within the joint modeling frame, some work
has been done by Rizopoulos (2011) [7] and Proust-Lima
and Taylor (2009) [8]. Specifically, they focus on predictive
accuracy measures that compare the actual value of pre-
dictions with the observed data using simulated data. Finally, a dataset consisting of more patients that are
followed for a longer time period may provide better pre-
dictions for future patients. From the analysis we obtained a non-significant asso-
ciation between aortic valve intervention and the evolu-
tion of BNP (Table 3). References 1. Katz M, Tarasoutchi F, Pesaro AE, Lopes RD, Spina GS, Vieira ML, et al. Natriuretic peptides and long-term mortality in patients with severe aortic
stenosis. J Heart Valve Dis. 2012;21:331–6. 2. Lancellotti P, Moonen M, Magne J, O’Connor K, Cosyns B, Attena E, et al. Prognostic effect of long-axis left ventricular dysfunction and B-type
natriuretic peptide levels in asymptomatic aortic stenosis. Am J Cardiol. 2010;105:383–8. 3. Otto CM, Burwash IG, Legget ME, Munt BI, Fujioka M, Healy NL, et al. Prospective study of asymptomatic valvular aortic stenosis. Clinical,
echocardiographic, and exercise predictors of outcome. Circulation. 1997;95:2262–70. Authors’ contributions
h f
d f
d Authors contributions
Writing the first draft and data analysis: ERA. Data interpretation, discussion
and preparation of the final manuscript: ERA, DR, MLG, EL, AJJCB, JJMT. All authors read and approved the final manuscript. Authors contributions
Writing the first draft and data analysis: ERA. Data interpretation, discussion
and preparation of the final manuscript: ERA, DR, MLG, EL, AJJCB, JJMT. All authors read and approved the final manuscript. Conclusions In conclusion, this paper has shown that temporal ad-
justment of risk prediction models for patients with se-
vere
AS,
as
more
measurements
of
BNP
become
available over time, provide the physician with an evi-
dence based understanding of the prognostic implication
of changes in the patient’s disease condition. With the
cardiovascular medical practice increasingly moving to-
wards personalized medicine [12], joint models may pro-
vide an attractive tool for subject-specific predictions. The proposed joint model that was built and used to
predict prognosis of patients suffering from severe AS,
can be easily extended to other chronic disease entities
that employ both longitudinal and survival data to dy-
namically assess patient prognosis. Competing interests Competing interests
The authors declare that they have no competing interests. Author details
1
f 1Department of Biostatistics, Erasmus MC, Rotterdam, The Netherlands. 2Department of Cardiology, Erasmus MC, Rotterdam, The Netherlands. 3KU
Leuven, L-Biostat, Leuven, Belgium. 4Department of Cardiothoracic Surgery,
Erasmus MC, Rotterdam, The Netherlands. Received: 26 May 2014 Accepted: 21 April 2015 Received: 26 May 2014 Accepted: 21 April 2015 Introduction to joint models
* 4. Heuvelman HJ, van Geldorp MW, Kappetein AP, Geleijnse ML, Galema TW,
Bogers AJ, et al. Clinical course of patients diagnosed with severe aortic
stenosis in the Rotterdam area: insights from the AVARIJN study. Neth Heart
J. 2012;20:487–93. Let Ti
* denote the true failure time for the i-th individual
(i = 1,…, n), and Ci the censoring time, then Ti = min(Ti
*, Ci)
represents the observed failure time for the i-th patient. Moreover, δi = 0,1 is the event indicator where 0 indicates
censoring. For the longitudinal part, we let yi consist of
longitudinal responses that may be obtained at different
time points tij and have length ni. To describe the subject-
specific evolutions over time of the longitudinal outcome
we utilize a linear mixed-effects model. Specifically, it
takes the form, 5. Rizopoulos D. Joint models for longitudinal and time-to-event data with
applications in R. Boca Raton: Chapman and Hall/CRC Biostatistics
Series; 2012. 6. Andrinopoulou ER, Rizopoulos D, Jin R, Bogers AJ, Lesaffre E, Takkenberg JJ. An introduction to mixed models and joint modeling: analysis of valve
function over time. Ann Thorac Surg. 2012;93:1765–72. 6. Andrinopoulou ER, Rizopoulos D, Jin R, Bogers AJ, Lesaffre E, Takkenberg JJ. An introduction to mixed models and joint modeling: analysis of valve
function over time. Ann Thorac Surg. 2012;93:1765–72. 7. Rizopoulos D. Dynamic predictions and prospective accuracy in joint
models for longitudinal and time-to-event data. Biometrics. 2011;67:819–29. 8. Proust-Lima C, Taylor JM. Development and validation of a dynamic
prognostic tool for prostate cancer recurrence using repeated measures of
posttreatment PSA: a joint modeling approach. Biostatistics. 2009;10:535–49. 9. Rizopoulos D. JM: A package for the joint modelling of the longitudinal and
time-to-event data. J Stat Software. 2010;35:1–33. g
7. Rizopoulos D. Dynamic predictions and prospective accuracy in joint
models for longitudinal and time-to-event data. Biometrics. 2011;67:819–29. 8. Proust-Lima C, Taylor JM. Development and validation of a dynamic
prognostic tool for prostate cancer recurrence using repeated measures of
posttreatment PSA: a joint modeling approach. Biostatistics. 2009;10:535–49. 8. Proust-Lima C, Taylor JM. Development and validation of a dynamic
prognostic tool for prostate cancer recurrence using repeated measures of
posttreatment PSA: a joint modeling approach. Biostatistics. 2009;10:535–49. yi tð Þ ¼ f i tð Þ þ ∈i ¼ xT
i tð Þβ þ zT
i tð Þbi þ ∈i; p
j
g pp
;
9. Rizopoulos D. Discussion Hence, the validation showed that
for the target group of patients the BNP as a marker for
intervention
does
not
exhibit
great
discrimination
power. BNP levels have been previously found to be pre-
dictors of reoperation. Therefore, although BNP profile
is not a good predictor of intervention in our case, it is
reliable in predicting mortality and thus can be very
helpful in planning an intervention to prevent mortality
due to AS disease progression. This non-significant re-
sult could be explained by the fact that additional car-
diovascular risk factors were not taken into account
because either there were not available or the patients
were not enough to include more factors in the model. Although all analysis was performed using standard
statistical packages, a level of expertise in programming
may be required. Thus, interactive web applications with
friendly controls that easily incorporate plots and sum-
maries are essential for adequate implementation of the
proposed models in clinical practice may be interesting In this paper we assumed linear trajectories for the
BNP biomarker since we did not have a big range of
values per patient. However, in a different setting where
more information would be available per patient it may
be of interest to investigate for non-linear profiles. Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 Page 8 of 8 corresponding vector of regression coefficients γ, and α
denotes the strength of association between the longitu-
dinal and survival outcome. Moreover, a Weibull base-
line hazard h0(t) = ψtψ–1 was assumed. Specifically, patients could have highly non-linear evolu-
tion that could not be described by a simple structure,
such as linear one. Even though the interpretation then
becomes more complex, it is evident that misspecifica-
tion of the evolution of the biomarkers could lead to
bias. In order to obtain valid results, it is important to
postulate a mixed-effects model that is capable of appro-
priately capturing such non-linear evolutions. Introduction to joint models
* JM: A package for the joint modelling of the longitudinal and
time-to-event data. J Stat Software. 2010;35:1–33. 10. van Geldorp MWA, Heuvelman HJ, Kappetein AP, Busschbach JJV,
Takkenberg JJM, Bogers AJJC. The effect of aortic valve replacement on
quality of life in symptomatic patients with severe aortic stenosis. Neth
Heart J. 2013;21:28–35. where xi(t) denotes the design vector for the fixed effects
regression coefficients β and zi(t) the design vector for
the random effects bi. 11. van Geldorp MWA, Heuvelman HJ, Kappetein AP, Busschbach JJ, Cohen DJ,
Takkenberg JJ, et al. Quality of life among patients with severe aortic
stenosis. Neth Heart J. 2013;21:21–7. Finally, we assume that a normal distribution for the
random effects describes the evolution of the longitu-
dinal outcomes, i.e., 12. Joint Task Force on the Management of Valvular Heart Disease of the
European Society of Cardiology (ESC); European Association for Cardio-
Thoracic Surgery (EACTS), Vahanian A, Alfieri O, Andreotti F, Antunes MJ,
Barón-Esquivias G, et al. Guidelines on the management of valvular heart
disease (version 2012). Eur Heart J. 2012;33:2451–96. bi∼N 0; σb
ð
Þ; Where σb is the variance of the random intercept. For the survival process we have Where σb is the variance of the random intercept. F
h
i
l
h For the survival process we have hi t; θs
ð
Þ ¼ h0 tð Þe γTωiþaf i tð Þ
f
g; where θs is the parameter vector for the survival out-
comes, ωi is a vector of baseline covariates with a where θs is the parameter vector for the survival out-
comes, ωi is a vector of baseline covariates with a
|
W4377824843.txt
| null |
de
|
Zur Bewertung verbaler und physischer Gewalt im Amateurfußball. Eine computergestützte qualitative Inhaltsanalyse am Beispiel von Sportgerichtsurteilen des Bayerischen Fußballverbandes
|
Zeitschrift für Fußball und Gesellschaft
| 2,023
|
cc-by
| 6,464
|
Zur Bewertung verbaler und physischer Gewalt im
Amateurfußball. Eine computergestützte qualitative
Inhaltsanalyse am Beispiel von Sportgerichtsurteilen des
Bayerischen Fußballverbandes
Florian Koch, Clemens Bernd
To cite this version:
Florian Koch, Clemens Bernd. Zur Bewertung verbaler und physischer Gewalt im Amateurfußball.
Eine computergestützte qualitative Inhaltsanalyse am Beispiel von Sportgerichtsurteilen des Bayerischen Fußballverbandes. FuG – Zeitschrift für Fußball und Gesellschaft, 2023, 4 (1), pp.27-42.
�10.3224/fug.v4i1.03�. �hal-04108585�
HAL Id: hal-04108585
https://u-bourgogne.hal.science/hal-04108585
Submitted on 27 May 2023
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.
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.
Distributed under a Creative Commons Attribution 4.0 International License
Zur Bewertung verbaler und physischer Gewalt im Amateurfußball.
Eine computergestützte qualitative Inhaltsanalyse am Beispiel von Sportgerichtsurteilen des
Bayerischen Fußballverbandes.
The evaluation of verbal and physical aggressions in amateur soccer. A computer-assisted
qualitative content analysis using the example of a sports court ruling of the Bavarian Football
Association.
Florian Koch1 und Clemens Bernd
Florian Koch (ORCID: 0000-0002-4897-7174) studierte Politikwissenschaften an der Universität
Leipzig und den IEPs in Lille und Straßburg. Anschließend absolvierte er das binationale
Promotionsprogramm Cotutelle de thèse zwischen der Universität Leipzig und der Université de
Paris. Zurzeit lehrt er als Maître de conférences an der Université de Bourgogne (uB) sowie am
IEP de Paris (Sciences Po) und forscht am Forschungslabor Centre Interlangues - Texte, Image,
Langage (TIL) zum alltäglichen Sprachverhalten rund um den Sport/Fußball.
Eine aktuelle Veröffentlichung:
Koch, Florian (2019): Die Abwertung des Anderen mittels Sprache im Amateurfußball. Ein
soziolinguistischer Vergleich in Deutschland und Frankreich. Berlin: Peter Lang (Sprache - Kultur
- Gesellschaft).
Clemens Bernd studierte Politikwissenschaften mit dem Schwerpunkt Internationale Beziehungen
an der Johannes Gutenberg-Universität Mainz. Zurzeit arbeitet er als Projektmanager in der
Hauptabteilung Innovationsmanagement und Digitale Transformation beim Südwestrundfunk
(SWR). Er forscht über die alltägliche Diskriminierung im Sport/Fußball.
1 Korrespondierender Autor, florian.koch@u-bourgogne.fr
Florian Koch und Clemens Bernd
Zur Bewertung verbaler und physischer Gewalt im Amateurfußball.
Eine computergestützte qualitative Inhaltsanalyse am Beispiel von Sportgerichtsurteilen
des Bayerischen Fußballverbandes.
Zusammenfassung
Bisherige Studien über verbale und/oder physische Gewalt im Amateurfußball und deren
Bewertung durch Sportrichter*innen legten Ihren Fokus auf rassistisch/fremdenfeindlich
motivierte Konflikte. Nicht untersucht wurden Einflussfaktoren sowohl bei der Ausübung als auch
bei der Bewertung verbaler und/oder physischer Gewalt aus – weiter gefassten –
menschenfeindlichen Motiven. Ziel dieses Artikels ist es daher einen Beitrag zu leisten diese
Forschungslücke zu schließen.
Dafür werden mittels einer qualitativen Inhaltsanalyse 25 ausgewählte saisonübergreifende Urteile
aus den Jahren 2016 bis 2020 des Sportgerichts des Bayerischen Fußball-Verband (BFV)
softwaregestützt explorativ untersucht.
Die Auswertung zeigt, dass Täter*innen und Opfer überwiegend männlich sind und sich Vorfälle
verbaler und/oder physischer Gewalt zumeist bei Spielen der aktiven Herrenmannschaften
ereigneten. Täter*innen waren dabei zumeist Trainer*innen/Betreuer*innen, gefolgt von
Spieler*innen und Fans. Zudem hierarchisierten Sportrichter*innen verbale und/oder physische
Gewalt. Während behindertenfeindlich motivierte verbale und/oder physische Gewalt milde
bestraft wurde und bei sexistisch motivierter Gewalt nahezu gleichverteilt leichte/mittelschwere
bzw. schwere/schwerste Strafen verhängt wurden, sprachen Sportgerichte des BFVs bei
rassistisch/fremdenfeindlich motivierter verbaler und/oder physischer Gewalt mehrheitlich
schwere/schwerste Strafen aus.
Zukünftig sollte der BFV daher darauf achten, dass neben den bereits durchgeführten
Präventionskampagnen und Anti-Gewalt-Kursen, die hauptsächlich auf Spieler*innen und
Trainer*innen/Betreuer*innen abzielen, auch Kurse/Seminare angeboten werden, die gezielt für
Amateurschiedsrichter*innen
und/oder
Sportrichter*innen
konzipiert
werden,
um
eine
unterschiedliche Bewertung verbaler und/oder physischer Gewalt, die auf menschenfeindliche
Einstellungen schließen lassen, zu vermeiden.
Schlüsselwörter: verbale und/oder physische Gewalt; menschenfeindliche Einstellungen;
Amateurfußball; Sportgerichtsbarkeit;
1
Abstract
Previous studies on verbal and/or physical aggression in amateur football and its evaluation by
sports judges have focused on conflicts motivated by racism and/or xenophobia. However, other
factors that influence both the practice and evaluation of verbal and/or physical aggression from
misanthropic motives have not yet been examined. The aim of this article is therefore to contribute
to the closure of this gap in current research.
To this end, 25 selected cross-seasonal rulings from 2016 to 2020 of the Bavarian Sports Court are
explored utilising computer-assisted qualitative data analysis software (CAQDAS) by means of a
qualitative content analysis.
The analysis shows that perpetrators and victims are predominantly male, and that incidents of
verbal and/or physical aggression mostly occur during matches involving teams of men. The
perpetrators were found to be mostly coaches/staff, followed by players and spectators. In addition,
the study shows that sports referees did hierarchically structure verbal and/or physical aggression.
While verbal and/or physical aggression motivated by hostility towards persons with disabilities
was punished leniently, and slight/medium, or severe/most severe penalties were imposed almost
equally for aggression motivated by sexism, the majority of penalties imposed by the Bavarian
Sports Court for verbal and/or physical aggression motivated by racism/xenophobia were
severe/most severe.
In future, the Bavarian Football Association (BFV) should therefore ensure that, in addition to their
prevention campaigns and anti-violence courses that they already carry out, which are mainly
aimed at players and coaches/staff, courses are also offered that are specifically designed for
amateur referees and/or sports judges in order to avoid a hierarchical structure in the assessment
of verbal and/or physical aggression that suggests group-focused enmity.
Keywords: verbal and/or physical aggression; group-focused enmity; amateur football; sports
court ruling;
2
Aktualität und Relevanz
Der über sieben Millionen Mitglieder zählende Deutsche Fußball-Bund (DFB) zeichnete in seinem
6. Lagebericht des Amateurfußballs ein ambivalentes Bild (DFB 2020). 2 Danach kam es in der
Saison 2019/2020 in 0,45% aller 60.000 erfassten Fußballspiele zu Gewalt- oder
Diskriminierungsvorfällen. Das war – allerdings vor dem Hintergrund der Corona-Pandemie, die
zahlreiche Absagen von saisonentscheidenden Spielen verursachte – ein Rückgang um ca. 0,05%
an gemeldeten Gewalt- oder Diskriminierungsvorfällen im Vergleich zu den beiden vorherigen
Saisons. Ähnlich verhält es sich mit Spielabbrüchen. Mussten in der Saison 2018/2019 noch 0,05%
der Spiele abgebrochen werden, waren es in der Folgesaison lediglich 0,03%.
Dieser Rückgang kann durchaus auch als (kurzfristiger) Erfolg der Strategie gewertet werden, die
darauf abzielt, Druck auf verbandliche und politische Entscheidungsträger*innen durch verstärkte
mediale Berichterstattung auszuüben. So wurden Brandbriefe von Amateurschiedsrichter*innen
veröffentlicht,
Schiedsrichterstreiks
organisiert
und
somit
Spielabsagen
verursacht
(Schulz/Teevs/Heier 2016). Zudem berieten sich die Innenminister*innen der Länder 2019, um
verbale aber auch physische Gewalt v.a. gegen Amateurschiedsrichter*innen wirksam
einzudämmen (BMI 2019). Der DFB veröffentlichte daraufhin einen offenen Brief, in dem allen
aktiven Schiedsrichter*innen – auch aufgrund eines Verlustes um fast 22.000 in den letzten zehn
Jahren
(DFB
2018)
–
umfassende
Unterstützung
zugesichert
wird
(Curtius/Keller/Koch/Zimmermann 2019). Schließlich verweist der DFB (2021) auf sein
Gewaltpräventionskonzept „Fair ist mehr“. Es bündelt bereits bestehende Angebote und
Maßnahmen zur Gewaltprävention und Intervention bei Gewaltvorfällen. Ein wesentlicher
Baustein befasst sich mit der effizienten Bearbeitung von Vorfällen und alternativen
Sanktionsmaßnahmen der Sportgerichtsbarkeit, auch „im Sinne des Statuierens eines Exempels“
mit dem Ziel die Täter*innen zu einer Einstellungs- und Verhaltensänderung zu bewegen.
Dennoch beklagt der 6. Lagebericht weiterhin das Auftreten gravierender Einzelfälle (DFB 2020).
Ein besonders eindrückliches Beispiel, das darüber hinaus aufzeigt, dass aktuelle politische
Konflikte mitunter auch auf dem Amateurfußballplatz ausgetragen werden, ereignete sich Mitte
Oktober 2019 – drei Tage nach dem Einmarsch türkischer Truppen in Syrien – beim Kreisliga C
Spiel in Essen (Poppe 2019). Die mehrheitlich aus syrischen Flüchtlingen bestehende zweite
Mannschaft des BV Altenessen spielte gegen den türkischen Migrantenverein Barisspor.
Aufgeheizt durch die politischen Ereignisse hetzten am Spielende Spieler des BV Altenessen den
2 Die Autoren bedanken sich herzlich bei den beiden anonymen Gutachter*innen für ihre zahlreichen kritischen
Kommentare und konstruktiven Hinweise.
3
Schiedsrichter über den Platz und verletzten ihn. Der alteingesessene Verein mit mehr als 300
Mitgliedern aus 30 Nationen regierte prompt und meldete die Mannschaft umgehend ab, auch weil
das zuständige Sportgericht ungewöhnlich hart durchgriff und fast alle Spieler mit einer Sperre
von mindestens einem Jahr belegt hatte.
Konkretes Ziel dieses Artikels ist es daher alltägliche verbale und/oder physische Gewalt im
Amateurfußball anhand von 25 ausgewählten saisonübergreifenden Sportgerichtsurteilen des
Bayrischen Fußball-Verbands (BFV) softwaregestützt explorativ zu untersuchen. Dabei
konzentriert sich die vorliegende Analyse sowohl auf Einflussfaktoren bei der Ausübung konkreter
alltäglicher verbaler und/oder physischer Gewalt im Amateurfußball als auch auf deren Bewertung
durch Sportrichter*innen des BFVs. Ein besonderes Augenmerk wird dabei auf menschfeindliche
Einstellungen gelegt.
Dafür wird zunächst der theoretische Rahmen, der Forschungsstand sowie das methodische
Vorgehen vorgestellt, um daran anschließend Einflussfaktoren bei der Ausübung verbaler und/oder
physischer Gewalt und deren Bewertung durch Sportrichter*innen des BFVs herauszuarbeiten.
Zuletzt werden die Ergebnisse diskutiert und konkrete Handlungsempfehlungen gegeben.
Theoretischer Rahmen und thematische Hinführung
Laut Delhom (2007: 229) lässt sich physische Gewalt nicht trennscharf von verbaler Gewalt
unterscheiden. Vielmehr ist Gewalt nie „stumm“ und „wird meistens durch Sprache begleitet: Sie
wird geplant und beschlossen, erzählt und kommentiert, gerechtfertigt oder legitimiert. Und
darüber hinaus wird sie durch die Sprache und in der Sprache vollzogen.“ 3 Ähnlich argumentiert
Krämer (2007: 31ff.). Sie definiert Gewalt ebenso als ein Phänomen, das sowohl auf den
physischen Gewaltakt abzielt, aber auch den sprachlichen Gewaltakt einschließt mit dem Ziel die
betroffene Person physisch oder symbolisch schmerzhaft zu verletzen.4
Verbale Gewalt ist ein Phänomen, das in allen Gesellschaften in vielfältiger Form auftritt (Krämer
2007: 32ff.). Klinker, Scharloth und Szczek (2018: 1ff.) verstehen darunter alltägliche sprachliche
Normüberschreitungen wie Beleidigungen, Herabwürdigungen und zeichenhafte Aggressionen.
Anders als physische Gewalt kann jedoch sprachlich-symbolische Gewalt dem Betroffenen nicht
aufgezwungen werden und benötigt sowohl die Mitwirkung der Betroffenen als auch die
3 Lobenstein-Reichmann (2012: 215ff.) beschreibt detailliert die historische Entwicklung der einstigen Antipoden
Gewalt und Sprache. Wesentlich ist dabei, dass in Anlehnung an Norbert Elias verbale Gewalt weitgehend physische
Gewalt ersetzt und dieser stets vorausgeht.
4 Herrmann, Krämer und Kuch (2007: 7ff.) geben einen einführenden Überblick über das Konzept „Verletzende
Worte“.
4
legitimierende Kraft Dritter (Lobenstein-Reichmann 2012: 219).
Der Amateurfußballplatz ist nun ein spezifischer sozialer Raum, auf dem Normabweichungen eine
weitaus höhere Toleranz erfahren und mögliche Sanktionen weniger konsequent durchgesetzt
werden. Aggressives Verhalten wird vielmehr gefordert und gefördert, um in eingeübten
unhinterfragten Routinen zu gerinnen (Dunning 1999: 30). Mennell (2006: 515) unterstreicht, dass
übermäßige Gewaltausübung vor allem in Mannschaftssportarten vorkommt, die körperliche
Robustheit mit einem komplexen Spiel kombinieren. Zahlreiche Autor*innen weisen zudem
darauf hin, dass diese eingeübten Routinen zunehmend in den unteren Ligen in verbale und/oder
physische Gewalt umschlagen (v.a. Ribler 2010: 13; Winands 2016: 7).
Das sozialpsychologische Konzept der Gruppenbezogenen Menschenfeindlichkeit (GMF)
untersucht nun den Zusammenhang zwischen der Ausübung alltäglicher Diskriminierung und
menschenfeindlichen Einstellungen (v.a. Zick 2010: 19ff.). Kern des Konzepts ist die auf
Vorurteilen
beruhende
Ideologie
der
Ungleichwertigkeit.
Diese
drückt
sich
durch
menschenfeindliche Einstellungen gegenüber als fremd wahrgenommenen Gruppen aus. Die
Zuschreibung zu den Gruppen erfolgt dabei aufgrund von invariablen Eigenschaften – wie der
Hautfarbe, der (vermeintlichen) ethnischen Herkunft und/oder religiösen Zugehörigkeit, dem
Geschlecht, der sexuellen Orientierung sowie der körperlichen Verfasstheit – entlang derer sich
einzelne Elemente der GMF – insbesondere Rassismus und Fremdenfeindlichkeit, Islamophobie
und Antisemitismus, Sexismus, Homophobie sowie Behindertenfeindlichkeit – konstituieren
(Zick/Hövermann/Küpper 2011: 27ff.). Da die Abwertung einer als fremd wahrgenommenen
Gruppe mit der Abwertung weiterer Gruppen einhergeht, bezeichnet Heitmeyer (2012: 15),
Begründer und Vordenker des Ansatzes, die GMF als ein Syndrom.
Zick, Scherer und Winands (2010: 133ff.) argumentieren, dass diese menschenfeindlichen
Einstellungen nun durch eine Vielzahl von Akteur*innen – wie Spieler*innen, Fans,
Funktionär*innen – in die Fußballarena hineingetragen werden. Dieses sei vor allem bei
unparteiisch
urteilenden
verbandlichen
Akteur*innen
wie
Schiedsrichter*innen
und
Sportrichter*innen höchst problematisch und hoch relevant, da dadurch menschenfeindliche
Einstellungsmuster legitimiert und zementiert werden können.5
Koch (2019: 93) verbindet das reale alltägliche Sprachverhalten mit dem auf Vorurteilen
basierenden Konzept der GMF. Er schlägt vor nunmehr von verbaler Abwertung zu sprechen,
wenn die tatsächlich ausgeübte verbale Gewalt auf gruppenbezogene menschenfeindliche
5 Aufgrund der institutionellen Benachteiligung vor Gericht von Sportrichter*innen mit Migrationshintergrund,
bezeichnen Zick, Scherer und Winands (2010: 133ff.) das Sportgericht gar pointiert als „ethnische Kampfarena“.
5
Einstellungen schließen lässt.
Sportrichter*innen im Amateurfußball urteilen basierend auf der verbandlichen Rechts- und
Verfahrensordnung (RVO) (BFV 2022a). Um als Sportrichter*in an ein unabhängiges bayrisches
Sportgericht berufen zu werden, reicht es demnach aus älter als 30 Jahre alt zu sein, über
ausreichend Erfahrung im Sport zu verfügen und sich im sportlichen Alltag bewährt zu haben. Die
Befähigung zum Richteramt benötigen zwingend lediglich Vorsitzende des Sportgerichts sowie
des Verbands-Sportgerichts (§ 9 RVO). Sanktioniert werden alle Formen unsportlichen Verhaltens
(§ 47a RVO) in der Regel in nicht öffentlichen Sitzungen (§ 11 RVO). Dazu zählt insbesondere
politisch extremistisches, obszön anstößiges oder provokativ beleidigendes Verhalten. § 47a RVO
nimmt den Kern der Ideologie der Ungleichwertigkeit auf und definiert besonders schwere Fälle
von Unsportlichkeit als solche, die durch herabwürdigende Äußerungen die Menschenwürde einer
Person oder einer Gruppe von Personen verletzen. Das ist insbesondere dann der Fall, wenn
Menschen aufgrund ihrer Herkunft, Hautfarbe, Sprache, Religion, Behinderung, Alter,
geschlechtlichen oder sexuellen Identität diskriminiert werden. Aufgrund des verbandspolitischen
Interesses leitet bei schweren Fällen unsportlichen Verhaltens in aller Regel der Verbandsanwalt
das Verfahren (§ 35 RVO). Zudem erhöht sich die vorgesehene Mindeststrafe bei Fällen von
festgestellten schweren Unsportlichkeiten auf eine Sperrstrafe von nicht unter fünf Wochen oder
fünf Spielen bis hin zum Ausschluss oder einer Mindestgeldstrafe von 300 € (§ 47a RVO).
Forschungsstand
Obwohl
verbale
und/oder
physische
Gewalt
im
Amateurfußball
vor
allem
gegen
Amateurschiedsrichter*innen (Vester 2013; Rullang/Emrich/Pierdzioch 2015a, 2015b), auch als
transnationales Phänomen (Koch 2019, 2021), wissenschaftlich untersucht und ebenso
Einflussfaktoren bei der Ausübung von menschenfeindlicher Gewalt und deren Bewertung durch
Amateurschiedsrichter*innen herausgearbeitet wurden (Koch 2020), ist der Sportgerichtsbarkeit
in den letzten Jahren nur wenig Aufmerksamkeit geschenkt worden.
Eine der wenigen Ausnahmen ist die Untersuchung von Pilz (2002). Anhand von ca. 4000
Sportgerichtsurteilen des Niedersächsischen Fußballverbandes (NFV) aus der Saison 1998/1999
analysiert dieser Konflikte im Jugendfußball, die auf rassistische/fremdenfeindliche Einstellungen
schließen lassen. Er stellt fest, dass bei vergleichbaren Taten Spieler*innen nicht deutscher
Herkunft härter bestraft wurden. So erhielten Spieler*innen nicht deutscher Herkunft bei
„Tätlichkeiten ohne Verletzungen“ in rund zwei Drittel der Vorfälle eine Sperre von bis zu vier
Wochen, 25,7% bis zu sechs Wochen und 9,2% über sechs Wochen, wohingegen deutsche
6
Spieler*innen in 78,9% der Vorfälle mit einer Sperre bis zu vier Wochen, 14,2% bis zu sechs und
5,8% über sechs Wochen belegt wurden. Noch deutlicher ist die unterschiedliche
Strafmaßbemessung bei „Tätlichkeiten mit Verletzungen“. Während 54% der deutschen
Spieler*innen für bis zu vier Wochen gesperrt wurden, waren es bei den nicht deutschen
Spieler*innen nur 25%. Demgegenüber wurden 50% der nicht deutschen Spieler*innen und
lediglich 20% der deutschen Spieler*innen mit einer Sperre von über sechs Wochen belegt.
Bei den Ursachen dieser messbaren Diskriminierung von deutschen und nicht-deutschen
Spieler*innen unterscheidet Pilz (2002: 11ff.) zwischen oberflächigen und tieferliegenden
Ursachen. Zu ersteren zählt Pilz u.a. Erklärungen von Vertretern „deutscher Vereine und
Verbände“, die auf das südländische Temperament und der damit einhergehenden
Disziplinlosigkeit der Migrant*innen vor allem bei Entscheidungen von Schiedsrichter*innen
abzielen, aber auch Klagen von Mitgliedern „ethnischer Vereine“, die sich (gezielt eingesetzter)
verbaler Gewalt durch ihre deutschen Gegenspieler*innen und institutionelle Benachteiligung
aufgrund
vermeintlich
(latent)
rassistischer/fremdenfeindlicher
Einstellungen
durch
Schiedsrichter*innen sowie Sportrichter*innen ausgesetzt sehen. Der Autor verweist ferner darauf,
dass der sportliche Wettstreit auf dem Fußballplatz zwischen Jugendlichen deutscher und nichtdeutscher Herkunft nunmehr eine Stellvertreterfunktion einnimmt. Im Kern geht es um soziale
Anerkennung und das Recht auf soziale Mobilität von Minderheiten in einer sich
herausgeforderten Mehrheitsgesellschaft, die sich gegen eine Neuverteilung der Ressourcen und
Privilegien wehrt. Die hohe Sensibilität der Migrant*innen auf dem Fußballplatz, die diese auch
für
Provokationen
Teilhabemöglichkeiten
anfällig
macht,
ist
sowie
herrschender
Ausdruck
ungleicher
Diskriminierung
gesellschaftlicher
aufgrund
von
(latent)
rassistischen/fremdenfeindlichen Einstellungen in der Mehrheitsgesellschaft.
Scherer und Winands (2010: 47ff.) untersuchen ebenso Konflikte im Amateurfußball, die auf
gruppenbezogene menschenfeindliche Einstellungen schließen lassen. Anhand von 1205
Sportgerichtsurteilen des Hessischen Fußball-Verbandes (HFV) aus der Saison 2007/2008 stellen
sie fest, dass Täter*innen zumeist Spieler*innen aus dem Seniorenbereich und Opfer zumeist
Schiedsrichter*innen waren. Zudem bestätigen sie, dass Spieler*innen anderer Herkunft vor dem
Sportgericht überrepräsentiert waren und sowohl überproportional häufiger als auch öfter für
härtere Vergehen verurteilt wurden. Darüber hinaus erhielten sie für vergleichbare Vergehen
härtere Strafen. Obwohl die Autor*innen einräumen, dass ihre Ergebnisse keinen eindeutigen
Zusammenhang zwischen den analysierten Sportgerichtsurteilen und GMF belegen, nehmen sie
an, dass auch Sportrichter*innen mit einem selektiven Blick urteilen.
7
Laerum (2010: 55ff.) arbeitete mittels einer qualitativen Befragung von Sportrichtern heraus, dass
diese, auch wenn kein direkter Zusammenhang zwischen Einstellung und Strafbeimessung
nachgewiesen wurde, durchaus latent rassistische/fremdenfeindliche und islamophobe Stereotype
und Vorurteile in die Sportgerichte hintragen. Das äußert sich auch dadurch, dass männliche
Jugendliche mit islamischer Prägung, sowie die Rolle von Migrantenvereinen als besonders
problematisch beschrieben wurden. Die Autorin sieht eine wesentliche Ursache dafür in der
homogenen Zusammensetzung der Sportgerichte. So waren die Befragten ausschließlich männlich,
deutscher Herkunft und über 60 Jahre alt.6
Die drei vorgestellten Studien legten ihren Fokus insbesondere auf Konflikte im Amateurfußball,
die im Kern auf rassistische/fremdenfeindliche und islamophobe Einstellungen schließen lassen.
Nicht untersucht wurden weitere Einflussfaktoren sowohl bei der Ausübung als auch bei der
Bewertung verbaler und/oder physischer Gewalt von Sportrichter*innen im Amateurfußball sowie
aus – weiter zu fassenden – menschenfeindlichen Motiven. Die vorliegende Untersuchung hat
somit zum Ziel einen Beitrag zu leisten diese Forschungslücke zu schließen.
Methodisches Vorgehen
Aufgrund des explorativen Charakters und vorliegenden Datenmaterials nutzen wir für unsere
Untersuchung die qualitative Inhaltsanalyse. Im Kern zielt diese spezifische Auswertungstechnik
darauf ab, Textdokumente – wie die vorliegenden Sportgerichtsurteile – systematisch zu
analysieren und zu interpretieren, indem aus dem vorhandenen Datenmaterial Zusammenhänge in
Form eines Kategoriensystems induktiv herausgearbeitet werden (Mayring/Fenzl 2014: 543ff.;
Mayring 2016: 114ff.).7
Die konkrete Zuordnung (Codieren) einzelner Textsegmente (Codings) – zumeist handelt es sich
dabei um (unbestimmte) Rechtsbegriffe aus dem Sport, dem Strafmaß und wenigen
kontextualisierenden Informationen (Ort, Datum, Liga, Spielphase, Opfer usw.) – zu den
Kategorien (Codes) erfolgte ganz überwiegend durch Clemens Bernd. Um die Zuverlässigkeit des
Codierens zu gewährleisten, wurden die sich entwickelnden Haupt- und Subkategorien (Subcodes)
gemeinsam definiert, zur besseren Orientierung mit Ankerbeispielen versehen und fortlaufend
6 Zick, Hövermann und Küpper (2011: 14) belegen, dass gruppenbezogene menschenfeindliche Einstellungen mit
zunehmendem Alter stärker ausgeprägt sind und mit dem Bildungsniveau und dem Einkommen korrelieren. Allerdings
weisen die Autor*innen auch daraufhin, dass das Geschlecht keinen bedeutenden Effekt auf die GMF hat.
7 Mayring und Fenzl (2014: 544) unterscheiden zwischen induktiver und deduktiver Textanalyse. Während für erstere
Variante ein Kategoriensystem genutzt wird, welches anhand des Datenmaterials entwickelt wird, nutzt die zweite
Variante ein vorab entwickeltes theoriegeleitetes Kategoriensystem.
8
überprüft. Zudem erfolgte schließlich von beiden Autoren eine kritische Überprüfung des codierten
Datenmaterials. Um das Codieren zu vereinfachen aber auch das spätere Korrelieren von Variablen
sowie deren Präsentation zu unterstützen (Kuckartz 2014: 99ff.), verwendeten wir für die erste
Analyse die QDA-Software MAXQDA Plus Version 2020, für die Überarbeitung des Datensatzes
die Version 2022. Mayring unterstreicht ferner (2016: 140ff.), dass durch die Nutzung von QDASoftware ebenso wichtige Gütekriterien einer qualitative Inhaltsanalyse erfüllt werden, da diese
einfacher intersubjektiv nachvollzogen, reproduziert und überprüft werden können.8
Als Datengrundlage nutzen wir öffentlich zugängliche, saisonübergreifende Sportgerichtsurteile
des BFVs aus den Jahren 2016 bis 2020.9 Dieser mit ca. 1,6 Millionen Mitglieder größte der
insgesamt 21 Landesverbände des DFBs verbindet ländliche und urbane Regionen und bietet mit
ca. 13.000 Spielen an einem Spieltag die größtmögliche Grundgesamtheit. Zudem veröffentlicht
dieser Verband – aus Datenschutzgründen lediglich eine begrenzte Auswahl – Sportgerichtsurteile
auf seiner Homepage (BFV 2022b).10
Der Großteil dieser ca. 300 veröffentlichten Urteile sind für unsere Untersuchung irrelevante
Vorfälle wie Freisprüche aber auch Spielsperren nach regulären roten Karten, Fragen zur
Spielberechtigung einzelner Spieler*innen oder Anfragen zur Änderung von Spielterminierungen.
Vor diesem Hintergrund haben wir folgende Kriterien entwickelt, die sicherstellen, dass die Urteile
für unsere Untersuchung verwertbare Daten enthalten: (1) Die geahndeten Vergehen müssen
losgelöst vom unmittelbaren Spielgeschehen sein. Dazu zählen u.a. Beleidigungen jeglicher Art
sowie tätliche Angriffe auf Schiedsrichter*innen. Nicht dazu zählen somit grobe Fouls, selbst wenn
diese den Spielausschluss und somit ein Sportgerichtsurteil zur Folge hatten. (2) Außerdem müssen
aus dem Urteil die beteiligten individuellen Akteur*innen und das verhängte Strafmaß eindeutig
hervorgehen. Nicht berücksichtigt werden daher u.a. Urteile gegen Vereine aufgrund von
mangelnden Sicherheitsvorkehrungen. Vor diesem Hintergrund berücksichtigen wir für unsere
nachfolgende Analyse 25 der ca. 300 gesichteten Urteile.
Ergebnispräsentation der qualitativen Inhaltsanalyse von Urteilen des BFVs
Die Ergebnispräsentation der qualitativen Inhaltsanalyse erfolgt in drei Schritten. Zuerst werden
Einflussfaktoren – (1) Akteur*innenbezogene Faktoren (75 Codings), (2) Spielbezogene Faktoren
(50 Codings), (3) Art der Vergehen (25 Codings) und (4) Verhängte Strafen (25 Codings)
8 So kann auf Anfrage der vorliegende Datensatz eingesehen und für weitere Untersuchungen genutzt werden.
9 Pandemiebedingt lagen in der Saison 2019/2020 lediglich zwei Urteile vor.
10 Unbegrenzter Zugang zu den Sportgerichtsurteilen ist lediglich Vereinsmitgliedern möglich, die über ein VereinsLogin verfügen.
9
zusammen mit zehn Subkategorien – bei der Ausübung verbaler und/oder physischer Gewalt und
deren Bewertung durch Sportgerichte des BFVs isoliert vorgestellt, um daran anschließend in
einem zweiten Schritt bedeutsame Einflussfaktoren miteinander zu kombinieren. Eine kritische
Einordnung dieser Ergebnisse wird in der abschließenden Diskussion vorgenommen.
Akteur*innenbezogene Faktoren
Die erste Kategorie umfasst die Subkategorie Beteiligte Akteur*innen. Diese beschreibt, von wem
das Vergehen ausging (Täter*in) und wer ihr Adressat war (Opfer). Zudem umfasst diese
Subkategorie soziodemographische Faktoren wie das Geschlecht und Alter der Beteiligten
Akteur*innen.
Tabelle 1: Täter*innen- und Opfergruppen
Täter*innengruppe
Relative Häufigkeit
Opfergruppe
Relative Häufigkeit
Trainer*innen/
Betreuer*innen
Spieler*innen
40%
Schiedsrichter*innen
68%
28%
Spieler*innen
32%
Fans
28%
Schiedsrichter*innen
4%
Gesamt
100%
100%
Tabelle 1 zeigt, dass die Täter*innengruppe vier beteiligte Akteur*innen umfasst, wohingegen zur
Opfergruppe lediglich zwei beteiligte Akteur*innen zählen. Des Weiteren springen drei prägnante
Auffälligkeiten hervor: Erstens sind Schiedsrichter*innen in über zwei Drittel der Vorfälle Opfer
von verbaler und/oder physischer Gewalt. Zweitens sind Spieler*innen in rund ein Viertel
Täter*innen (28%) und in fast ein Drittel Opfer (32%). Drittens sind Trainer*innen/Betreuer*innen
am häufigsten in der Täter*innenrolle zu finden (40%), gefolgt von den Spieler*innen (28%) und
den Fans (28%).
Analysiert man darüber hinaus die direkte Täter*innen-Opfer Konstellation, dann fällt auf, dass
Trainer*innen/Betreuer*innen (28%), Spieler*innen (24%) und Fans (16%) in mehr als zwei
Drittel der Vorfälle jeweils den Schiedsrichter*innen verbal und/oder physisch attackiert haben.
Eigentlich erwartbare Vorfälle zwischen Spieler*innen sind dagegen äußerst selten (4%).
Die Analyse zeigt zudem, dass – allerdings vor dem Hintergrund einer extrem ungleichen
Verteilung der insgesamt ca. 300 gesichteten Sportgerichtsurteile – sowohl die Täter*innen (96%)
als auch die Opfer (84%) ganz überwiegend männlich sind und sich die übergroße Mehrheit der
Vorfälle nicht bei Spielen von Junior*innen ereignete (8%), sondern bei Spielen der aktiven
10
Herren- und/oder Damenmannschaften (92%).
Spielbezogene Faktoren
Diese Kategorie unterteilt sich in die Subkategorien Spielphase und Spielklasse. Erstere bezieht
sich auf den Zeitpunkt – vor, während und/oder nach dem Spiel – der Ausübung verbaler und/oder
physischer Gewalt. 65% der zuordenbaren Vorfälle fanden in der zweiten Halbzeit, mehr als 35%
in der letzten Viertelstunde und rund ein Viertel (24%) nach dem Spiel, statt. Vor dem Spiel kam
es dagegen nie und in der ersten Halbzeit eher selten zu verbaler und/oder physischer
Gewaltanwendung (18%). Knapp die Hälfte der Vorfälle (48%) ereignete sich in der untersten
Spielklasse, der Kreisliga, ein Fünftel (20%) in der ebenso unterklassigen Bezirksliga. In den
höheren Ligen, wie Landesliga, Oberliga und der höchsten Spielklasse, der Regionalliga, wurde
lediglich ein Vorfall vor dem Sportgericht abgeurteilt.
Art der Vergehen
Die Kategorie Art der Vergehen gliedert sich in die zwei Subkategorien Art der Attacke und Art
der verbalen Abwertungen. Erstere beschreibt die Häufigkeitsverteilung der Vorfälle von verbaler
und/oder physischer Gewalt mit dem Ziel deren Verknüpfung aufzuzeigen. In der Tat zeigt die
Verteilung, dass in fast der Hälfte der Vorfälle (44%) verbale Gewalt Vorläufer für physische
Gewalt war. In 56% der analysierten Urteile wurde ausschließlich verbale Gewalt angewandt. Bei
keinem einzigen verhandelten Vorfall fand eine physische Attacke isoliert statt.
Die Analyse zeigt zudem, dass sexistisch motivierte Gewalt am häufigsten vorkam (40%). Die
meisten verbalen Abwertungen richten sich dabei gegen die Mütter der betroffenen Personen
(„Mutterficker“, „Hurensohn“). Fast ein Viertel der Beleidigungen (24%) haben eine
behindertenfeindliche Motivation, wobei allerdings keine Worthäufung auszumachen ist.
Lediglich die Beleidigung der Opfer als „blind“ kam mehrmals vor. Die aufgrund der thematischen
Nähe zu einer Subkategorie zusammengefassten rassistischen/fremdenfeindlichen Beleidigungen
traten selten auf (12%). Konkret wurden die Formulierungen „Ihr braucht euch nicht zu wundern,
wenn ihr mit solch einem Pack verliert“ in Verbindung mit „Ich muss nicht mit Ausländern
reden“,11 „Raus aus Deutschland“ und „Scheiß Neger“ verurteilt. Nicht in unserem Datensatz
enthalten sind eigentlich aufgrund medialer Berichterstattung anzunehmende homophobe,
11 Im konkreten Fall wurde ein Schiedsrichter letztlich wegen provokativen Beleidigungen und einem Verhalten, das
auf rassistische/fremdenfeindliche Einstellungen schließen lässt, gegenüber mehreren Spielern zu einer Geldstrafe in
Höhe von 1000€ verurteilt.
11
antisemitische oder islamfeindliche Abwertungen.
Verhängte Strafen
Diese Kategorie bildet die Schwere der ausgesprochenen Strafe und deren Häufigkeit ab. Unterteilt
ist diese in vier Subkategorien: (1) leichte Strafe, (2) mittelschwere Strafe, (3) schwere Strafe und
(4) schwerste Strafe. In 28% der Vorfälle wurde eine leichte Strafe verhängt, d.h. es wurde eine
Geldstrafe von bis zu 150€ oder ein Platzverweis mit maximal zwei Spielen Sperre ausgesprochen.
In 24% der Vorfälle wurden die Täter*innen zu einer mittelschweren Strafe verurteilt, d.h. die
verhängte Geldstrafe lag zwischen 150€ bis 500€ und Spieler*innen wurden für zwei Spieltage
gesperrt. Eine schwere Strafe wurde in 36% der Vorfälle ausgesprochen. Diese lag vor, wenn eine
Spielsperre von mehr als zwei Spielen oder eine Geldstrafe von bis zu 1.000€ verhängt wurde. In
12% der vorliegenden Vorfälle wurde die schwerste Strafe, d.h. der Ausschluss aus dem Verband
ausgesprochen.
Deskriptive bivariate Analyse
Da die Präsentation einzelner Variablen und deren Häufigkeitsverteilung hinsichtlich unserer
Forschungsfragen lediglich von begrenzter Aussagekraft ist, werden im Folgenden einzelne
prägnante
Variablen
miteinander
kombiniert,
um
deren
Zusammenhänge
deskriptiv
herauszuarbeiten. Konkret wird zuerst gezeigt, wie sich das verhängte Strafmaß nach Art der
Attacke verteilt.
Tabelle 2: Das Strafmaß nach Art der Attacke
Leichte &
mittelschwere Strafe
Verbale Gewalt
8
Schwere &
schwerste Strafe
5
Gesamt
13
Verbale & physische
Gewalt
Absolute Häufigkeit
5
7
12
13
12
25
Relative Häufigkeit
52%
48%
100%
Tabelle 2 zeigt, dass verbale Gewalt im Verhältnis milder bestraft wurde als verbale Gewalt, die
in physische Gewaltanwendung mündete. Während ausschließlich verbale Gewalt in fast zwei
Drittel der Vorfälle mit leichten/mittelschweren Strafen sanktioniert wurde (62%), verhängten
Sportgerichte des BFVs bei Vorfällen verbaler Gewalt, die in physischer Gewalt überging, in fast
12
60% der Vorfälle schwere/schwerste Strafen. Schließlich wurde die schwerste Strafe, der
Ausschluss aus dem Verband, nur bei Anwendung verbaler Gewalt in Verbindung mit physischer
Gewalt ausgesprochen.
Allerdings variiert das verhängte Strafmaß nach beteiligten Akteur*innen teils beträchtlich.
Während Trainer*innen/Betreuer*innen, die verbale und/oder physische Gewalt gegen den
Schiedsrichter*innen anwandten, ausschließlich zu leichten bis mittelschweren Strafen verurteilt
wurden, verhängte das Sportgericht ausschließlich schwere bis schwerste Strafen gegen
Spieler*innen, die verbale und/oder physische Gewalt gegen Schiedsrichter*innen ausübten.
Trainer*innen/Betreuer*innen, die Spieler*innen verbal und/oder physisch attackierten, wurden
stets mit schwersten Strafen belegt, während Zuschauer, die Spieler*innen attackierten mit
mittelschweren und schweren Strafen sanktioniert wurden. Bei allen Verbandsausschlüssen lag ein
tätlicher Übergriff von Spieler*innen auf Schiedsrichter*innen vor.
Tabelle 3: Das Strafmaß nach Art der verbalen Abwertung
Leichte &
Schwere &
mittelschwere Strafe
schwerste Strafe
Art der verbalen
Abwertung
Sexistisch
6
4
Gesamt
10
Behindertenfeindlich
5
1
6
Rassistisch/
fremdenfeindlich
Ohne Zuordnung
1
2
3
1
5
6
Absolute Häufigkeit
13
12
25
Relative Häufigkeit
52%
48%
100%
Die Tabelle 3 stellt den Zusammenhang zwischen dem Strafmaß und der Art der verbalen
Abwertung dar. Fasst man wiederum die Kategorien leichte und mittelschwere Strafe sowie jene
der schweren und schwersten Strafe zusammen, fällt auf, dass das Strafmaß über alle Arten der
verbalen und/oder physischen Gewalt hinweg nahezu gleich verteilt ist. Das gilt insbesondere für
sexistisch
motivierte
Gewalt.
Allerdings
ist
ebenso
festzustellen,
dass
während
behindertenfeindlich motivierte Gewalt nahezu ausschließlich mit leichten und mittelschweren
Strafen sanktioniert wurde, Sportgerichte des BFVs – allerdings bei einer geringen Fallzahl –
mehrheitlich schwere und schwerste Strafen bei rassistisch/fremdenfeindlich motivierter Gewalt
verhängten.
Auffällig ist zudem, dass – wenngleich stark kontextspezifisch – das Strafmaß bei vergleichbaren
13
verbalen Abwertungen durchaus variiert. So wurde die behindertenfeindlich motivierte Abwertung
„blind“ sowohl mit einer leichten Strafe als auch mit einer mittelschweren Strafe sanktioniert.
Ähnlich verhält es sich bei der sexistisch motivierten Abwertung „Hurensohn“, die sowohl mit
einer leichten, mittelschweren als auch schweren Strafe sanktioniert wurde. Erstaunlich ist zudem,
dass – zumindest nach Meinung der Autoren – heftige verbale Abwertungen wie „du Mongo“
weniger hart bestraft wurden als vergleichbar harmlose Abwertung wie „du bist doch behindert“.
Diskussionswürdig ist schließlich, dass die sexistisch und rassistisch/fremdenfeindlich motivierte
Abwertung „Halt die Fresse du kubanische Hure“ lediglich mit einer mittelschweren Strafe
verhältnismäßig milde sanktioniert wurde.
Diskussion und praktische Handlungsempfehlung
Ziel dieses Artikels war es Einflussfaktoren bei der Ausübung konkreter verbaler und/oder
physischer Gewalt und deren Bewertung durch Sportrichter*innen des BFVs anhand von 25
ausgewählten saisonübergreifenden Urteilen aus den Jahren 2016 bis 2020 explorativ zu
untersuchen. Da bisherige Studien Ihren Fokus auf rassistisch/fremdenfeindlich motivierte
Konflikte im Amateurfußball und deren Bewertung durch Sportrichter*innen legten, konzentriert
sich die vorliegende Untersuchung auf das Herausarbeiten von – weiter zu fassenden –
menschfeindlichen Einstellungen und leistet somit einen ersten Beitrag diese Forschungslücke zu
schließen.
Die softwaregestützte qualitative Inhaltsanalyse zeigt die herausragende Bedeutung der Beteiligten
Akteur*innen bei der Ausübung verbaler und/oder physischer Gewalt. Opfer wurden zumeist
Schiedsrichter*innen,
Täter*innen
waren
mehrheitlich
Trainer*innen/Betreuer*innen,
Spieler*innen und Fans. Beide Befunde bestätigen vorherige Studien, die die prekäre Situation der
Schiedsrichter*innen
belegen
(siehe
vor
allem
Vester
2013:
35ff.)12
und
den
konfliktverschärfenden Einfluss der Trainer*innen/Betreuer*innen herausarbeiten (Koch 2019:
161ff.). Bestätigt werden kann ebenso, dass verbale und/physische Gewalt zuvörderst ein
Phänomen ist, das bei Männermannschaften auftritt und nicht bei Spielen von Junior*innen
(Scherer und Winands 2010: 47ff.).
Bedeutsam bei der Ausübung verbaler und/oder physischer Gewalt ist zudem die Spielphase. Dass
fast zwei Drittel (65%) der Vorfälle in der zweiten Halbzeit und mehr als 35% der Vorfälle in der
12 Vester (2013: 35ff.) untersucht detailliert in einer Vollerhebung die Opferwerdung von
Amateurschiedsrichter*innen im Württembergischen Fußball-Verband (wfv). Sie stellt fest, dass Schiedsrichter*innen
geradezu „Zielscheibe“ verbaler und/oder physischer Gewalt sind.
14
letzten Viertelstunde stattfanden, deutet darauf hin, dass die Zunahme verbaler und/oder physischer
Gewalt mit zunehmender körperlicher Erschöpfung und/oder sich aufstauender negativer
Emotionen, wie zunehmende Resignation und Frustration über die sich abzeichnende Niederlage,
einhergeht. Bestätigt werden kann zudem, dass sich verbale und/oder physische Gewalt tatsächlich
in die unteren Ligen verlagert, sicherlich auch deshalb, weil diese weitaus weniger im Fokus der
Öffentlichkeit stehen und medial begleitet werden als die höheren (Profi-)Ligen (Ribler 2010: 13;
Winands 2016: 7).
Das häufige Auftreten sexistisch motivierter Abwertungen (40%) bestätigt wiederum, dass im
Amateurfußball, in dem nach wie vor mehrheitlich männliche Akteure agieren, sexistische
Einstellungen eine höhere Akzeptanz erfahren als in anderen Bereichen der Gesellschaft
(Degele/Janz 2011: 10ff.). Bestätigt werden kann zudem, dass verbale Gewalt weitgehend
physische Gewalt ersetzt und dieser stets vorausgeht. Daraus kann geschlussfolgert werden, dass
verbale Gewalt keineswegs nur harmlose Provokation ist, sondern als Vorbote das Potential einer
Eskalation hin zur Ausübung physischer Gewalt in sich trägt (Lobenstein-Reichmann 2012:
215ff.). Ratsam ist daher ein frühzeitiges Eingreifen auch bei vermeintlich harmloser verbaler
Gewalt, um ein sich gegenseitiges Hochschaukeln zu vermeiden.
Die vorgenommene Einteilung nach der Schwere der Strafe zeigt auf, dass Sportrichter*innen –
wie Amateurschiedsrichter*innen – einzelne Vergehen stark kontextspezifisch bewerten (Koch
2021: 205ff.). Erwartungsgemäß wurde verbale Gewalt milder bestraft als verbale Gewalt, die in
physische Gewaltanwendung mündete. Die schwerste Strafe, der Ausschluss aus dem Verband,
wurde nur bei Anwendung verbaler Gewalt in Verbindung mit physischer Gewalt ausgesprochen.
Allerdings variiert das verhängte Strafmaß nach beteiligten Akteur*innen beträchtlich. Während
verbale und/oder physische Gewalt von Trainer*innen/Betreuer*innen gegenüber den
Schiedsrichter*innen ausschließlich mit leichten bis mittelschweren Strafen sanktioniert wurde,
belegte das Sportgericht Spieler*innen, die verbale und/oder physische Gewalt gegen den
Schiedsrichter*innen ausübten, mit schweren bis schwersten Strafen. Bei der schwersten Strafe,
dem
Verbandsausschluss,
lag
stets
ein
tätlicher
Angriff
von
Spieler*innen
auf
Schiedsrichter*innen vor. Beide Befunde lassen darauf schließen, dass der BFV darauf bedacht ist,
die Sicherheit seiner Spielleiter*innen zu gewährleisten, auch durch die Verhängung schwerster
Strafen, insbesondere bei physischen Übergriffen von Spieler*innen gegen Schiedsrichter*innen.
Das ist konsequent, da der 6. Lagebericht des DFBs zeigt, dass Amateurschiedsrichter*innen, wenn
auch äußerst selten, durchaus das Ziel von körperlichen Angriffen mit teilweise schweren
gesundheitlichen Folgen sind. Andererseits belegen die vergleichsweise milden Strafen für
Trainer*innen/Betreuer*innen und Fans, dass das konfliktverschärfende Eingreifen ins
15
Spielgeschehen
durch
Dritte
als
weniger
gravierend
bewertet
wird,
obwohl
Amateurschiedsrichter*innen selbst über die bedeutende Rolle der Trainer*innen als Schlichter
einerseits oder andererseits als Konfliktauslöser und/oder Verstärker berichten (Koch 2019: 181).
Schließlich zeigt die Analyse, dass das Strafmaß und die Art der verbalen Gewalt über alle Arten
verbaler und/oder physischer Gewalt hinweg nahezu gleich verteilt sind. Das trifft insbesondere
auf sexistisch motivierte Gewalt zu. Demgegenüber steht der Befund, dass behindertenfeindlich
motivierte Gewalt fast ausschließlich mit leichten und mittelschweren Strafen sanktioniert wurde,
wohingegen Sportgerichte des BFVs – allerdings vor dem Hintergrund einer geringen Fallzahl –
mehrheitlich schwere und schwerste Strafen bei rassistisch/fremdenfeindlich motivierter Gewalt
verhängte. Daraus kann geschlussfolgert werden, dass nicht nur Amateurschiedsrichter*innen
(Koch 2019: 172ff.), sondern eben auch Sportrichter*innen durchaus eine unterschiedliche
Bewertung von verbalen Abwertungen abhängig von der ihnen zugrunde liegenden Motivation
vornehmen. Darüber hinaus kann festgehalten werden, dass – wenn auch stark kontextspezifisch –
das Strafmaß bei vergleichbaren verbalen Abwertungen variiert. So wurde die behindertenfeindlich
motivierte Abwertung „blind“ und die sexistisch motivierte Abwertung „Hurensohn“
unterschiedlich bestraft. Schließlich konnte aufgezeigt werden, dass einige heftige verbale
Abwertungen teils milder bestraft wurden als vergleichbar harmlose verbale Abwertungen. Ratsam
ist daher – in Anlehnung an Hilpert (2009: 128) – die Erarbeitung einer Orientierung gebenden
Kasuistik, nun auch von verbalen Abwertungen, also von verbaler Gewalt, die menschenfeindliche
Einstellungen begründen.
Darüber hinaus sollte der BFV zukünftig darauf bedacht sein, dass die verschiedenen Präventionsund Konfliktmanagementstrategien, wie das Einsetzen von Konfliktmanager*innen, aber auch das
Anbieten von Anti-Gewalt-Kursen und das Durchführen von Anti-Rassismus-Kampagnen (BVF
2022), um Kurse erweitert werden, die gezielt für Amateurschiedsrichter*innen und/oder
Sportrichter*innen konzipiert werden, um eine unterschiedliche Bewertung von verbaler und/oder
physischer Gewalt, die auf menschenfeindliche Einstellungen schließen lassen, insbesondere
gegenüber Menschen mit Behinderung, zu vermeiden.
Die vorliegende Untersuchung ist lediglich ein erster thematisch begrenzter Beitrag
Einflussfaktoren sowohl bei der Ausübung konkreter verbaler und/oder physischer Gewalt aus
menschenfeindlichen Motiven als auch deren Bewertung durch neutrale Dritte herauszuarbeiten.
Weiterer Forschungsbedarf besteht insbesondere aufgrund des geringen verwertbaren
Datenmaterials. Ebenso kann in Analogie zu vergleichbaren Studien ein hohes Dunkelfeld
angenommen werden, da oftmals verbale Abwertungen als Banalität, dem (Fußball-)Sport
16
innewohnend und/oder dem alltäglichen Stress geschuldet abgetan und somit erst gar nicht
dokumentiert
werden
kontextualisierende
(Koch
im
Informationen,
Erscheinen:
die
zur
16).
Zudem
fehlen
Herausarbeitung
oftmals
von
wichtige
vermeintlich
menschenfeindlichen Einstellungen allerdings wesentlich sind. Wünschenswert ist es daher, dass
grundlegende Annahmen und Ergebnisse der vorliegenden Untersuchung aufgenommen, mit
Erkenntnissen weiterer Studien angereichert, auf vergleichbare Forschungsfelder übertragen und
um quantitative Folgestudien erweitert werden.
17
Literatur
BfV. 2022. “Das ist der Bayerische Fußball-Verband e.V.” (https://www.bfv.de/der-bfv/wer-wirsind/wer-wir-sind-und-standpunkte/bfv---wer-wir-sind-und-wofur-wir-stehen).
BMI. 2019. Innenminister von Bund und Ländern geschlossen beim Kampf gegen
Rechtsextremismus und Antisemitismus Bundesinnenminister Horst Seehofer zieht positive
Bilanz zum Abschluss der Innenministerkonferenz in Lübeck. Berlin.
Curtius, Friedrich, Fritz Keller, Rainer Koch, and Ronny Zimmermann. 2019. Angriffe auf Schiris:
„Das muss aufhören!“. Frankfurt am Main.
Degele, Nina and Caroline Janz. 2011. “Hetero, weiß und männlich? Fußball ist viel mehr!: Eine
Studie der Friedrich-Ebert-Stiftung zu Homophobie, Rassismus und Sexismus im Fußball”.
Berlin: Friedrich-Ebert-Stiftung. Retrieved April 14, 2018.
Delhom, Pascal. 2007. “Die geraubte Stimme” Pp. 229–48 in Verletzende Worte: Die Grammatik
sprachlicher Missachtung, edited by Steffen K. Herrmann, Sybille Krämer, and Hannes Kuch.
Bielefeld: Transcript.
DFB. “Schiedsrichter-Einsatzstatistik.: Saison 2018/2019”. Frankfurt am Main: DFB
(https://www.dfb.de/fileadmin/_dfbdam/216014-SR18-19_neu.pdf).
DFB. 2020. Gravierende Einzelfälle bei insgesamt weniger Störungsfällen. Frankfurt a.M.
DFB. 2021. „Fair ist mehr“ – Das Gewaltpräventionskonzept des Deutschen Fußball-Bundes.
Frankfurt am Main.
Dunning, Eric. 1999. Sport matters. Sociological studies of sport, violence and civilization.
London, New York: Routledge.
Herrmann, Steffen K., and Hannes Kuch. 2007. “Verletzende Worte. Eine Einleitung” Pp. 7–30 in
Verletzende Worte: Die Grammatik sprachlicher Missachtung, edited by Steffen K. Herrmann,
Sybille Krämer, and Hannes Kuch. Bielefeld: Transcript.
Klinker, Fabian, Joachim Scharloth, and Joanna Szczek. 2018. “Editorial” Pp. 1–5 in Sprachliche
Gewalt: Formen und Effekte von Pejorisierung, verbaler Aggression und Hassrede, edited by
Fabian Klinker, Joachim Scharloth, and Joanna Szczek. Stuttgart: J.B. Metzler.
Koch, Florian. 2019. Die Abwertung des Anderen mittels Sprache im Amateurfußball. Ein
soziolinguistischer Vergleich in Deutschland und Frankreich. Sprache - Kultur - Gesellschaft.
Berlin: Peter Lang.
Koch, Florian. 2020. “Die Abwertung des Anderen mittels Sprache: Einflussfaktoren bei der
Bewertung alltäglicher verbaler Gewalt im Amateurfußball”. Romanistik in Geschichte und
Gegenwart 26 (2):195–212.
Koch, Florian. 2021. “Le dénigrement de l’autre par la langue dans le foot amateur en Allemagne
et en France”. Allemagne d‘aujourd‘hui N° 237 (3):200–16. doi: 10.3917/all.237.0200.
Krämer, Sybille. 2007. “Sprache als Gewalt oder: Warum verletzen Worte?” Pp. 31–48 in
Verletzende Worte: Die Grammatik sprachlicher Missachtung, edited by Steffen K. Herrmann,
Sybille Krämer, and Hannes Kuch. Bielefeld: Transcript.
Kuckartz, Udo. 2014. Mixed Methods. Methodologie, Forschungsdesigns und Analyseverfahren.
Wiesbaden: VS Verlag für Sozialwissenschaften.
Laerum, Eva. 2010. “Sportrichter als Akteur in interkulturellen Konflikten - eine qualitative
Befragung” Pp. 55–60 in Konfliktmanagement im Fußball, edited by Angelika Ribler and
Astrid Pulter. Frankfurt am Main: HFV.
18
Lobenstein-Reichmann, Anja. 2012. “Verbale Gewalt: Ein Forschungsgegenstand der
Sprachgeschichtsschreibung”. Jahrbuch für Germanistische Sprachgeschichte 3 (1):215–38.
doi: 10.1515/jbgsg-2012-0014.
Mayring, Philipp. 2016. Einführung in die qualitative Sozialforschung. Eine Anleitung zu
qualitativem Denken, 6th edn. Weinheim, Basel: Beltz.
Mayring, Philipp, and Thomas Fenzl. 2014. “Qualitative Inhaltsanalyse” Pp. 543–56 in Handbuch
Methoden der empirischen Sozialforschung, vol. 3, edited by Nina Baur and Jörg Blasius.
Wiesbaden: VS Verlag für Sozialwissenschaften.
Mennell, Stephen. 2006. “The Contribution of Eric Dunning to the Sociology of Sport: The
Foundations”. Sport in Society 9 (4):514–32. doi: 10.1080/17430430600768728.
Pilz, Gunter A. 2002. Rote Karten statt Integration? Eine Untersuchung über Fußball und
ethnische Konflikte. Sport als Mittel der Integration, Kultur- und Stadthistorischen Museum
Duisburg, 25 June 2002. Retrieved May 5, 2017.
Poppe, Thorsten. 2019. “Gewalt im AmateurfußballSinnlose Präventionskonzepte?”.
Deutschlandfunk. 30 November 2019 (https://www.deutschlandfunk.de/gewalt-imamateurfussball-sinnlose-praeventionskonzepte-100.html).
Ribler, Angelika. 2010. “„Zustände wie in Wildwest“: Auf den Fußballplätzen der Amateurligen
nehmen Pöbeleien und Prügeleien rapide zu, immer mehr Spiele enden vor Gericht.” Pp. 13–
18 in Konfliktmanagement im Fußball, edited by Angelika Ribler and Astrid Pulter. Frankfurt
am Main: HFV.
Rullang, Christian, Eike Emrich, and Christian Pierdzioch. 2015a. “Mit Zuckerbrot und Pfeife –
Die Bedeutung unterschiedlicher Autoritätsformen im Rollenselbstbild von Schiedsrichtern /
With a Carrot and a Whistle – The Importance of Different Forms of Authority in the Role
Self-Perception of Referees”. Sport und Gesellschaft 12 (3):215–39. doi: 10.1515/sug-20150304.
Rullang, Christian, Eike Emrich, and Christian Pierdzioch. 2015b. “Wie häufig werden
Schiedsrichter Opfer von Beleidigungen, Drohungen und Gewalt? : Ergebnisse einer
bundesweiten Umfrage unter aktiven Schiedsrichtern”. Leipziger sportwissenschaftliche
Beiträge (Lehmanns Media) 56:S. 44-66.
Scherer, Judith, and Martin Winands. 2010. “Konfliktbelastungen im Amateurfußball” Pp. 47–54
in Konfliktmanagement im Fußball, edited by Angelika Ribler and Astrid Pulter. Frankfurt am
Main: HFV.
Schulz, Benjamin, Christian Teevs, and Sebastian Heier. 2016. “Gewalt gegen Unparteiische:
Schiri, du Arschloch”. Spiegel Online. 28 August 2016. Retrieved April 27, 2017
(http://www.spiegel.de/sport/fussball/gewalt-gegen-schiedsrichter-schiri-du-arschloch-a1109141.html).
Vester, Thaya. 2013. Zielscheibe Schiedsrichter. Zum Sicherheitsgefühl und zur Opferwerdung
von Unparteiischen im Amateurfußball, 1st edn. Schriften zum Sportrecht, vol. 31. BadenBaden: Nomos.
Winands, Martin. 2016. “Fußball als Diskriminierungsagent: Die Situation im Bundesligafußball”.
Dossier Bundesliga: Spielfeld der Gesellschaft. 22 April 2016. Retrieved August 27, 2016
(http://www.bpb.de/gesellschaft/sport/bundesliga/156634/fussball-alsdiskriminierungsagent?p=all).
Zick, Andreas. 2010. “Achtung Menschenfeindlichkeit - Vorurteile innerhalb und außerhalb der
Sportplätze” Pp. 19–24 in Konfliktmanagement im Fußball, edited by Angelika Ribler and
19
Astrid Pulter. Frankfurt am Main: HFV.
Zick, Andreas, Andreas Hövermann, and Beate Küpper. 2011. Intolerance, prejudice and
discrimination. A European report. Projekt gegen Rechtsextremismus / FES. Berlin: FriedrichEbert-Stiftung, Forum Berlin.
Zick, Andreas, Judith Scherer, and Martin Winands. 2010. “Der Fußballplatz und das Sportgericht
als ethnische Kampfarena” Pp. 133–39 in Der Ball ist bunt: Fussball, Migration und die Vielfalt
der Identitäten in Deutschland, edited by Diethelm Blecking and Gerd Dembowski. Frankfurt
am Main: Brandes & Apsel.
20
|
|
https://openalex.org/W3174951177
|
https://www.revistas.usp.br/rlae/article/download/188063/173644
|
Spanish; Castilian
| null |
Validation of a questionnaire on the use of Interactive Response System in Higher Education
|
Revista latino-americana de enfermagem
| 2,021
|
cc-by
| 8,157
|
Validación del cuestionario sobre uso de Mandos Interactivos de
Respuesta en la Educación Superior Ángel Custodio Mingorance-Estrada1
https://orcid.org/0000-0003-4478-3011
Juan Granda-Vera2
https://orcid.org/0000-0001-6888-7785
Gloria Rojas-Ruiz1
https://orcid.org/0000-0001-8541-383X
Inmaculada Alemany-Arrebola3
https://orcid.org/0000-0002-4127-3502 Objetivo: el presente estudio tiene como objetivo diseñar y
validar un cuestionario que permita medir la percepción de
los estudiantes universitarios del uso de los MIR como recurso
tecnopedagógico en el aula. Método: se aplicó el cuestionario
Mandos Interactivos de Respuesta para la Mejora del Proceso de
Enseñanza-Aprendizaje, creado ad hoc para esta investigación,
que está formado por 24 ítems, aplicado a 142 estudiantes
universitarios. Resultados: tanto el análisis factorial
exploratorio como el confirmatorio arrojan tres dimensiones:
ambiente de clase, los procesos de enseñanza-aprendizaje y la
evaluación. Los resultados obtenidos tanto en la fiabilidad (alfa
de Cronbach 0.955) como en el análisis factorial confirmatorio
(χ2/gl=1.944, CFI=0.97; GFI=0.78; RMR=0.077; RMSEA=0.08)
arrojan índices altamente satisfactorios. Conclusión: los
análisis estadísticos muestran que este instrumento es una
herramienta válida, fiable y de fácil aplicación para el docente
que le permitirá evaluar la experiencia del alumnado sobre el
aprendizaje centrado en el estudiante. Descriptores: Educación; Educación Superior; Aprendizaje;
Estudios de Validación; Encuestas y Cuestionarios; Tecnología
Educacional. Cómo citar este artículo
Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. Validation of a questionnaire
on the use of Interactive Response System in Higher Education. Rev. Latino-Am. Enfermagem. 2021;29:e3418. [Access
día
mes
año
]; Available in:
URL
. DOI: http://dx.doi.org/10.1590/1518-8345.3374.3418. 1 University of Granada, Department of Didactics and School
Organization, Melilla, ES, España.
2 University of Granada, Department of Didactics of Corporal
Expression, Melilla, ES, España.
3 University of Granada, Department of Developmental and
Educational Psychology, Melilla, ES, España. Ángel Custodio Mingorance-Estrada1
https://orcid.org/0000-0003-4478-3011
Juan Granda-Vera2
https://orcid.org/0000-0001-6888-7785
Gloria Rojas-Ruiz1
https://orcid.org/0000-0001-8541-383X
Inmaculada Alemany-Arrebola3
https://orcid.org/0000-0002-4127-3502 Rev. Latino-Am. Enfermagem
2021;29:e3418
DOI: 10.1590/1518-8345.3374.3418
www.eerp.usp.br/rlae Artículo Original Introducción Los estudios consultados muestran cómo la
utilización de los MIR en las aulas universitarias permiten
mejorar tres grandes ámbitos: el ambiente de clase, los
procesos de enseñanza-aprendizaje y la evaluación de los
alumnos(15-20), siendo cada vez más importante definir las
posibilidades y limitaciones de esta herramienta para la
mejora de la calidad de la Educación Superior(6). La convergencia de la Educación Superior debe
establecer un espacio de intercambio de experiencias
prácticas que permitan avanzar en el conocimiento e
investigación desarrollados en el marco común del Espacio
Europeo e Iberoamericano de Educación Superior(1-2). La puesta en marcha del aprendizaje centrado
en el estudiante conlleva el interés por establecer un
modelo que integre eficazmente la tecnología con los
conocimientos de mediación didáctica, evolucionando
hacia el Modelo Tecnopedagógico (TPACK), que combina
los conocimientos disciplinar, pedagógico y tecnológico,
pero siempre teniendo presente el contexto en el que se
interviene(3-4), con mayor interacción entre profesores y
estudiantes desde un análisis dialógico-crítico(2). Centrándonos en el factor ambiente del aula, los
MIR aumentan la asistencia(21) y la participación de
los estudiantes(22-25), ya que se produce una mayor
participación en clase que con la metodología más
tradicional(23,26-27). Dentro del factor aprendizaje, algunos
estudios establecen que la interacción frecuente y
positiva dinamizan las clases cuando se usan los MIR(13,28-
29) promoviendo el aprendizaje activo(30-32). Además,
se fomenta la atención(33-34), la concentración(35) y la
memoria(36-37) en el estudio. Existe una amplia investigación
que sugiere que la mejora en el rendimiento es fruto del
uso de los MIR tal como indican algunos estudios(31-32,38-41),
otros trabajos no encuentran dicho efecto(42). En relación
con el factor evaluación, los hallazgos de la literatura
apoyan la capacidad de los MIR como herramienta para la
evaluación y la retroalimentación(43-44). Se considera que
tanto estudiantes como docentes consiguen beneficios
a través de la retroalimentación que reciben con el uso
de esta tecnopedagogía(20,27). Todo ello, conlleva a un
proceso de aprendizaje clave para la interacción del saber
y saber hacer(45). Algunos autores muestran la necesidad de establecer
el conocimiento didáctico mediante la relación entre
los distintos tipos de conocimiento (de la disciplina,
pedagógico general y del alumnado) y la biografía del
profesor(5). Cómo citar este artículo Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. Validation of a questionnaire
on the use of Interactive Response System in Higher Education. Rev. Latino-Am. Enfermagem. 2021;29:e3418. [Access
día
mes
año
]; Available in:
URL
. DOI: http://dx.doi.org/10.1590/1518-8345.3374.3418. 2 Rev. Latino-Am. Enfermagem 2021;29:e3418. Introducción Así, se pone en evidencia que en la formación
inicial de los enfermeros tanto los aspectos pedagógicos,
usando la monitorización mediante los Mandos Interactivos
de Respuesta (en adelante MIR), como la importancia
del aprendizaje con docentes expertos, puede conllevar
el desarrollo de una enseñanza de calidad, entre otras
cuestiones(6). En este sentido, es importante estimular a los
docentes a integrar la tecnología en las aulas, tal y como
se pone de manifiesto en el Informe Horizon(7), logrando
un impacto importante sobre la educación en los próximos
años. Para ello, el profesorado universitario debe utilizar
las herramientas tecnológicas que más conocen y utilizan,
así como acceder a los nuevos recursos tecnológicos para
la mejora de los procesos docentes(8). El presente estudio tiene como objetivo diseñar y
validar un cuestionario que permita medir la percepción
de los estudiantes universitarios del uso de los MIR como
recurso tecnopedagógico en el aula. www.eerp.usp.br/rlae www.eerp.usp.br/rlae Resultados En primer lugar, se analizó la confiabilidad del
cuestionario MIR-MPEA con 35 elementos a través del
coeficiente de consistencia interna α de Cronbach, siendo
0.965. Aunque este índice era elevado se procedió a
eliminar aquellos ítems cuya correlación ítem-total fue
menor a 0.20. De tal forma, el cuestionario final quedó
formado por 24 ítems con un α=0.955, oscilando los
índices de homogeneidad entre 0.42 a 0.85. La experiencia tuvo lugar en diferentes Grados
de la Universidad de Granada (España), en materias
concernientes a la formación básica del alumnado. Para
ello, se pide la colaboración del profesorado de forma
voluntaria para participar en dicho estudio y que utilice
los MIR en sus clases. Posteriormente se midió las medias, desviaciones
típicas, asimetría y las correlaciones ítem-total de cada
uno de los ítems que componen el cuestionario. Tal como
se observa en la Tabla 1, la asimetría es negativa en
todos los ítems, lo que muestra mayor concentración de
respuestas que denotan puntuaciones altas en esos ítems. El uso de los MIR en estas materias básicas se llevó
a cabo a lo largo de todo el semestre del curso académico
2016-17, antes, durante y al finalizar las clases. A la
finalización del semestre, se aplicó el cuestionario MIR-
MPEA para conocer la percepción de la experiencia llevada Tabla 1 - Valores descriptivos de los ítems del cuestionario MIR-MPEA. Granada, España, 2017
Nº
Ítems
M*
DT†
Asimetría
Correl. ítem-total
1
Con el uso de los MIR estoy más concentrado en las clases. 3.40
1.15
-0.477
0.689
2
Con el uso de los MIR mido si estoy siguiendo correctamente los
contenidos dados de la asignatura durante las clases. 3.81
1.01
-0.658
0.619
5
Durante mi experiencia con los MIR lo paso bien aprendiendo. 3.52
1.18
-0.559
0.566
9
Los MIR se utilizan para conocer los conocimientos iniciales de los
alumnos. 3.54
1.258
-0.595
0.488
10
El uso de los MIR se lleva a cabo por docentes experimentados
para proporcionar una buena retroalimentación (feedback). 3.97
0.891
-0.616
0.610
14
La utilización de los MIR me ayuda a desarrollar mi razonamiento
sobre los contenidos trabajados. 3.59
1.162
-0.666
0.639
15
El uso de los MIR hace que las clases sean amenas y dinámicas. 3.82
1.119
-0.877
0.656
18
El uso de los MIR permite aumentar mi rendimiento en el
aprendizaje. 3.76
1.129
-0.653
0.762
19
El uso continuado de los MIR mejora mi asistencia a las clases. Método Del mismo modo se manifiesta que los cambios
tecnológicos en los docentes universitarios se orientan a la
variación dentro de la estabilidad, introduciendo las que son
coherentes con sus prácticas docentes en las actividades
de aprendizaje que normalmente desarrollan(9). El tipo de diseño fue transeccional descriptivo, ya
que se recolectaron los datos en un tiempo único con el
fin de poder describir el fenómeno y analizar su incidencia
en un momento determinado. La investigación se desarrolla en la Universidad de
Granada (España) en su Campus de Melilla, situado en el
Norte de África, cuyo alumnado pertenece a las Facultades
de Ciencias de la Salud, Ciencias Sociales y Jurídicas, y
Ciencias de la Educación y del Deporte. Para ello, se ha
utilizado un muestreo no probabilístico de tipo intencional. Los criterios de selección han sido: en primer lugar,
docentes que utilicen tecnometodología en sus aulas,
los Mandos Interactivos de Respuesta en sus clases y en
segundo lugar, la voluntariedad del alumnado en participar
en este estudio. Por todo ello, la muestra está formada
por 142 estudiantes, 110 son mujeres (77.5%) y 32 son
hombres (22.5%). En relación con el curso académico, Para afrontar estos retos tecnológicos es importante
invertir el aprendizaje, proporcionando materiales a los
alumnos, en diversos formatos, para que desarrollen un
trabajo previo antes de llegar al aula, incorporando el
uso de los MIR para constatar las mejoras del proceso de
aprendizaje centrado en el estudiante(10-12). A su vez, se
expone que los MIR son parte de la experiencia que se
está llevando a cabo en las aulas universitarias hoy día(13). Así, la literatura actual en la Educación Superior sobre el
uso de esta tecnología en los últimos años se centra en los
ámbitos de Ciencia, Tecnología, Ingeniería y Matemáticas,
Sociología, Humanidades, Salud (Medicina y Enfermería),
Administración de Empresas y Negocios e Inglés(13-14). 3 Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. 17 alumnos son de primero (12%), 95 de segundo curso
(66.9%) y 30 de tercero (21.1%). a cabo. Dicho cuestionario tuvo lugar la última semana
del semestre, con una duración de unos 15 minutos,
aproximadamente. Se pide conformidad a los alumnos
para participar voluntariamente y de forma anónima en
esta experiencia, siguiendo las normas del Committee on
Publication Ethics (COPE). Método Para la realización de esta investigación se ha
elaborado un cuestionario ad hoc para esta investigación,
“Mandos Interactivos de Respuestas para la Mejora del
Proceso de Enseñanza-Aprendizaje (MIR-MPEA)”. En relación con los ítems del MIR-MPEA, fueron
redactados después de una amplia revisión bibliográfica
sobre los 3 factores destacados anteriormente(21,46-47),
que permitió establecer la validez de contenido, ya que
no se disponía de expertos en esta materia. Se partió de
un banco inicial de 65 ítems agrupados por categorías(48),
después del análisis de los ítems, se obtuvieron 35 ítems
que fueron agrupados en las tres dimensiones: ambiente,
proceso y evaluación. En cuanto al formato de respuesta
del cuestionario se utilizó una escala tipo Likert, con 5
alternativas de respuesta, que abarca desde 1 Totalmente
en Descuerdo a 5 Totalmente de Acuerdo. Para el tratamiento estadístico de los datos se ha
utilizado el programa informático SPSS en su versión
20.0. Para conocer la fiabilidad de cada bloque se utilizó
el alfa de Cronbach y para la validez del cuestionario se
realizó análisis factorial exploratorio. Así como el programa
LISREL 8.8 para el análisis factorial confirmatorio. (continúa en la página siguiente...) Resultados Además, las comunalidades de los ítems
están por encima de h2=0.40, oscilando entre 0.421
“El uso continuado de los MIR mejoram mi asistencia
a las clases” a 0.791 “El uso de los MIR le permitiría
conocer y comparar sus respuestas con las respuestas
de los compañeros”. El AFE muestra la existencia de 3 factores que explican
el 61.61% de la varianza total, siendo esta proporción
aceptable. Además, las comunalidades de los ítems
están por encima de h2=0.40, oscilando entre 0.421
“El uso continuado de los MIR mejoram mi asistencia
a las clases” a 0.791 “El uso de los MIR le permitiría
conocer y comparar sus respuestas con las respuestas
de los compañeros”. Debido a que previamente no se había realizado
ninguna publicación acerca del análisis factorial del
cuestionario MIR-MPEA, antes de realizar un Análisis
Factorial Confirmatorio era conveniente llevar a cabo
un Análisis Factorial Exploratorio (AFE) con el objetivo
de conocer cómo se agrupan los ítems en factores. Para garantizar que los datos se ajustan a un modelo
de análisis factorial se sometieron los datos a la
prueba de Kaiser, Meyer y Olkin (KMO = 0.941) y a
la prueba de esfericidad de Barlett (c2 = 2446.206;
gl = 300; p<0.001), encontrando valores que permiten
la utilización del análisis factorial como técnica idónea
para interpretar la información contenida en esta matriz. En la Tabla 2 se muestran los factores, los ítems, los
pesos factoriales y la fiabilidad de cada dimensión, así
como la interpretación de dichos factores. Para determinar
las dimensiones se ha seguido el criterio de las cargas
factoriales, siendo este valor de 0.30(49). Tabla 2 - Factores, ítems, pesos obtenidos en el Análisis Factorial Exploratorio del MIR-MPEA. Granada, España, 2017
Alfa,
Peso
factorial
Ítems, factores y varianza explicada
1
2
3
h2*
α†
FACTOR 1: Ambiente
F1
51.07%
15.El uso de los MIR hace que las clases sean amenas y dinámicas. 0.759
0.625
0.926
30.El uso de los MIR permite debatir activamente las ideas erróneas para construir el
conocimiento. 0.748
0.303
0.736
48.El uso de los MIR mejora mi participación en las clases desde el anonimato. 0.731
0.650
42.El uso de los MIR permite tener más confianza para hacer preguntas durante las clases. 0.685
0.403
0.673
53.Las respuestas proporcionadas a través de los MIR aumentan mi confianza en las
clases al ver que contesta de manera acertada. Resultados 3.70
1.266
-0.793
0.579
20
El uso de los MIR permite conocer y comparar mis respuestas con
las respuestas de los compañeros. 3.42
1.234
-0.646
0.441
21
El uso de los MIR permite corregir un error o falta de comprensión
sobre los contenidos del tema durante las clases. 3.71
1.121
-1.032
0.558
22
Podrías estar más interesado/a en las clases cuando se utilizan los
MIR. 3.65
1.066
-0.698
0.768
24
El uso de los MIR me gusta como control de la asistencia. 3.65
1.005
-0.605
0.699
29
El uso de los MIR mejora la motivación durante las clases. 3.83
1.111
-0.825
0.753
(continúa en la página siguiente...) www.eerp.usp.br/rlae 4 Rev. Latino-Am. Enfermagem 2021;29:e3418. Nº
Ítems
M*
DT†
Asimetría
Correl. ítem-total
30
El uso de los MIR permite debatir activamente las ideas erróneas
para construir el conocimiento. 3.80
1.168
-0.771
0.792
35
El uso de los MIR evalúa mi conocimiento comprensivo de los
contenidos en cada uno de los temas tratados durante las clases. 3.99
0.971
-1.211
0.788
36
El uso de los MIR promueve el estudio regular de la asignatura para
ir mejor. preparados a las clases. 3.64
1.094
-0.762
0.715
42
El uso de los MIR permite tener más confianza para hacer
preguntas durante las clases. 3.76
1.254
-0.739
0.787
46
La utilización de los MIR se hace al finalizar las clases para repasar
los contenidos dados cada día. 3.80
1.100
-0.736
0.772
47
La utilización de los MIR hace que las clases tengan un ambiente
más agradables e interactivo en comparación con las clases
tradicionales. 3.86
1.082
-0.804
0.769
48
El uso de los MIR mejoran mi participación en las clases desde el
anonimato. 3.87
1.104
-0.773
0.712
53
Las respuestas proporcionadas a través de los MIR aumentan mi
confianza en las clases al ver que contesta de manera acertada. 3.44
1.164
-0.420
0.700
59
Los MIR proporcionan una información valiosa para mejorar mi
proceso de aprendizaje. 4.09
1.017
-1.007
0.687
65
El uso de los MIR mejora la comprensión de los contenidos
explicados en clase. 3.87
1.160
-0.911
0.727
*M = Media; †DT = Desviación típica
(continuación...) *M = Media; †DT = Desviación típica *M = Media; †DT = Desviación típica El AFE muestra la existencia de 3 factores que explican
el 61.61% de la varianza total, siendo esta proporción
aceptable. *M = Media; †DT = Desviación típica Resultados 0.640
0.537
5.Durante mi experiencia con los MIR lo paso bien aprendiendo. 0.602
0.347
0.484
22.Estoy más interesado/a en las clases cuando se utilizan los MIR. 0.577
0.497
0.641
47.La utilización de los MIR hace que las clases tengan un ambiente más agradables e
interactivo en comparación con las clases tradicionales. 0.562
0.491
0.644
24.El uso de los MIR me gusta como control de la asistencia
0.538
0.480
0.590
1.Con el uso de los MIR estoy más concentrado en las clases. 0.483
0.560
0.564
(continúa en la página siguiente
) s, ítems, pesos obtenidos en el Análisis Factorial Exploratorio del MIR-MPEA. Granada, España, 2017 5 Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. Alfa,
Peso
factorial
Ítems, factores y varianza explicada
1
2
3
h2*
α†
FACTOR 2: Proceso Enseñanza-aprendizaje
F2
5.47%
18.El uso de los MIR permite aumentar mi rendimiento en aprendizaje. 0.404
0.726
0.740
0.869
9.Los MIR se utiliza en la evaluación inicial de los contenidos que se van a empezar a
explicar para saber los conocimientos iniciales de los alumnos. 0.725
0.550
2.El uso de los MIR mide si estoy siguiendo correctamente los contenidos dados de la
asignatura durante las clases. 0.702
0.599
59. Los MIR proporcionan una información valiosa para mejorar su proceso de aprendizaje. 0.628
0.512
10.El uso de los MIR se lleva a cabo por docentes experimentados para proporcionar una
buena retroalimentación (feedback). 0.324
0.576
0.483
46.La utilización de los MIR se hace al finalizar las clases para repasar los contenidos
dados cada día. 0.402
0.687
19.El uso continuado de los MIR mejora mi asistencia a las clases. 0.516
0.422
FACTOR 3: Evaluación
F3
5.07%
20.El uso de los MIR permite conocer y comparar sus respuestas con las respuestas de
los compañeros. 0.864
0.755
0.871
36.El uso de los MIR promueve el estudio regular de la asignatura para ir mejor preparados
a las clases. 0.403
0.678
0.686
21.El uso de los MIR permite corregir un error o falta de comprensión sobre los contenidos
del tema durante las clases. 0.361
0.641
0.576
35.El uso de los MIR evalúa mi conocimiento comprensivo de los contenidos en cada uno
de los temas tratados durante las clases. 0.604
0.523
0.715
29.El uso de los MIR mejora la motivación durante las clases. 0.391
0.491
0.680
65.El uso de los MIR mejora la comprensión de los contenidos explicados en clase. *h2 = Comunalidad; †α = Alfa de Cronbach Resultados 0.332
0.486
0.602
(continuación...) (continuación...) (RMSEA) de 0.080, ambos índices se consideran aceptables
ya que se encuentran entre 0.5 y .08(49). Otros indicadores
fueron el índice de bondad de ajuste (GFI) y el índice de
ajuste comparativo (CFI) (con valores de 0.78 y 0.97,
respectivamente), se encuentran dentro de los límites de
la tolerancia. Estos resultados confirman que el modelo de
los 3 factores se ajusta a los datos, por esto, consideramos
que el modelo puede mantenerse como una explicación
plausible a la estructura dimensional que se propone. Posteriormente, se realizó el Análisis Factorial
Confirmatorio y se sometieron a prueba el modelo de 3
factores. Se empleó el método de estimación de máxima
verosimilitud para analizar la matriz de correlaciones. La
bondad de ajuste del modelo propuesto se evaluó mediante
diversos indicadores, se utilizó la χ2/gl (484.13/249)
siendo de 1.944, este índice se encuentra dentro de los
estándares establecidos. El Residuo Cuadrático Medio (RMR)
es de 0.077 y el Error de Aproximación Cuadrático Medio www.eerp.usp.br/rlae www.eerp.usp.br/rlae 6 Rev. Latino-Am. Enfermagem 2021;29:e3418. 6 Figura 1 - Análisis factorial confirmatorio del cuestionario “Mandos Interactivos de Respuestas para la Mejora del
Proceso de Enseñanza-Aprendizaje” Figura 1 - Análisis factorial confirmatorio del cuestionario “Mandos Interactivos de Respuestas para la Mejora del
Proceso de Enseñanza-Aprendizaje” Tabla 3 - Fiabilidad Compuesta y Varianza Media Extraída
de los factores del cuestionario MIR-MPEA. Granada,
España, 2017
Factores
FC*
VME†
F1: Ambiente de Aprendizaje
0.955
0.544
F2: Proceso de enseñanza-aprendizaje
0.930
0.508
F3: Evaluación del aprendizaje
0.863
0.540
*FC = Fiabilidad compuesta; †VME = Varianza media extraída En cuanto a la fiabilidad del instrumento se realiza
el alfa de Cronbach, tanto total con un α= 0.955 como
de las dimensiones que lo componen, obteniendo valores
que oscilan entre α=0,922 para el factor 1, “Ambiente”,
hasta α= 0,869 en el factor 2, “Proceso de enseñanza-
aprendizaje”. Estos datos demuestran que la fiabilidad del
cuestionario es buena en todos los factores, siendo más
bajo en el Factor 3, “Evaluación”. Aunque este estadístico
ha sido ampliamente utilizado en la investigación social
se debe complementar con otro tipo de análisis como
los índices de Fiabilidad Compuesta (FC) y la Varianza
Media Extractada (VME). Los resultados obtenidos se
muestran en la Tabla 3, siendo ambos en todos los casos
muy adecuados. www.eerp.usp.br/rlae Discusión En relación con la
calidad de los ítems, se midió a través de la correlación
ítem-total, los datos indican índices altos, que oscilan de
0.488 a 0.787, lo que muestra una alta consistencia interna
apoyando la idea de que los ítems están correlacionados
y la escala es precisa. Además, en el análisis descriptivo
realizado de los ítems que componen el cuestionario, los
datos muestran que existe asimetría negativa, lo que
revela que los universitarios tienen tendencia a responder
de acuerdo o muy de acuerdo. En cuanto a la primera dimensión, el factor “Ambiente
de aprendizaje”, el alfa de fiabilidad medido a través
del alfa de Cronbach es de 0.926, por lo que muestra
alta consistencia interna y el AFE explica el 51.7% de
la varianza total, siendo el factor con mayor peso en
este cuestionario. Entre los ítems que componen esta
dimensión se encuentran: ítem 15: el uso de los MIR
hace que las clases sean amenas y dinámicas, ítem 48:
el uso de los MIR mejora mi participación en las clases
desde el anonimato, ítem 47: la utilización de los MIR
hace que las clases tengan un ambiente más agradable
e interactivo en comparación con las clases tradicionales,
entre otros. Este Factor es determinante ya que el
alumnado universitario con un ambiente adecuado de
clase aumentará la asistencia a esta asignatura(22) y mejora
su participación(13,17,44). Además, incrementa la interacción
entre el profesorado y el alumnado(13), influye de forma
positiva en la atención(33-34) y en su concentración(35), tal
como avalan los estudios analizados. En cuanto a la fiabilidad de la escala, los datos arrojan
un alfa de Cronbach de 0.965, lo que indica una alta
fiabilidad. Estos datos están en consonancia con los índices
de fiabilidad compuesta de los factores que componen MIR-
MPEA, que alcanzan unos niveles óptimos en los 3 factores
(Ambiente, Proceso de enseñanza-aprendizaje y Evaluación)
que oscilan entre 0.955, 0.930 y 0.863, respectivamente,
siendo el valor mínimo recomendado de 0.70. La segunda dimensión, que corresponde al factor
“Proceso de enseñanza-aprendizaje”, muestra un índice
de consistencia interno de 0.869 y explica el 5.4% de
la varianza total, bajando de forma significativa el peso
del factor. Discusión El objetivo del presente estudio es la construcción
de un cuestionario válido y fiable para medir el uso de
los MIR en el aprendizaje centrado en el estudiante 7 Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. 61.61% de la varianza total. Posteriormente, se procedió
con el AFC para confirmar el modelo de los tres factores. Los índices de bondad utilizados han sido χ2/gl siendo de
1.944, este índice se encuentra dentro de los estándares
establecidos, el RMR es 0.077 y el RMSEA es de 0.080,
índices que se consideran aceptables ya que se encuentran
entre 0.5 y 0.08(49). Además, el GFI que representa el
ajuste conjunto es de 0.78 y CFI con un valor de 0.97,
se encuentran dentro de los límites de la tolerancia. Estos
resultados confirman que el modelo de los 3 factores se
ajusta a los datos, considerando que el modelo puede
mantenerse como una explicación plausible a la estructura
dimensional que se propone. universitario. Los resultados muestran que este trabajo
constituye un aporte empírico a la validez de este modelo
de intervención en el uso de los MIR, permitiendo a
los profesores universitarios en general, y de manera
particular, a los docentes de Ciencias de la Salud
transformar el proceso de enseñanza-aprendizaje para
incentivar e involucrar a los estudiantes a través de un
enfoque más activo para profesores del siglo XXI, que
puede medir la percepción del alumnado en el uso de los
MIR, habiendo avanzado a pasos agigantados, además
de ser un recurso tecnopedagógico, se ha convirtiendo
en una forma de juego para los estudiantes, adaptándolo
al contexto que no son juego, incorporándose como una
metodología emergente denominada gamificación, que
proporciona la oportunidad de transformar drásticamente
las aulas tradicionales de manera que puedan mejorar
el ambiente de clase, el proceso de aprendizaje y su
rendimiento académico de manera lúdica y divertida. Por todo lo anterior, estos resultados permiten confiar
en la fiabilidad y validez de este instrumento, por lo que
con la construcción y validación de este cuestionario se
hace posible medir la percepción del estudiante en el uso
de los MIR en el proceso de aprendizaje. El cuestionario MIR-MPEA está formado por 24 ítems
que se agrupan en 3 factores: Ambiente, Proceso de
enseñanza-aprendizaje y Evaluación. www.eerp.usp.br/rlae Discusión Esta dimensión hace referencia a aquellos
elementos que son básicos para acceder al conocimiento,
tales como los debates, la interacción entre el profesorado-
alumnado, que inciden positivamente en su proceso de
aprendizaje, ya que ayuda al repaso y a la comprensión
de los contenidos. Entre los ítems que lo componen se
encuentran, ítem 47: la utilización de los MIR hace que las
clases tengan un ambiente más agradable e interactivo
en comparación con las clases tradicionales, ítem 59: los
MIR proporcionan una información valiosa para mejorar mi
proceso de aprendizaje, ítem 46: la utilización de los MIR
se hace al finalizar las clases para repasar los contenidos Pero, además, se realizó la validez del constructo,
tanto a través del Análisis Factorial Exploratorio (AFE)
como del Confirmatorio (AFC). En primer lugar, se procedió
al AFE, ya que no había instrumentos similares validados
que midieran la percepción del alumnado universitario
hacia el aprendizaje y solo conocíamos las dimensiones
que conforman el constructo objeto de estudio fruto de
la bibliografía consultada. Para ello, en primer lugar,
los datos se sometieron a prueba de de Kaiser, Meyer y
Olkin, obteniendo un valor significativo (KMO = 0.941)
y a la prueba de esfericidad de Barlett (c2 = 2446.206;
gl = 300; p < 0.001), estos valores permiten la utilización
del análisis factorial como técnica idónea para interpretar
la información contenida en esta matriz. Este análisis
arrojó tres factores claramente delimitados que explican el Rev. Latino-Am. Enfermagem 2021;29:e3418. 8 del Campus de Melilla, de la Universidad de Granada,
que utilizan recursos tecnopedagógicos como los MIR
en sus aulas, adaptándolos a procesos de metodología
activa gamificada, por lo que se hace necesario impulsar
su uso e investigarlo. Por esta razón, es fundamental
concienciar y formar al profesorado universitario en
el modelo TPACK (Technological Pedagogical Content
Knowledge) como un marco conceptual que puede
orientar al profesorado para la integración de la tecnología
en los procesos de aprendizaje de los estudiantes, lo
que promoverá las metodologías emergentes, tales
como la gamificación. En segundo lugar, se debe seguir
mejorando el instrumento para que pueda ser utilizado
por el profesorado, de cualquier ámbito de conocimiento
y en cualquier circunstancia que acontezca, tal y como
se ha producido en el confinamiento del COVID-19,
pudiéndose trabajar, en épocas venideras, según los
planteamientos gubernamentales y universitarios, en
entornos presenciales, B-Learning y E-Learning con
su alumnado. Discusión Por lo que se tendrá que mejorar el
instrumento, incluyendo nuevos ítems que valoren este
nuevo modelo de enseñanza virtual. Además, debemos
profundizar la influencia de este instrumento en función
de las distintas disciplinas de las Ciencias de la Salud,
hecho que pretendemos llevar a cabo en el futuro. La
aplicación de los avances tecnológicos en el ámbito
universitario mejora el proceso aprendizaje activo de los
estudiantes, siendo necesario conocer la opinión de los
mismos para lograr un avance y mejora en la docencia. En
tercer lugar, es necesario realizar más investigación cuasi-
experimental en el ámbito de la docencia universitaria,
en sus diversas ramas de conocimiento, para analizar la
influencia de la metodología activa gamificada a través
de estos recursos tecnopedagógicos y la metodología
tradicional, teniendo muy presente su alcance o relación
con el rendimiento académico. dados cada día. Estos elementos coinciden con los estudios
que consideran que esta metodología de trabajo mejora
el rendimiento(31-32,38-41), con el cambio de paradigma que
establece el aprendizaje activo de los estudiantes a través
de la conexión entre el saber y el saber hacer(45), fruto de
la interacción frecuente y positiva que puede dinamizar
las clases cuando se usan los MIR(13,28). Todo esto permite
un razonamiento sobre el material, adquiriendo un
conocimiento más profundo, ayudando al repaso y a la
comprensión de los contenidos y mejora de la retención
a largo plazo(36-37), con la consiguiente optimización del
proceso de aprendizaje. En relación con la tercera dimensión, “Evaluación
del aprendizaje” presenta un alfa de Cronbach de 0.871,
y el AFE explica el 5.07% de la varianza total, siendo un
porcentaje similar al factor anterior. Este dimensión se
relaciona con la retroalimentación y evaluación formativa,
que ayudan a corregir errores o falta de comprensión
sobre los contenidos del tema trabajados, tal como indican
las investigaciones consultadas(4,20,40). Está formado por el
ítem 20: el uso de los MIR permite conocer y comparar sus
respuestas con las respuestas de los compañeros, el ítem
36: el uso de los MIR promueve el estudio regular de la
asignatura para ir mejor preparados a las clases, el ítem
35: el uso de los MIR evalúa mi conocimiento comprensivo
de los contenidos en cada uno de los temas tratados
durante las clases, entre otros. Discusión Esta retroalimentación
inmediata incide de forma positiva tanto en el alumnado
en su proceso de aprendizaje como en el docente en
su proceso de enseñanza, lo que permite la evaluación
formativa de los alumnos. De este modo, el mundo global en el que vivimos, sin
fronteras académicas, los Espacios de Educación Superior
Europeo e Iberoamericano cumplen la premisa en la que se
pone de manifiesto que la calidad del sistema universitario
debe estar en continuo proceso de transformación con
el cambio de paradigma hacia el aprendizaje activo de
los alumnos y al aprendizaje permanente a lo largo de
toda la vida. Además, es necesario establecer procesos
de evaluación y acreditación del trabajo que se realiza en
las universidades, con la consiguiente transferencia del
conocimiento de las investigaciones realizadas entre las
distintas investigaciones en las diversas ramas del saber,
estableciendo un entendimiento interdisciplinar que resalte
la realidad y cuestione el pensamiento tradicional de las
distintas ramas del saber como compartimentos aislados
y separados de las fronteras disciplinares. www.eerp.usp.br/rlae Conclusión La contribución de este estudio al conocimiento
es el diseño y desarrollo de un instrumento y su
transferencia, que permita medir y valorar la percepción
de los estudiantes en el uso de los MIR con respecto al
desarrollo de la docencia universitaria para el dominio de
la materia de cualquier rama del conocimiento, ya sean
Ciencias de la Salud, Ciencias Sociales y Jurídicas, Artes
y Humanidades, e Ingeniería y Arquitectura, mediante un
recurso tecnopedagógico, en tres factores fundamentales:
ambiente, proceso de enseñanza-aprendizaje y evaluación. Este cuestionario puede ser aplicado en clase durante el
tiempo que dure la formación, lo que ayudará a mejorar
los procesos de enseñanza-aprendizaje. En este sentido,
los profesores de Ciencias de la Salud deben establecer Entre las limitaciones del presente estudio
enumeramos las siguientes: en primer lugar, es necesario
seguir aumentando el número de participantes, ya que la
muestra no es excesivamente amplia, pero permite tener
un punto de partida para transferirlo a otras ramas de
conocimiento. La razón es que son escasos los docentes 9 9 Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. Literature Review and Case Studies, EUR 29882. Luxembourg: European Commission; 2019. doi:
https://doi.org/10.2760/552774 y explorar nuevas formas innovadoras de involucrar
a los estudiantes y estimular el aprendizaje activo,
siendo importante la incorporación de metodologías
activas a través del uso de los mandos interactivos de
respuestas en disciplinas como Enfermería, Medicina,
Farmacia, Educación Paramédica, Psicología, Odontología,
Fisioterapia, Logopedia, Biotecnología, Epidemiología,
Genética, Bioquímica, Terapia Ocupacional, Nutrición
Humana y Dietética, entre otras. Este instrumento
proporciona un enfoque pedagógico positivo en el proceso
de enseñanza-aprendizaje para docentes y estudiantes
de Ciencias de la Salud, lo que conlleva una mejora del
rendimiento con el propósito de adquirir un conocimiento
profundo de las materias. 9. Marcelo C, Yot C, Mayor C. Enseñar con tecnologías
digitales en la Universidad. Comunicar. 2015;45:117-24. doi: https://doi.org/10.3916/C45-2015-12 10. Hinojo FJ, Mingorance AC, Trujillo JM, Aznar I, Cáceres
MP. Incidence of the Flipped Classroom in the Physical
Education Students’ Academic Performance in University
Contexts. Sustainability. 2018;10(5):1334-46. doi:
https://doi.org/10.3390/su10051334 10. Hinojo FJ, Mingorance AC, Trujillo JM, Aznar I, Cáceres pp
y
Education Students’ Academic Performance in University
Contexts. Sustainability. 2018;10(5):1334-46. doi:
https://doi.org/10.3390/su10051334 11. Njie-Carr VP, Ludeman E, Lee MC, Dordunoo D,
Trocky NM, Jenkins LS. An Integrative Review of Flipped
Classroom Teaching Models in Nursing Education. J Prof Nurs. 2017;33(2):133-44. doi: 10.1016/j. profnurs.2016.07.001 Referencias 12. Wilson J, Walker S. Turning a crisis into an interactive
drama: the introduction of a ‘clickers theatre’ in nurse
education. Nurse Educ Today. 2017;51:109-11. doi:
10.1016 / j.nedt.2016.11.023 1. Sotillo JA, Rodrígue I, Echart E, Ojeda T. El Espacio
Iberoamericano de Educación Superior. Diagnóstico y
propuestas institucionales. Madrid: Fundación Carolina,
CeALCI; 2009. 13. Carrino SS. Digital connection in a physical classroom:
Clickers and the student-teacher relationship [thesis]. Greensboro: University of North Carolina; 2015 [cited
Dec 12, 2019]. Available from: https://libres.uncg.edu/
ir/uncg/f/Carrino_uncg_0154D_11791.pdf 13. Carrino SS. Digital connection in a physical classroom:
Clickers and the student-teacher relationship [thesis]. Greensboro: University of North Carolina; 2015 [cited
Dec 12, 2019]. Available from: https://libres.uncg.edu/
ir/uncg/f/Carrino_uncg_0154D_11791.pdf 2. Mingorance AC, Granda J, Rojas G, Alemany I. Flipped Classroom to Improve University Student
Centered Learning and Academic Performance. Soc
Sci. 2019;8(11):315-28. doi: https://doi.org/10.3390/
socsci8110315 2. Mingorance AC, Granda J, Rojas G, Alemany I. Flipped Classroom to Improve University Student
Centered Learning and Academic Performance. Soc
Sci. 2019;8(11):315-28. doi: https://doi.org/10.3390/
socsci8110315 14. Zainuddin Z, Halili SH. Flipped classroom research
and trends from different fields of study. Int Rev Res
Open Distance Learn. [Internet]. 2016 [cited Jul 18,
2018];17(3):313-40. Available from: https://files.eric. ed.gov/fulltext/EJ1102721.pdf 3. Harris J, Mishra P, Koehler M. Teachers’ technological
pedagogical content knowledge and learning activity
types: curriculum-based technology integration refrained. J Res Technol Educ. [Internet]. 2009 [cited Oct 21,
2018];41(4):393-416. Available from: https://files.eric. ed.gov/fulltext/EJ844273.pdf 3. Harris J, Mishra P, Koehler M. Teachers’ technological
pedagogical content knowledge and learning activity
types: curriculum-based technology integration refrained. J Res Technol Educ. [Internet]. 2009 [cited Oct 21,
2018];41(4):393-416. Available from: https://files.eric. ed.gov/fulltext/EJ844273.pdf 15. Ali RA, Alnatour A, Alnuaimi K, Alzoubi F, Almomani
M, Othman A. Effects of interactive teaching on university
students’ knowledge and attitude toward reproductive
health: a pilot study in Jordan. J Multidiscip Healthc. 2018;11:211-21. doi: https://doi.org/10.2147/JMDH. S160135 15. Ali RA, Alnatour A, Alnuaimi K, Alzoubi F, Almomani
M, Othman A. Effects of interactive teaching on university
students’ knowledge and attitude toward reproductive
health: a pilot study in Jordan. J Multidiscip Healthc. 2018;11:211-21. doi: https://doi.org/10.2147/JMDH. S160135 4. Cheung G, Wan K, Chan K. Efficient Use of Clickers:
A Mixed-Method Inquiry with University Teachers. Educ. Sci. 2018;8(31):1-15. doi: https://doi.org/10.3390/
educsci8010031 4. Cheung G, Wan K, Chan K. Efficient Use of Clickers:
A Mixed-Method Inquiry with University Teachers. Educ. Sci. 2018;8(31):1-15. doi: https://doi.org/10.3390/
educsci8010031 5. Schubert V, Medina J, Lenise M. Referencias Proceso de construcción
del conocimiento pedagógico del docente universitario de
enfermería1. Rev. Latino-Am. Enfermagem. [Internet]. 2011 [cited Oct 22, 2018];19(2). Available from: http://
www.scielo.br/pdf/rlae/v19n2/es_26.pdf 5. Schubert V, Medina J, Lenise M. Proceso de construcción
del conocimiento pedagógico del docente universitario de
enfermería1. Rev. Latino-Am. Enfermagem. [Internet]. 2011 [cited Oct 22, 2018];19(2). Available from: http://
www.scielo.br/pdf/rlae/v19n2/es_26.pdf 16. Barcelo JM. Medical laboratory science and nursing
students’ perception of the academic learning environment
at a Philippine university using the Dundee Ready
Education Environment Measure. J Educ Eval Health
Prof. 2016;13(33):1-7. doi: https://doi.org/10.3352/
jeehp.2016.13.33 16. Barcelo JM. Medical laboratory science and nursing
students’ perception of the academic learning environment
at a Philippine university using the Dundee Ready
Education Environment Measure. J Educ Eval Health
Prof. 2016;13(33):1-7. doi: https://doi.org/10.3352/
jeehp.2016.13.33 6. Cheung G, Wan K, Chan K. Efficient Use of Clickers:
A Mixed-Method Inquiry with University Teachers. Educ. Sci. 2018;8(31):1-15. doi: https://doi.org/10.3390/
educsci8010031 6. Cheung G, Wan K, Chan K. Efficient Use of Clickers:
A Mixed-Method Inquiry with University Teachers. Educ. Sci. 2018;8(31):1-15. doi: https://doi.org/10.3390/
educsci8010031 17. Brady M, Seli H, Rosenthal J. “Clickers” and
metacognition: a quasi-experimental comparative study
about metacognitive self-regulation and use of electronic
feedback devices. Comput Educ. 2013;65:56-63. doi:
https://doi.org/10.1016/j.compedu.2013.02.001
18. Castro MJ, López M, Cao MJ, Fernández-Castro M,
García S, Frutos M, et al. Impact of educational games
on academic outcomes of students in the Degree in
Nursing. PLoS One. 2019;14(7):e0220388. doi: https://
doi.org/10.1371/journal.pone.0220388 17. Brady M, Seli H, Rosenthal J. “Clickers” and
metacognition: a quasi-experimental comparative study
about metacognitive self-regulation and use of electronic
feedback devices. Comput Educ. 2013;65:56-63. doi:
https://doi.org/10.1016/j.compedu.2013.02.001 17. Brady M, Seli H, Rosenthal J. “Clickers” and
metacognition: a quasi-experimental comparative study
about metacognitive self-regulation and use of electronic
feedback devices. Comput Educ. 2013;65:56-63. doi:
https://doi.org/10.1016/j.compedu.2013.02.001 7. Johnson L, Adams S, Estrada V, Freeman A. NMC
Horizon Report: 2016 Higher Education Edition. [Internet]. Austin: The New Media Consortium; 2016 [cited Sep 15,
2018]. Available from: http://www.aprendevirtual.org/
centro-documentacion-pdf/2016-nmc-horizon-report-
HE-ES.pdf 7. Johnson L, Adams S, Estrada V, Freeman A. NMC
Horizon Report: 2016 Higher Education Edition. [Internet]. 7. Johnson L, Adams S, Estrada V, Freeman A. NMC
Horizon Report: 2016 Higher Education Edition. [Internet]. 18. Castro MJ, López M, Cao MJ, Fernández-Castro M,
García S, Frutos M, et al. Impact of educational games
on academic outcomes of students in the Degree in
Nursing. PLoS One. 2019;14(7):e0220388. doi: https://
doi.org/10.1371/journal.pone.0220388 8. www.eerp.usp.br/rlae Referencias Enhancing student engagement and
immediate feedback with clickers and response cards. Int J Innov Learn. 2018;24(1):81-97. doi: https://doi. org/10.1504/IJIL.2018.092924
28. Banks D. Reflections on the use of ARS with small
groups. In: ______. Audience Response Systems in Higher
Education: Applications and Cases. Pennsylvania: IGI
Global; 2006. p. 373-86. 29. Wood A. Nurse Perceptions of Interactivity during Their
Onboarding Orientation: Effect of an Audience Response
System [thesis]. Boiling Springs: Hunt School of Nursing;
2017 [cited Dec 12, 2019]. Available from: https://
digitalcommons.gardner-webb.edu/cgi/viewcontent.cgi
?article=1268&context=nursing_etd
30. Ismaile S, Alhosban F, Hawamdeh S. Making learning
fun to increase nursing students’ success: Formative
feedback in communication learning. Australas Med J. 2017;10(12):1014-21. doi: https://doi.org/10.21767/
AMJ.2017.3228 27. Mohan R. Enhancing student engagement and
immediate feedback with clickers and response cards. Int J Innov Learn. 2018;24(1):81-97. doi: https://doi. org/10.1504/IJIL.2018.092924 38. Rana NP, Dwivedi YK. Can clicking promote learning? measuring student learning performance using clickers in
the undergraduate information systems class. J Int Educ
Bus. 2017;10(2):201-15. doi: http://doi.org/10.1108/
JIEB-06-2016-0010 38. Rana NP, Dwivedi YK. Can clicking promote learning? measuring student learning performance using clickers in
the undergraduate information systems class. J Int Educ
Bus. 2017;10(2):201-15. doi: http://doi.org/10.1108/
JIEB-06-2016-0010 org/10.1504/IJIL.2018.092924
28. Banks D. Reflections on the use of ARS with small
groups. In: ______. Audience Response Systems in Higher
Education: Applications and Cases. Pennsylvania: IGI
Global; 2006. p. 373-86. 28. Banks D. Reflections on the use of ARS with small
groups. In: ______. Audience Response Systems in Higher Education: Applications and Cases. Pennsylvania: IGI
Global; 2006. p. 373-86. Education: Applications and Cases. Pennsylvania: IGI
Global; 2006. p. 373-86. 39. Iwamoto D, Hargis J, Taitano E, Vuong K. Analyzing the
efficacy of the testing effect using kahoottm on student
performance. Turkish Online J Distance Educ. [Internet]. 2017;18(2):93-80. doi: https://doi.org/10.17718/
tojde.306561 29. Wood A. Nurse Perceptions of Interactivity during Their
Onboarding Orientation: Effect of an Audience Response
System [thesis]. Boiling Springs: Hunt School of Nursing;
2017 [cited Dec 12, 2019]. Available from: https://
digitalcommons.gardner-webb.edu/cgi/viewcontent.cgi
?article=1268&context=nursing_etd 29. Wood A. Nurse Perceptions of Interactivity during Their
Onboarding Orientation: Effect of an Audience Response
System [thesis]. Boiling Springs: Hunt School of Nursing;
2017 [cited Dec 12, 2019]. Available from: https://
digitalcommons.gardner-webb.edu/cgi/viewcontent.cgi
?article=1268&context=nursing_etd 40. George C, Gallegos C, Tesar AJ, Connor K, Martz K. The
use of a game-based learning platform to engage nurs-
ing students: A descriptive, qualitative study. Nurse Educ
Pract. 2017;27:101-6. doi: https://doi.org/10.1016/j. nepr.2017.08.019. 40. Referencias Kapsalis G, Ferrari A, Punie Y, Conrads J, Collado A,
Hotulainen R, et al. Evidence of Innovative Assessment: 8. Kapsalis G, Ferrari A, Punie Y, Conrads J, Collado A,
Hotulainen R, et al. Evidence of Innovative Assessment: www.eerp.usp.br/rlae 10 Rev. Latino-Am. Enfermagem 2021;29:e3418. 31. Castro MJ, López M, Cao MJ, Fernández-Castro M,
García S, Frutos M, et al. Impact of educational games
on academic outcomes of students in the Degree in
Nursing. PLoS One 2019;14(7):e0220388. doi: https://
doi.org/10.1371/journal.pone.0220388 19. Nishimura A. Effects of different methods of reflection
on nurses’ gaze and judgement in a task using a touch
panel. J Clin Nurs. 2018;27:569-77. doi: https://doi. org/10.1111/jocn.14096 19. Nishimura A. Effects of different methods of reflection
on nurses’ gaze and judgement in a task using a touch
panel. J Clin Nurs. 2018;27:569-77. doi: https://doi. org/10.1111/jocn.14096 20. Toothaker R. Millennial’s perspective of clicker
technology in a nursing classroom: A mixed methods
h
t d
N
Ed
T d
2018 62 80 4 d i 20. Toothaker R. Millennial’s perspective of clicker
technology in a nursing classroom: A mixed methods
research study. Nurse Educ Today. 2018;62:80-4. doi:
https://doi.org/10.1016/j.nedt.2017.12.027 32. Corell A, Regueras LM, Verdú E, Verdú MJ, Castro JP. Effects of competitive learning tools on medi- cal students:
A case study. PLoS One. 2018;13(3):e0194096. doi:
https://doi.org/10.1371/journal.pone.0194158 32. Corell A, Regueras LM, Verdú E, Verdú MJ, Castro JP. Effects of competitive learning tools on medi- cal students:
A case study. PLoS One. 2018;13(3):e0194096. doi:
https://doi.org/10.1371/journal.pone.0194158 https://doi.org/10.1016/j.nedt.2017.12.027 21. Chen TL, Lan YL. Using a personal response system as
an in-class assessment tool in the teaching of basic college 33. Oswald KM, Rhoten SE. Improving classroom
clicker practices: Effects of incentives and feedback on
retention. N Am J Psychol. [Internet]. 2014 [cited Dec
6, 2017];16(1):79-88. Available from: https://www. researchgate.net/publication/285958781_Improving_
classroom_clicker_practices_Effects_of_incentives_and_
feedback_on_retention chemistry. Australas J Educ Technol. 2013;29(1):32-40. doi: https://doi org/10 14742/ajet 95 chemistry. Australas J Educ Technol. 2013;29(1):32-40. chemistry. Australas J Educ Technol. 2013;29(1):32-40. doi: https://doi.org/10.14742/ajet.95 doi: https://doi.org/10.14742/ajet.95 22. Aktekin NÇ, Çelebi H, Aktekin M. Let’s kahoot! Anatomy. Int. J. Morphol. 2018;36(2):716-21. doi: http:// dx.doi.org/10.4067/S0717-95022018000200716 23. Barr ML. Encouraging college student active
engagement in learning: The influence of response
methods. Innov High Educ. 2013;39(4):307-19. doi:
https://doi.org/10.1111/jcal.12205 34. Marshall LL, Varnon AW. An Empirical Investigation
of Clicker Technology in Financial Accounting Principles. J Learn High Educ. [Internet]. 2012 [cited Jun 3,
2018];8(1):7-18. Available from: https://files.eric.ed.gov/
fulltext/EJ1144930.pdf 34. Marshall LL, Varnon AW. Referencias An Empirical Investigation
of Clicker Technology in Financial Accounting Principles. 34. Marshall LL, Varnon AW. An Empirical Investigation
of Clicker Technology in Financial Accounting Principles. 34. Marshall LL, Varnon AW. An Empirical Investigation
of Clicker Technology in Financial Accounting Principles. 34. Marshall LL, Varnon AW. An Empirical Investigation
of Clicker Technology in Financial Accounting Principles. J Learn High Educ. [Internet]. 2012 [cited Jun 3,
2018];8(1):7-18. Available from: https://files.eric.ed.gov/
fulltext/EJ1144930.pdf J Learn High Educ. [Internet]. 2012 [cited Jun 3,
2018];8(1):7-18. Available from: https://files.eric.ed.gov/
fulltext/EJ1144930.pdf J Learn High Educ. [Internet]. 2012 [cited Jun 3,
2018];8(1):7-18. Available from: https://files.eric.ed.gov/
fulltext/EJ1144930.pdf 24. Iskander M. Systematic review of the implementation 24. Iskander M. Systematic review of the implementation
of audience response systems and their impact on
participation and engagement in the education of
healthcare professionals. BMJ Simulation & Technology
Enhanced Learning. 2018;4(2):47-50. doi: http://dx.doi. org/10.1136/bmjstel-2017-000245 of audience response systems and their impact on participation and engagement in the education of 35. Bojinova ED, Oigara JN. Teaching and learning with
Clickers: Are Clickers good for students? Interdiscip J
E-Learning Learn Objects. 2011;7:169-83. doi: https://
doi.org/10.28945/1506 healthcare professionals. BMJ Simulation & Technology org/10.1136/bmjstel-2017-000245 25. Licorish SA, Owen HE, Daniel, B, George, JL. Students’
perception of Kahoot!’s in uence on teaching and learning. RPTEL. 2018;13(9):1-23. doi: https://doi.org/10.1186/
s41039-018-0078-8 36. Benson JD, Szucs KA, DeIuliis E, Leri A. Impact of
Student Response Systems on Initial Learning and Retention 36. Benson JD, Szucs KA, DeIuliis E, Leri A. Impact of
Student Response Systems on Initial Learning and Retention
of Course Content in Health Sciences Students. J Allied
Health. [Internet]. 2017 [cited Feb 12, 2018];46(3):158-
63. Available from: https://www.ingentaconnect.com/
content/asahp/jah/2017/00000046/00000003/art00008
37. Njie-Carr VP, Ludeman E, Lee MC, Dordunoo D,
Trocky NM, Jenkins LS. An Integrative Review of Flipped
Classroom Teaching Models in Nursing Education. J Prof Nurs. 2017;33(2):133-44. doi: 10.1016/j. profnurs.2016.07.001 RPTEL. 2018;13(9):1-23. doi: https://doi.org/10.1186/
s41039-018-0078-8 of Course Content in Health Sciences Students. J Allied
Health. [Internet]. 2017 [cited Feb 12, 2018];46(3):158- 63. Available from: https://www.ingentaconnect.com/
content/asahp/jah/2017/00000046/00000003/art00008 26. Oliveira C, Tirapelli C, Rodrigues CT, Domaneschi C,
Caldeira SA. Interactive audience response systems in
oral and maxillofacial radiology undergraduate lectures. Eur J Dent Educ. [Internet]. 2017;22:63-9. doi: https://
doi.org/10.1111/eje.12258 26. Oliveira C, Tirapelli C, Rodrigues CT, Domaneschi C,
Caldeira SA. Interactive audience response systems in
oral and maxillofacial radiology undergraduate lectures. Eur J Dent Educ. [Internet]. 2017;22:63-9. doi: https://
doi.org/10.1111/eje.12258 27. Mohan R. Referencias George C, Gallegos C, Tesar AJ, Connor K, Martz K. The
use of a game-based learning platform to engage nurs-
ing students: A descriptive, qualitative study. Nurse Educ
Pract. 2017;27:101-6. doi: https://doi.org/10.1016/j. nepr.2017.08.019. 30. Ismaile S, Alhosban F, Hawamdeh S. Making learning
fun to increase nursing students’ success: Formative
feedback in communication learning. Australas Med J. 2017;10(12):1014-21. doi: https://doi.org/10.21767/
AMJ.2017.3228 41. Kalaian SA, Kasim RM. Effectiveness of various
innovative learning methods in health science class- www.eerp.usp.br/rlae www.eerp.usp.br/rlae 11 Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. Copyright © 2021 Revista Latino-Americana de Enfermagem
Este es un artículo de acceso abierto distribuido bajo los términos
de la Licencia Creative Commons CC BY.
Esta licencia permite a otros distribuir, mezclar, ajustar y construir
a partir de su obra, incluso con fines comerciales, siempre que le
sea reconocida la autoría de la creación original. Esta es la licencia
más servicial de las ofrecidas. Recomendada para una máxima
difusión y utilización de los materiales sujetos a la licencia. Contribución de los Autores: rooms: a meta-analysis. Adv Health Sci Educ Theory
Pract. [Internet]. 2017;22(5):1151-67. doi: https://doi. org/10.1007/s10459-017-9753-6 Concepción y dibujo de la pesquisa: Ángel Custodio
Mingorance-Estrada. Obtención de datos: Ángel
Custodio Mingorance-Estrada, Gloria Rojas-Ruiz. Análisis
e interpretación de los datos: Ángel Custodio
Mingorance-Estrada, Juan Granda-Vera, Inmaculada
Alemany-Arrebola. Análisis estadístico: Juan Granda-
Vera, Inmaculada Alemany-Arrebola. Redacción del
manuscrito: Ángel Custodio Mingorance-Estrada, Gloria
Rojas-Ruiz. Revisión crítica del manuscrito en cuanto
al contenido intelectual importante: Ángel Custodio
Mingorance-Estrada. 42. Maloney LM, Dilger JP, Werfel PA, Cimino LM. Are
Audience Response Systems Worth the Cost ? Comparing
Question-Driven Teaching Strategies for Emergency
Medical Technician Education. Internet J Allied Health
Sci Pract. [Internet]. 2017 [cited May 7, 2018];16(1):1-
8. Available from: https://nsuworks.nova.edu/cgi/
viewcontent.cgi?article=1704&context=ijahsp Medical Technician Education. Internet J Allied Health 43. Ismail MA, Mohammad JA. Kahoot: A promising tool
for formative assessment in medical education. Educ
Med J. 2017;9(2):19-26 doi: https://doi.org/10.21315/
eimj2017.9.2.2 Todos los autores aprobaron la versión final del
texto. 44. Nikou SA, Economides AA. Mobile-based assessment:
A literature review of publications in major referred
journals from 2009 to 2018. Comput Educ. 2018;125:101-
19. doi: https://doi.org/10.1016/j.compedu.2018.06.006
45. Montejano-Lozoya R, Gea-Caballero V, Miguel-Montoya
I, Juárez-Vela R, Sanjuán-Quiles A, Ferrer-Ferrandiz E. Validation of a questionnaire designed to measure nursing
student satisfaction with practical training. Rev. Latino-
Am. Enfermagem. 2019;27:e3206. doi: https://doi. org/10.1590/1518-8345.3102.3206 44. Nikou SA, Economides AA. Mobile-based assessment:
A literature review of publications in major referred
journals from 2009 to 2018. Comput Educ. 2018;125:101-
19. doi: https://doi.org/10.1016/j.compedu.2018.06.006
45. Montejano-Lozoya R, Gea-Caballero V, Miguel-Montoya
I, Juárez-Vela R, Sanjuán-Quiles A, Ferrer-Ferrandiz E. Validation of a questionnaire designed to measure nursing
student satisfaction with practical training. Rev. Latino-
Am. Enfermagem. 2019;27:e3206. doi: https://doi. org/10.1590/1518-8345.3102.3206 Conflicto de intereses: los autores han declarado
que no existe ningún conflicto de intereses. Conflicto de intereses: los autores han declarado
que no existe ningún conflicto de intereses. 46. Miles NG, Soares TP. Acceptance of clickers in a
large multimodal biochemistry class as determined by
student evaluations of teaching: Are they just an annoying
distraction for distance students? Biochem Mol Biol Educ. 2016; Oct;44(1):99-108. doi: https://doi.org/10.1002/
bmb.20917 47. Yeh CR, Tao YH. How benefits and challenges of
personal response system impact students’ continuance
intention? A Taiwanese context. Educ Technol Soc. [Internet]. 2013 [cited Jan 10, 2018];16(2):257-70. Available from: https://pdfs.semanticscholar.org/283d/
f5d6cddabfc9abb8d2806efbf809975818cb.pdf
48. Lin YC, Liu TC, Chu CC. Implementing clickers to
assist learning in science lectures: The clicker-assisted
conceptual change model. Australas J Educ Technol. 2011;27(6):979-96. Contribución de los Autores: doi: https://doi.org/10.14742/
ajet.924 47. Yeh CR, Tao YH. How benefits and challenges of
personal response system impact students’ continuance
intention? A Taiwanese context. Educ Technol Soc. [Internet]. 2013 [cited Jan 10, 2018];16(2):257-70. Available from: https://pdfs.semanticscholar.org/283d/
f5d6cddabfc9abb8d2806efbf809975818cb.pdf
48. Lin YC, Liu TC, Chu CC. Implementing clickers to
assist learning in science lectures: The clicker-assisted
conceptual change model. Australas J Educ Technol. 2011;27(6):979-96. doi: https://doi.org/10.14742/
ajet.924 49. Kline R. Principles and Practice of Structural Equation
Modeling. New York: The Guilford Press; 2011. 49. Kline R. Principles and Practice of Structural Equation
Modeling. New York: The Guilford Press; 2011. Recibido: 12.12.2019
Aceptado: 13.08.2020 Editora Asociada:
Regina Aparecida Garcia de Lima Copyright © 2021 Revista Latino-Americana de Enfermagem
Este es un artículo de acceso abierto distribuido bajo los términos
de la Licencia Creative Commons CC BY. Esta licencia permite a otros distribuir, mezclar, ajustar y construir
a partir de su obra, incluso con fines comerciales, siempre que le
sea reconocida la autoría de la creación original. Esta es la licencia
más servicial de las ofrecidas. Recomendada para una máxima
difusión y utilización de los materiales sujetos a la licencia. Autor de correspondencia:
Ángel Custodio Mingorance Estrada
E-mail: amingoe@ugr.es
https://orcid.org/0000-0003-4478-3011 www.eerp.usp.br/rlae
|
https://openalex.org/W4392404203
|
https://www.oejournal.org/data/article/export-pdf?id=65e5952799d881090c5da732
|
English
| null |
Data-driven polarimetric imaging: a review
|
Opto-electronic science
| 2,024
|
cc-by
| 30,818
|
Data-driven polarimetric imaging: a review Citation: Yang K, Liu F, Liang SY, et al. Data-driven polarimetric imaging: a review. Opto-Electron Sci 3, 230042 (2024). https://doi.org/10.29026/oes.2024.230042 Received: 14 November 2023; Accepted: 19 January 2024; Published online: 29 February 2024 Received: 14 November 2023; Accepted: 19 January 2024; Published online: 29 February 2024 1School of Optoelectronic Engineering, Xidian University, Xi’an 710071, China; 2School of Physics, Xidian University, Xi’an 710071, China;
3Graduate School of Information, Production and Systems, Waseda University, Kitakyushu 808-0135, Japan; 4Department of Mechanical
Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; 5Hangzhou Institute of Technology, Xidian University,
Hangzhou 311200, China. Data-driven polarimetric imaging: a review Kui Yang1†, Fei Liu1†*, Shiyang Liang3†, Meng Xiang1, Pingli Han1,
Jinpeng Liu1, Xue Dong1, Yi Wei4, Bingjian Wang2, Koichi Shimizu3* and
Xiaopeng Shao1,5* This study reviews the recent advances in data-driven polarimetric imaging technologies based on a wide range of prac-
tical applications. The widespread international research and activity in polarimetric imaging techniques demonstrate
their broad applications and interest. Polarization information is increasingly incorporated into convolutional neural net-
works (CNN) as a supplemental feature of objects to improve performance in computer vision task applications. Polarimetric imaging and deep learning can extract abundant information to address various challenges. Therefore, this
article briefly reviews recent developments in data-driven polarimetric imaging, including polarimetric descattering, 3D
imaging, reflection removal, target detection, and biomedical imaging. Furthermore, we synthetically analyze the input,
datasets, and loss functions and list the existing datasets and loss functions with an evaluation of their advantages and
disadvantages. We also highlight the significance of data-driven polarimetric imaging in future research and develop-
ment. Keywords: deep learning; polarimetric imaging; image processing Yang K, Liu F, Liang SY et al. Data-driven polarimetric imaging: a review. Opto-Electron Sci 3, 230042 (2024). Yang K, Liu F, Liang SY et al. Data-driven polarimetric imaging: a review. Opto-Electron Sci 3, 230042 (2024) 1School of Optoelectronic Engineering, Xidian University, Xi’an 710071, China; 2School of Physics, Xidian University, Xi’an 710071, China;
3Graduate School of Information, Production and Systems, Waseda University, Kitakyushu 808-0135, Japan; 4Department of Mechanical
Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; 5Hangzhou Institute of Technology, Xidian University,
Hangzhou 311200, China.
†These authors contributed equally to this work.
*Correspondence: F Liu, E-mail: feiliu@xidian.edu.cn; K Shimizu, E-mail: shimizu@xidian.edu.cn;
XP Shao, E-mail: xpshao@xidian.edu.cn
Received: 14 November 2023; Accepted: 19 January 2024; Published online: 29 February 2024
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License.
To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
© The Author(s) 2024. Published by Institute of Optics and Electronics, Chinese Academy of Sciences.
230042-1 †These authors contributed equally to this work.
*Correspondence: F Liu, E-mail: feiliu@xidian.edu.cn; K Shimizu, E-mail: shimizu@xidian.edu.cn;
XP Shao, E-mail: xpshao@xidian.edu.cn
Received: 14 November 2023; Accepted: 19 January 2024; Published online: 29 February 2024
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License.
To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
© The Author(s) 2024. Published by Institute of Optics and Electronics, Chinese Academy of Sciences.
230042-1 Crosstalk-free achromatic full Stokes imaging polarimetry metasurface enabled by polarization-dependent
phase optimization Crosstalk-free achromatic full Stokes imaging polarimetry metasurface enabled by polarization-dependent
phase optimization Yaxin Zhang, Mingbo Pu, Jinjin Jin, Xinjian Lu, Yinghui Guo, Jixiang Cai, Fei Zhang, Yingli Ha, Qiong He, Mingfeng Xu, Xiong Li, Xiaoliang
Ma, Xiangang Luo http://www.oejournal.org/oes Review
February 2024, Vol. 3, No. 2 Review Short history Polarimetric imaging, first observed by Sir Isaac Newton
and Christiaan Huygens in the 17th century, is a tech-
nique used to capture and analyze the polarization prop-
erties of light. Moreover, they noticed that light could be
separated into two polarized lights when interacting with
calcite crystals. In the 19th century, scientists such as
Malus and Fresnel made significant contributions to the
understanding of polarization. Since its inception, polari-
metric imaging has progressed and been applied in three
essential fields in the 20th century: polarization micro-
scopy, which allows scientists to study the birefringence
properties of materials under polarized light, remote
sensing and polarimetry, enhancing the detection and
discrimination of objects based on their polarization
characteristics; medical imaging; and tissue optical prop-
erties. In recent years, digital cameras and other innovat-
ive imaging techniques have become capable of captur-
ing valuable information on surfaces, materials, and bio-
logical samples. Recent developments in computer vis-
ion have enabled the incorporation of polarization in-
formation into various tasks, offering potential advant-
ages over traditional RGB imaging. Deep learning, a subfield of machine learning, is in-
spired by the human brain. Artificial neural networks
emerged during the 1940s and 1950s. Research on neur-
al networks continued during the 1960s and 1980s, and
perceptron and backpropagation algorithms were de-
veloped. However, neural networks face limitations ow-
ing to computational constraints and insufficient data. In
the early 2000s, traditional machine learning techniques,
such as support vector machines and decision trees, out-
performed deep learning in many tasks, leading to a de-
cline in interest in deep learning. After 2010, the propos-
al of a new network structure and improvements in com-
putational power propelled the advancement of deep
learning. Key developments during this decade include
the use of CNN for computer vision and recurrent neur-
al networks for natural language processing. With ongo-
ing research, deep learning continues to evolve into more
efficient training algorithms, model architectures, and
applications across various domains, making it a central
part of the AI field of artificial intelligence. The remainder of this study is organized as follows:
First, we review the recent trends in data-driven polari-
metric imaging from four aspects. Second, seven exist-
ing research fields are categorized and analyzed with
their corresponding algorithms. Third, we discuss vari-
ous algorithmic approaches used in each field. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 theory and practice37−41. To overcome the lack of a physical theoretical model,
the use of deep learning technology to optimize the in-
formation processing procedure has been proposed. Sev-
eral experiments have proven its advantages in extract-
ing potential features from images and determining the
relationship between information transmission systems
based on massive datasets. However, intensity informa-
tion is commonly used in existing data-driven methods,
which lose other three-dimensional information. In ad-
dition, the unicity of information would make the ima-
ging performance suffer from various challenges; for ex-
ample, in target detection tasks, spoofing targets and
camouflaged targets can reduce the accuracy rate; in se-
mantic segmentation, water hazards and metallic sur-
faces are the key challenges in the segmentation of road
scenes; in pathological diagnosis, the only color informa-
tion in medical images would increase the risk of misdia-
gnosis; and for transparent objects, parts of imaging pro-
cesses would be challenging to implement, such as 3D re-
construction and segmentation in the intensity field. Therefore, the introduction of additional light-field
physical information, such as polarization, is gaining in-
creasing attention as a supplemental feature of objects
for improved performance in higher-level visual tasks,
expanding beyond intensity-only coverage. Therefore,
polarimetric imaging and deep learning will contribute
to future research and development. This review sum-
marizes the existing methods of combining polarimetric
imaging and deep learning and demonstrates the cur-
rent gains visually and comprehensively. Introduction gnosis24−29, remote sensing30−33, and semantic segmenta-
tion34−36. Furthermore, harnessing the features of polariz-
ation opens new possibilities for research and techno-
logy development. Polarization is a fundamental physical property of light
that expresses the characteristics of vector shear wave1,2. When light interacts with objects or media, it exhibits
various characteristics and representations of polariza-
tion that correspond to the intrinsic characteristics of the
material. This unique quality is an additional dimension
of information that has many applications in various
fields, such as polarimetric descattering3−9, 3D shape re-
construction10−13,
reflection
removal14,15,
target
detection16−20, biomedical imaging21−23, pathological dia- The conventional polarization method is limited in ac-
curately capturing and utilizing information owing to
complicated interactions in the transmission process. Moreover, convolutional neural networks (CNN) excel
at nonlinear expression and information extraction
based on large datasets, making them better suited for
modeling and interpreting polarization information than
traditional algorithms, which can bridge the gap between Short history Next, we
discuss three critical aspects of their existing datasets and
loss functions and their advantages and disadvantages. Next, we discuss the strengths and weaknesses of the
practical applications of data-driven polarimetric ima-
ging and possible opportunities to address these chal-
lenges. Finally, we provide conclusions, highlighting the
importance and potential of data-driven polarimetric
imaging in various applications and research areas that
could extend the current uses of data-driven polarimet-
ric imaging and provide insights to advance its future
development. Until the 21st century, the combination of deep learn-
ing and polarimetric imaging has become more pro-
nounced. In the mid-2010s, data-driven techniques were 230042-2 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 applied to polarimetric imaging. Machines and deep
learning have been used for tasks, including object recog-
nition in polarimetric images. Subsequently, researchers
explored the potential of deep learning for processing
and analyzing polarimetric data in more diverse do-
mains because deep learning helps extract valuable in-
formation from polarization data. Additionally, the syn-
ergy between deep learning and polarimetric imaging
continues to evolve with advancements in models, al-
gorithms, and applications, forming the significant field
called “data-driven polarimetric imaging”, which is the
novel method that combines the learning method and
polarimetric imaging. Data-driven polarimetric imaging
can revolutionize various fields and represents a prom-
ising interdisciplinary research area and technological
innovation. A diagram of this short history is shown in
Fig. 1. With the exploration of data-driven polarimetric ima-
ging, the application fields and utilization of polariza-
tion information have gradually increased. Regarding the
input and use of polarization information, directly cap-
tured images from detectors, such as division of focal
plane (DoFP) images42−45 or 0°, 45°, 90°, and 135°46−52 po-
larization images, are the most common inputs. It should
be noted that, despite having four components, the last
one is often neglected in practical applications, leading to
the predominant use of only the initial three parameters. This is because, theoretically, the last parameter can be
derived from the first three, thereby reducing the diffi-
culty in image acquisition. In addition, polarimetric
parameter features were calculated to represent the po-
larization information more intuitively, such as the de-
gree
of
polarization
(DoP)
and
angle
of
polarization(AoP)52−57, [S0, S1, S2]52,55,58,59, Mueller matrix
images60−63 and other combinations of these elements. Short history Features based on the corresponding physical model
during different tasks were also computed to guide net-
work training, such as zenith and azimuth angle maps
derived from specular and diffuse reflection64,65 in 3D
shape reconstruction tasks. With the exploration of data-driven polarimetric ima-
ging, the application fields and utilization of polariza-
tion information have gradually increased. Regarding the
input and use of polarization information, directly cap-
tured images from detectors, such as division of focal
plane (DoFP) images42−45 or 0°, 45°, 90°, and 135°46−52 po-
larization images, are the most common inputs. It should
be noted that, despite having four components, the last
one is often neglected in practical applications, leading to
the predominant use of only the initial three parameters. Trends Data-driven polarimetric imaging is a novel approach
aimed at compensating for the defects and difficulties of
a single-information interpretation model. Recently, the
advantages of combining polarimetric imaging and deep
learning have been applied in several fields, and dozens
of algorithms based on data-driven polarimetric ima-
ging have been proposed, covering several application
areas. This section describes more recent trends in data-
driven polarimetric imaging from four perspectives. Schematics of the trends in existing data-driven polari-
metric imaging are shown in Fig. 2. Furthermore, physical modes are crucial during net-
work training, such as polarimetric descattering models,
three-dimensional imaging models, Fresnel equations,
Mueller matrix interpretation models, and other tradi-
tional polarization models. The preliminary methods are
often end-to-end architectures43,46,47,56,64,66−70, suggesting
that the polarization information is input into the 17th century
19th century
20th century
21th century
More valuable
information
Mid-2010s
Birefringence of
calcite crystals
Malus law
Fresnel laws
Detector
Primary
phenomena
Proposal of theory
1940s-1950s
1960s-1980s
2010s
2010s
Neure
Perceptron
Backpropagation
algorithm
Advances of
hardware
Applications
Data-driven
polarization
imaging
Extraction of
potential features
Computer
Fig. 1 | Schematic of short history about data-driven polarimetric imaging. 230042-3 21th century
More valuable
information 17th century
Birefringence of
calcite crystals Malus law Detector Fresnel laws Data-driven
polarization
imaging Fig. 1 | Schematic of short history about data-driven polarimetric imaging. 230042-3 Yang K et al. Opto-Electron Sci 3, 230042 (2024)
https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 From raw polarimetric images to preprocessing features
DoFP image
[S0, S1, S2]
[S0, DoP, AoP]
Polarimetric
sub-images
Mueller matrix
images
Mueller matrix
parameters maps
Physical priors
Epoch
Loss
Epoch
Loss
Epoch
Loss
Epoch
Loss
Other features
Network
training
From end-to-end architecture to physical-model-combined structure
End-to-end network training
Physical
formulation
Physical
formulation
Physical-model-guided network training
Physical-model-integrated network training
Network
output
Network
input
Epoch
Loss
From single to multi physical properties
Polarimetric
information
Intensity
information
Pre-trained
network maps
Spectral
information
Phase
information
Speckle field
information
Network
training
From image processing to semantic tasks
Image
enhancement
Semantic
segmentation
Target detection
lmage
reconstruction
Biomedical
imaging
a
d
b
c
2 | Schematics of the trends of existing data-driven polarimetric imaging. The solid black arrows refer to the data flow, the dotted bla
ws refer to that the data may or not flow. The gradient blue arrows mean the trends of corresponding aspects. Trends (a) The utilization of polarim
nformation has been gradually deepened from raw polarimetric images to preprocessing features. (b) The physical modes have been cruc
ng the training of network from end-to-end architecture to physical-model-combined structure. (c) The various physical properties have be
ually fused to network. (d) The tasks have been developed from image processing to semantic tasks. Yang K et al. Opto-Electron Sci 3, 230042 (2024)
https://doi.org/10.29026/oes.2024.230042 From raw polarimetric images to preprocessing features
DoFP image
[S0, S1, S2]
[S0, DoP, AoP]
Polarimetric
sub-images
Mueller matrix
images
Mueller matrix
parameters maps
Physical priors
Epoch
Loss
Other features
Network
training
a a Epoch
Loss
Epoch
Loss
Epoch
Loss
From end-to-end architecture to physical-model-combined structure
End-to-end network training
Physical
formulation
Physical
formulation
Physical-model-guided network training
Physical-model-integrated network training
Network
output
Network
input
b From end-to-end architecture to physical-model-combined structure b Physical
formulation Physical
formulation Ep
Physical-model-integrated network training Ep
Physical-model-integrated network training Physical model integrated network training
Epoch
Loss
From single to multi physical properties
Polarimetric
information
Intensity
information
Pre-trained
network maps
Spectral
information
Phase
information
Speckle field
information
Network
training
From image processing to semantic tasks
Image
enhancement
Semantic
segmentation
Target detection
lmage
reconstruction
Biomedical
imaging
d
c From single to multi physical properties
c Phase
information Intensity
information Fig. 2 | Schematics of the trends of existing data-driven polarimetric imaging. The solid black arrows refer to the data flow, the dotted black
arrows refer to that the data may or not flow. The gradient blue arrows mean the trends of corresponding aspects. (a) The utilization of polarimet-
ric information has been gradually deepened from raw polarimetric images to preprocessing features. (b) The physical modes have been crucial
during the training of network from end-to-end architecture to physical-model-combined structure. (c) The various physical properties have been
gradually fused to network. (d) The tasks have been developed from image processing to semantic tasks. 230042-4 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) network to generate the desired outputs directly;
however, the physical models are gradually guided46,47,49,71
or integrated into network training48,72−74. Furthermore,
the physical model and its inverse process can form a
self-supervised closed loop to achieve better perform-
ance. Furthermore, data-driven polarimetric imaging en-
ables the physical interpretability of networks compared
with conventional deep network methods. Polarimetric
parameters were initially adopted to perform different
tasks without exploring the hidden mechanisms. Trends In more
in-depth research on data-driven polarimetric imaging,
the estimation of specific physical parameters based on
the nonlinear representation of deep networks36,58 and
the physical interpretation of network layers63 have been
gradually studied. This process contributes to future re-
search and development. Based on the polarimetric in-
formation fused into the network, other physical proper-
ties of light have also been introduced in network train-
ing, such as spectrum172 and phase71,75. Finally, the applic-
ation fields tend to be semantic tasks based on convolu-
tional enhancement or restoration imaging. Initially, im-
age processing was the main field applied by data-driven
polarimetric
imaging,
such
as
descattering
imaging46−49,66,70,72,76, denoising45,77,78, demosaicing43,44,68,
dynamic range enhancement79, reflection removal53,73,74,
low-light imaging42,50, and even 3D reconstruction
shape64,65,67,71,80,81. Next, semantic tasks appeared gradu-
ally similar to semantic segmentation56,57,69,82,83, camou-
flage object detection84, classification60,61, pathological
diagnosis62,63,85,86,87. larization parameters of the nonlinear operators are
sensitive to noise. Consequently, the effective restora-
tion and enhancement of polarization information are
crucial for subsequent applications. In this section, we
analyze the limitations of polarimetry techniques and re-
view the existing restoration and enhancement of accur-
ate polarization information methods. Restoration and enhancement of accurate After capturing the images using the aforementioned
polarimetric techniques, Stokes vectors were adopted to
display the polarization characteristics. The relationship
between them can be characterized by the follow
equation: Applications of data-driven polarimetric
imaging According to the application field, existing data-driven
polarimetric imaging methods can be classified into sev-
en categories: polarimetric descattering, 3D shape recon-
struction, reflection removal, restoration and enhance-
ment of polarization information, target detection, bio-
medical imaging and pathological diagnosis, and se-
mantic segmentation, as shown in Fig. 3. Polarimetry techniques Polarimetry techniques are crucial in obtaining polariza-
tion information. Several sub-polarized direction images
(0°, 45°, 90°, and 135° or 0°, 60°, and 120°) must be cap-
tured to obtain the polarization characteristics based on
the Stokes vector model. However, the obtaining and cal-
culating process, which acquires polarization informa-
tion indirectly, introduces extra imaging noise to sharply
reduce the accuracy of the polarization information. To
date, there are four typical methods for measuring polar-
ization images: division of time/rotating elements, divi-
sion of amplitude88−90, division of aperture91,92, and divi-
sion of the focal plane93−95. The division of time/rotating elements is the most
common method, depending on the time-sequential
activity. The polarizers and retarders were rotated, and
measurements were made at different positions of the
polarimetry elements. Furthermore, the time gap
between operations may cause misregistration of polariz-
ation images in a dynamic scene or the motion of the
camera case. The division of amplitude could capture
multiple images simultaneously; however, the inherent
drawback is that the intensity of images would decrease
to less than a quarter of the intensity of the original sig-
nal, which causes the image contrast to decrease sharply
and amplify the image noise. Moreover, misregistration
must be solved. The aperture captures images simultan-
eously using four coaxial cameras with different polariza-
tion directions. Distinctly, it is expensive and has a fixed
misregistration. The division of the focal plane is the
method used in real-time polarimetric imaging, even in
dynamic scenes. However, instantaneous field-of-view
errors (i.e., mosaicking and low-resolution problems) af-
fect the calculation of polarization parameters. Table 1
presents a comparison of the polarimetric elements. polarization information Accurate polarization information is the foundation of
imaging and its applications. In the real world, theoretic-
al constraints and technological limitations lead to dis-
tortion of polarization information. Additionally, the po- 230042-5 230042-5 Yang K et al. Opto-Electron Sci 3, 230042 (2024)
https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Pathological diagnosis
Species classification
Spoofing target
Camouflaged target
Pseudo-color target
Target
detection
Biomedical imaging and
pathological diagnosis
Data-driven
polarimetric imaging
Semantic
segmentation
Polarimetric
descattering
3D shape
reconstruction
Reflection
removal
Polarization
channels
Hypers-
pectral
imaging
Remote sensing
Medicine
Transparent object
Scene in wild
Underwater
descattering
Dehazing
Remote sensing
Indoor object
Scene in wild
Transparent
object
Human face
MMPD
Holographic
amplitude &
phase
Stokes
vectors
Low-light
imaging
Restoration and
enhancement of
polarization
information
HDR reconstruction
MMPD denoising
Polarimetric
parameters
denoising
Demosaicing
lndoor & outdoor
Real & synthetic
Fig. 3 | Applications of data-driven polarimetric imaging. Holographic
li
d
& Stokes
vectors MMPD denoising Fig. 3 | Applications of data-driven polarimetric imaging. P =
√
S2
1 + S2
2 + S2
3
S0
, PL =
√
S2
1 + S2
2
S0
,
PC = S3
S0
, ϕpol = 1
2arctan
(S2
S1
)
,
(3) Iφ = (S0 + S1cos2φ + S2sin2φ)/2 ,
(1) Iφ = (S0 + S1cos2φ + S2sin2φ)/2 ,
(1) (1) where Iφ is the image with the polarization direction φ. Then, the Stokes vectors can be calculated from the sub-
polarization direction images96,97: (3) ϕpol
where P, PL, PC are DoP, degree of linear polarization
(DoLP), degree of circular polarization (DoCP), respect-
ively. is the AoP. S0 = 1
2 (I0 + I45 + I90 + I135) = 2
3 (I0 + I60 + I120) ,
S1 = I0 −I90 = 4
3
(
I0 −1
2I60 −1
2I120
)
,
S2 = I45 −I135 = 2
√
3
3
(I0 −I120) ,
S3 = I45, π
2 −I135, π
2 = 2
√
3
3
(
I0, π
2 −I120, π
2
)
,
(2 Furthermore, the Mueller matrix is another common
but comprehensive parameter that describes the modula-
tion of light after interaction with a material or
media98−100. The Mueller matrix contained 16 elements
using a 4×4 matrix. https://doi.org/10.29026/oes.2024.230042 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) Table 1 | Comparisons of various polarimetric elements. Advantages
Disadvantages
Division of time
● Robust
● Small system size and inexpensive
● Easiest to implement
● Not suitable for dynamic scenes
● Not simultaneously imaging
● Probable linear misregistration
Division of amplitude
● Simultaneous imaging
● Largest system size
● Expensive and complex
● Nonlinear misregistration
● Loss of image intensity
● Highly mechanical flexibility required
Division of aperture
● Simultaneous imaging
● Robust
● Expensive
● Loss of field of view
● Fixed misregistration
Division of focal plane
● Simultaneous imaging
● No misregistration
● Small and portable
● Loss of spatial resolution
● Mosaicking problem
● Fabrication difficult
● Expensive Table 1 | Comparisons of various polarimetric elements. Advantages
Disadvantages
Division of time
● Robust
● Small system size and inexpensive
● Easiest to implement
● Not suitable for dynamic scenes
● Not simultaneously imaging
● Probable linear misregistration
Division of amplitude
● Simultaneous imaging
● Largest system size
● Expensive and complex
● Nonlinear misregistration
● Loss of image intensity
● Highly mechanical flexibility required
Division of aperture
● Simultaneous imaging
● Robust
● Expensive
● Loss of field of view
● Fixed misregistration
Division of focal plane
● Simultaneous imaging
● No misregistration
● Small and portable
● Loss of spatial resolution
● Mosaicking problem
● Fabrication difficult
● Expensive the other 15 elements encode the vectorial properties of
the object. Furthermore, Mueller matrix polar decom-
position (MMPD)101, Mueller matrix transformation
(MMT)102, Mueller matrix anisotropy coefficients
(MMAC)103, and other decompositions of the Mueller
matrix104−106 have been proposed to quantitatively charac-
terize the properties of an object. tion parameters amplify errors and negatively affect sub-
sequent imaging performance. tion parameters amplify errors and negatively affect sub-
sequent imaging performance. In summary, the acquisition device for raw sub-polar-
ization images, the use of nonlinear operators to calcu-
late polarization characteristic parameters, and special
imaging environments also decrease the inaccuracy of
the polarization information. Therefore, polarization in-
formation with high accuracy must be restored and en-
hanced. In this section, we examine photostarvation, mo-
saicking, and noising to demonstrate the restoration and
enhancement of polarization information, as depicted in
Fig. 4. The estimation of these parameters, which represent
different polarizations, is always based on nonlinear op-
erations, which are unavoidable when introducing er-
rors and noise. https://doi.org/10.29026/oes.2024.230042 To achieve the desired performance, the
calculated polarization parameters must be refined or re-
stored to obtain highly accurate information. In addi-
tion, obtaining precise polarization information can be
challenging when operating in special imaging environ-
ments, such as low light and noise, or with special ima-
ging devices. In these scenarios, the disturbed polariza- polarization information The Stokes vector of the output light
Sout can be expressed by the Mueller matrix after the in-
cident light Sin propagates through the medium as follows: (2) where S0, S1, S2, S3 are the components of Stokes vectors. The parameters [S0, S1, S2] are the most common repres-
entations of the linear polarization components. S3
shows the circular polarization component. Therefore,
the polarization characteristic parameters can be ob-
tained using Eq. (3):
Sout
0
Sout
1
Sout
2
Sout
3
=
m00
m01
m02
m03
m10
m11
m12
m13
m20
m21
m22
m23
m30
m31
m32
m33
Sin
0
Sin
1
Sin
2
Sin
3
, (4) where m00 represents the transformation of intensity and where m00 represents the transformation of intensity and 230042-6 information methods In photostarved environments, imaging always suffers Demosaicing
Holographic
amplitude &
phase
Stokes
vectors
Low-light imaging
MMPD
images
Muller matrix denoising
HDR reconstruction
Polarimetric parameters
denoising
Polarization
channels
L1
S1
S2
S3
25 μm
0°
45°
135°
90°
Fig. 4 | The considered tasks of restoration and enhancement of polarization information, including low-light imaging, Muller matrix de-
noising, high dynamic range (HDR) reconstruction, polarimetric parameters denoising, demosaicing, transformation between holo-
graphic amplitude & phase and polarization channels, transformation between Stokes vectors and MMPD images. 230042-7 D
i i Holographic
lit d
& Fig. 4 | The considered tasks of restoration and enhancement of polarization information, including low-light imaging, Muller matrix de-
noising, high dynamic range (HDR) reconstruction, polarimetric parameters denoising, demosaicing, transformation between holo-
graphic amplitude & phase and polarization channels, transformation between Stokes vectors and MMPD images. 230042-7 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) from a low signal-to-noise ratio, which affects the ima-
ging quality, making low-light imaging challenging. In
polarimetric imaging, the accuracy of polarization in-
formation is degraded. Existing methods consider de-
noising, correction of color bias, and exposure time in
intensity imaging107,108. However, the difference between
polarimetric and conventional intensity imaging has not
been fully considered. Based on the power of being data
driven, Hu et al. presented a one-to-three (intensity,
DoLP, and AoP) hybrid network called IPLNet to simul-
taneously enhance the image quality of intensity and po-
larization information42, as shown in Fig. 5(a). The en-
hanced RGB image generated by the chromatic RGB
subnetwork is divided into three channels, and each
channel is fed into the polarization subnetwork to pre-
dict the polarization information. Enhanced results and
corresponding comparisons with mainstream methods
are shown in Fig. 5(b) with corresponding values of
structural similarity index (SSIM) of each image. However, many parameters are generated in this net-
work, which sharply reduces the operational efficiency. The image color is also inaccurate. Therefore, Xu et al. first performed initial denoising and color deviation cor-
rection of four polarization orientation images by net-
work named ColorPolarNet and used the polarization
difference network to enhance intensity details, DoLP,
and AoP maps50. The results demonstrate that the pro-
posed methods have faster processing speed and better
performance regarding signal fidelity, contrast enhance-
ment, and color reproduction50,109. information methods Compared with IPL-
Net, ColorPolarNet demonstrates slightly superior per- formance and achieves higher peak signal-to-noise ratio
(PSNR), SSIM and patch-based contrast quality index
(PCQI) in terms of S0, DoLP, and AoP. Additionally,
ColorPolarNet achieves notably lower color difference
(CD), indicating reduced distortion compared to IPLNet. In processing speed, ColorPolarNet (2.88 s) is more than
twice as fast as the IPLNet model (6.10 s). Division of the focal plane polarimeter is one of the
most common polarimetric imaging sensors that can in-
stantaneously capture dynamic polarization information. Each individual pixel with different polarization orienta-
tions is situated in a 2×2 superpixel, recording only one
from the four essential intensity measurements; there-
fore, demosaicing and reconstruction of full-resolution
and accurate polarization information are indispensable. Zhang et al. proposed a convolutional demosaicing net-
work called the PDCNN to learn end-to-end mapping
between coarse interpolation results and full-resolution
polarization images68, which is the first typical demosa-
icing architecture, as shown in Fig. 6(a). The bicubic-in-
terpolated results are used as the input, which intro-
duces an interpolation bias, resulting in inaccurate re-
constructed results. After comparison with several main-
stream methods, the reconstruction results of DoLP and
AoP outperformed those of other methods, as shown in
Fig. 6(b) and 6(c) with corresponding values of PSNR of
each image110−113. Zeng et al. proposed a four-layer end-
to-end fully convolutional neural network that directly
learns mapping from DoFP to three polarization proper-
ties: intensity, DoLP, and AoP43. However, the noise in
the AoP images remained significant. Wu et al. provided R
G
B
Polar-Net
RGB-Net
S0
DoLP
AoP
S0
DoLP
AoP
Detail
Residual dense network
Residual dense block (RDB)
Convolutional layer
Concatenate layer
0
0
0.50
1 −π/2
π/2
SSIM=0.66
SSIM=0.24
SSIM=0.15
SSIM=0.86
SSIM=0.62
SSIM=0.40
SSIM=0.71
SSIM=0.61
SSIM=0.36
SSIM=0.91
SSIM=0.71
SSIM=0.44
SSIM=0.95
SSIM=0.76
SSIM=0.48
lPLNet
CBM3D
SIDNet
ColorPolarNet
lnput
a
b
Fig. 5 | Enhanced method of polarization information. (a) Architecture of the Hu et al.’s method. (b) Enhanced results and corresponding
comparisons with mainstream methods. Figure reproduced with permission from ref.42, Optical Society of America Publishing AG. 230042-8 S0
DoLP
AoP
Detail
0
0
0.50
1 −π/2
π/2
SSIM=0.66
SSIM=0.24
SSIM=0.15
SSIM=0.86
SSIM=0.62
SSIM=0.40
SSIM=0.71
SSIM=0.61
SSIM=0.36
SSIM=0.91
SSIM=0.71
SSIM=0.44
SSIM=0.95
SSIM=0.76
SSIM=0.48
th d (b) E h
d
lt
d
di a Fig. 5 | Enhanced method of polarization information. (a) Architecture of the Hu et al.’s method. information methods (b) Enhanced results and corresponding
comparisons with mainstream methods. Figure reproduced with permission from ref.42, Optical Society of America Publishing AG. 230042-8 Yang K et al. Opto-Electron Sci 3, 230042 (2024)
https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 PSNR=37.23 dB
PSNR=36.59 dB
PSNR=36.95 dB
PSNR=42.85 dB
Ground truth
PSNR=40.36 dB
PSNR=25.34 dB
PSNR=22.95 dB
PSNR=25.27 dB
PSNR=30.61 dB
Ground truth
PSNR=24.31 dB
Block
Block
Block
Block
Block
Block
Conv
Concatenation
Input
OutputN
Output2
Output1
Output
Skip connection
S0
S1
S2
Stokes
layer
a
b
b1
b2
b3
b4
b5
b6
c1
c2
c3
c4
c5
c6
c
Fig. 6 | The convolutional demosaicing network proposed by Zhang et al. and results68. (a) Architecture of the Zhang et al. method. (b) Re-
constructed DoLP images of different methods on the test images. (b1) The results of method113. (b2) The results of method110. (b3) The results of
method111. (b4) The results of method112. (b5) and (b6) are respectively PDCNN and ground truth. (c) Reconstructed AoP images of different
methods on the test images. (c5) and (c6) are respectively PDCNN and ground truth. Figure reproduced with permission from ref.68, Optical Soci-
ety of America Publishing AG. Fig. 6 | The convolutional demosaicing network proposed by Zhang et al. and results68. (a) Architecture of the Zhang et al. method. (b) Re-
constructed DoLP images of different methods on the test images. (b1) The results of method113. (b2) The results of method110. (b3) The results of
method111. (b4) The results of method112. (b5) and (b6) are respectively PDCNN and ground truth. (c) Reconstructed AoP images of different
methods on the test images. (c5) and (c6) are respectively PDCNN and ground truth. Figure reproduced with permission from ref.68, Optical Soci-
ety of America Publishing AG. quality45. However, all channel-wise features are treated
equally, resulting in a lack of flexibility. Inspired by the
attention mechanism, Liu et al. proposed an attention-
based residual neural network to remove noise and re-
store the polarization information of polarimetric im-
ages77, as Fig. 7. Therefore, the proposed methods can
suppress noise more effectively and restore polarization
information more accurately, as shown in Fig. 7(b)109. Additionally, SSIM is used to compare the quality of im-
ages obtained by different methods. Focusing on the de-
noising of Mueller matrix images, Yang et al. information methods The intensity image
describes the reflectivity and transmissivity of the object,
whereas the polarization image describes the texture de-
tails, material properties, shape, shading, and roughness. These two types of images provide complementary in-
formation from different aspects to obtain images with
rich physical features and improve the performance of
practical tasks. Conventional fusion methods are challen-
ging to handle various scenes because a manually de-
signed fused factor is adopted. Based on the excellent
performance of CNN, Zhang et al. proposed an unsuper-
vised deep network called PFNet to fuse the intensity and
DoLP images126. The feature extraction module trans-
forms the images of S0 and DoLP into high-dimensional
nonlinear feature maps using two Dense Blocks, and the
concatenation operator is used to fuse the feature maps
to reconstruct the fused image using the reconstruction
module. The architecture of PFNet is shown in Fig. 8(a). Therefore, the method based on deep learning outper-
forms other state-of-the-art methods, as shown in Fig. 8(b)122−125. SSIM is used to compare the quality of images
obtained by different methods. Contrarily, they modi-
fied the architecture to enhance performance. A Dense
Block is used to encode the input images and the fusion
subnetwork rather than a concatenation operator to fuse
the feature maps127. New loss function strategies are ad-
opted, such as the loss between fused and input images
and the loss between fused and encoded features. The
proposed architecture can also be used for infrared and
visible image fusion, and multi-focus image fusion. the camera, a digital camera always captures only a lim-
ited fraction of the range, resulting in low-dynamic-
range images with over- or underexposed areas that can-
not reflect real-world scenes in high-dynamic-range im-
ages128,129. Ting et al. studied the relationship between po-
larization parameters and the exposure time of a pixel in
a polarization image and trained the reconstruction
framework to recover a high-dynamic-range image us-
ing polarization images79. Other applications of restoration and enhancement
are based on data-driven polarimetric imaging. The po-
larization parameters and other physical properties are
interconvertible. Liu et al. used a deep neural network to
transform holographic images reconstructed from a
single state of polarization into images equivalent to
those captured using a single-shot computational polar-
ized light microscope75. Si et al. information methods built a deep
residual U-Net that incorporated channel attention with
many paired low- and high-SNR Mueller matrix
images78. The ground truth is obtained based on the low
equally weighted variance (EWV), which can be ex-
pressed as: a more physically relevant loss function for the angle of
linear polarization (AoLP) reconstruction, establishing a
two-stage lightweight approach for reconstructing the
intensity and polarization information in real time44. The
improved version meets the demand for real-time infer-
ence. Wen et al.114, Sargent et al.115, Sun et al.116 and Pis-
tellato et al.117 also proposed the data-driven demosa-
icing methods to ensure the fidelity of polarization signa-
tures and enhance image resolution. Besides deep learn-
ing-based methods, other approaches also yield favor-
able results in this domain, such as the sparse tensor fac-
torization-based model, which introduces a combina-
tion of tensor factorization and sparse coding for the first
time118. Because the polarimetric parameters are always de-
rived from the measured intensities through nonlinear
operators, which would amplify the noise for the AoP,
removing noise to precisely restore the polarization in-
formation is a significant task107,119,120. CNN have distinct
advantages regarding extracting image features and hid-
den structures; thus, they are suitable for image restora-
tion and enhancement in noisy environments. Li et al. employed deep neural networks to significantly suppress
the noise in polarimetric images and enhance the image EWV = 220
N
(
σ2 + m00
4
)
,
(5) (5) σ2
where
is the variance of Gaussian noise and N denotes
the number of states of polarization. The larger the value
of N, the higher is the signal-to-noise ratio. The pro-
posed method can effectively resolve the conflict between
the measurement accuracy and acquisition time. The fused images contain more information than The fused images contain more information than 230042-9 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Fd
Fd
ωFd
ω
δ
σ
ˆ
ˆ
ˆ
ω
CARDB d−1 Channel attention
Channel attention
Global residual learning
1
2
d
d
d−1
d−2
Local residual learning
Concatenation
(0°,45°,90°,135°)
(0°,45°,90°,135°)
DoLP
DoLP
AoP
AoP
3D Conv (5×3×3)
Reshape
2D Conv (3×3)
Concatenation
2D Conv (1×1)
Global pooling
Dense
SSIM=0.60 SSIM=0.12 SSIM=0.05
SSIM=0.89 SSIM=0.77 SSIM=0.16
SSIM=0.97 SSIM=0.81 SSIM=0.22
SSIM=0.92 SSIM=0.84 SSIM=0.25
Ours
GT
BM3D
PDRDN
lnput
a
b
S0
Sample A: polyresin statue
=
Rescale
Fig. 7 | Liu et al. method. (a) Architecture of the proposed method. information methods fed Stokes images to a
well-designed deep learning network to generate Mueller
matrix-based parameter images, such as linear retard-
ance and diattenuation parameters58. information methods (b) Enhanced results and corresponding comparisons with mainstream
methods. Figure reproduced with permission from ref.77, Optical Society of America Publishing AG. DoLP
AoP
3)
SSIM=0.60 SSIM=0.12 SSIM=0.05
SSIM=0.89 SSIM=0.77 SSIM=0.16
SSIM=0.97 SSIM=0.81 SSIM=0.22
SSIM=0.92 SSIM=0.84 SSIM=0.25
Ours
GT
BM3D
PDRDN
lnput
b
S0
Sample A: polyresin statue Global residual learning Fig. 7 | Liu et al. method. (a) Architecture of the proposed method. (b) Enhanced results and corresponding comparisons with mainstream
methods. Figure reproduced with permission from ref.77, Optical Society of America Publishing AG. single physical properties because the mixed informa-
tion describes various characteristics. The fusion of po-
larization and intensity is the most common method
used in practical applications121−125. The intensity image
describes the reflectivity and transmissivity of the object,
whereas the polarization image describes the texture de-
tails, material properties, shape, shading, and roughness. These two types of images provide complementary in-
formation from different aspects to obtain images with
rich physical features and improve the performance of
practical tasks. Conventional fusion methods are challen-
ging to handle various scenes because a manually de-
signed fused factor is adopted. Based on the excellent
performance of CNN, Zhang et al. proposed an unsuper-
vised deep network called PFNet to fuse the intensity and
DoLP images126. The feature extraction module trans-
forms the images of S0 and DoLP into high-dimensional
nonlinear feature maps using two Dense Blocks, and the
concatenation operator is used to fuse the feature maps
to reconstruct the fused image using the reconstruction
module. The architecture of PFNet is shown in Fig. 8(a). Therefore, the method based on deep learning outper-
forms other state-of-the-art methods, as shown in Fig. 8(b)122−125. SSIM is used to compare the quality of images
obtained by different methods. Contrarily, they modi-
fied the architecture to enhance performance. A Dense
Block is used to encode the input images and the fusion
subnetwork rather than a concatenation operator to fuse
the feature maps127. New loss function strategies are ad-
opted, such as the loss between fused and input images
and the loss between fused and encoded features. The
proposed architecture can also be used for infrared and
visible image fusion, and multi-focus image fusion. single physical properties because the mixed informa-
tion describes various characteristics. The fusion of po-
larization and intensity is the most common method
used in practical applications121−125. Polarimetric descattering Clear vision in scattering media is critical for various ap-
plications such as industrial and civil fields130, traffic sur-
veillance
systems131,
automatic
drives132,
remote
sensing133, rescue operations134, seabed mapping135, mon-
itoring of marine species migration and coral reefs, and
scene analysis136. However, when capturing images in a
scattered environment, the visibility of objects is typic-
ally sharply degraded, which is caused by the scattering
of suspended particles such as clouds, water, haze,
smoke, smog, fog, and mist in the air, soil particles, float-
ing excrement of marine animals, algae, and mineral salt
in underwater scenes. The backscattered light was mixed Because of the limitations of the response function of 230042-10 Yang K et al. Opto-Electron Sci 3, 230042 (2024)
https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 DoLP
Input
S0
Dense
block
Dense
block
Concatenation
Fusion
Feature
map
Feature extraction
Conv+ReLU
Conv
Reconstruction
Output
Fused image
DoLP
GF
GTF
S0
SR
MSVD
PFNet
CVT
a
b
SSIM=0.672
SSIM=0.676
SSIM=0.701
SSIM=0.712
SSIM=0.661
SSIM=0.673
Fig. 8 | The unsupervised deep network PFNet proposed by Zhang et al.126 (a) The architecture of PFNet. (b) Fused results compared with
conventional methods. Figure reproduced with permission from ref.126, Optical Society of America Publishing AG. Fig. 8 | The unsupervised deep network PFNet proposed by Zhang et al.126 (a) The architecture of PFNet. (b) Fused res
conventional methods. Figure reproduced with permission from ref.126, Optical Society of America Publishing AG. ervised deep network PFNet proposed by Zhang et al.126 (a) The architecture of PFNet. (b) Fused results compared with
ds. Figure reproduced with permission from ref.126, Optical Society of America Publishing AG. with the object signal during its propagation towards the
camera. Because the physical properties of imaging in
haze and underwater environments are similar, we ana-
lyzed the dehazing and descattering processes based on
deep learning and polarimetric imaging. where z is the distance between the object and the cam-
era; β is the degraded coefficient, and Lobject is the origin-
al object signal not attenuated by the scattering media
along the line of sight. The transmitted process e–βz is the
delay of exponential function, which also expressed as t. Second, backscattered light A is an undesired com-
ponent that veils the object light to reduce the contrast of
the image. The backscattered light can be expressed as: Polarimetric imaging model in scattering media Based on the atmospheric transport model137,138, image I
captured by the camera after propagation in scattering
media consists of two components: (1) direct transmis-
sion signal D, which represents the light that an object
reflection is scattered by the media during propagation
towards the camera; and (2) backscattered light A, which
denotes the light backscattered by the particles in the ob-
ject light line of sight without an object signal139. The
model can be expressed as: A = A∞
(
1 −e−βz)
,
(8) (8) where A∞ is the saturated backscattering light as the dis-
tance increases. We aimed to reconstruct the original ob-
ject signal Lobject by combining Eq. (6) and (7). Then, Lob-
ject can be expressed as: Lobject =
I −A
1 −A/A∞
,
(9) (9) I⊥
I∥
as a result, the estimation of two unknown components
A and A∞ is key to reconstructed Lobject. Schechner et al. proposed a polarization descattering model based on an
atmospheric scattering model. The proposed method ob-
tains two orthogonally polarized images,
and
through two orthogonal polarization states of the polar-
izer139. This method assumes that the air light is usually I⊥
I∥
as a result, the estimation of two unknown components
A and A∞ is key to reconstructed Lobject. Schechner et al. proposed a polarization descattering model based on an
atmospheric scattering model. The proposed method ob-
tains two orthogonally polarized images,
and
through two orthogonal polarization states of the polar-
izer139. This method assumes that the air light is usually I = D + A . (6) (6) First, as the light reflected from the object propagates
towards the camera via the medium, the object radiance
suffers from absorption and scattering, yielding a de-
graded signal. This process is described as follows: D = Lobjecte−βz ,
(7) (7) https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) backscattered light by extrapolation fitting to overcome
the first and third assumptions. However, selecting the
fitting function when the light was irregular was challen-
ging142. Wei et al. considered the difference between the
AoP of backscattering light and the object signal, using
independent component analysis (ICA) to estimate the
object signal with nonuniform polarization characterist-
ics to avoid the limitation of assumptions (3) and (4)143. Polarimetric imaging model in scattering media These methods are based on physical models that lack
the robustness and effectiveness of a single method in
complex scenes because accurate estimation of key para-
meters may not be achieved. However, the latent factors
influencing image quality have not been explored. partially polarized, whereas the object is not polarized. Thus, the captured images can be described by: Ii = D
2 + Ai, i ∈[⊥, ∥] . (10) (10) Because the object is not polarized, the DoP of the im-
ages is equal to that of the backscattered light. Further-
more, the backscattered light is estimated using the po-
larization model: ˆA = IP
PA
,
(11) (11) where PA and P are the DoPs of the backscattered light
and captured images, respectively. The saturated backs-
cattering light A∞ is the mean of the background region
selected in the image where there is no object and the PA
is calculated by the selected region which is the DoP of
total backscattering light. Finally, the reconstructed
Lobject can be expressed as: Deep learning methods based on CNN are adept at ex-
tracting hidden features and fitting the nonlinear rela-
tionships between backscattering light and object signals. Thus, it is a promising choice for descattering and dehaz-
ing. Polarimetric data-driven descattering methods com-
bine polarization information and deep learning. Moreover, the existing model can guide the training of
the descattering network to combine its advantages. Three different pipelines were used in the data-driven
polarization descattering method. First, the end-to-end
architecture without the physical model, which always
uses the polarization images to feed into the network. Second, the physical-model-guided network methods,
which are guided by the existing or proposed but non-
participation in network training; Third, the physical-
model-integrated network methods, which integrate the
physical model into the network to train the descattering
network together. The following section provides a de-
tailed explanation of these three perspectives. Lobject =
I −A
1 −A/A∞
=
I (1 −P)
1 −IP/A∞PA
. (12) (12) A polarimetric imaging model is a physical, low-cost,
and effective method for restoring clear images in a scat-
tering environment. However, the method is based on
the following assumptions: 1) The backscattered light at infinity is uniform, but in
the real world, clouds, the solar radiation angle, and oth-
er factors may influence the distribution. 2) The DoP of the object is not polarized, but it does
not make sense. Polarimetric imaging model in scattering media 3) The DoP of the backscattered light is constant;
however, it must be spatially variable. 4) The polarized direction of the image is equal to that
of the backscattered light and the object signal, but there
are possible differences. End-to-end descattering network 5) The degraded coefficients of backscattering light
and object signal are similar; however, Akkaynak et al.’s
study has proved that attenuation coefficient of object
signal depends on the distance z, reflectance ρ, spectrum
of ambient light E, spectral response of the camera Sc,
and beam attenuation coefficients of the water body βb;
however, the backscattering light is related to E, Sc, βb
and scattering of the water body b84,140,141. The end-to-end architecture is a common structure that
enhances image quality and relies on higher-order non-
linear representations. The descattering process of a net-
work is the fitting of the descattering transmission of a
high-order function, which has been successfully applied
to complete scattering removal using intensity informa-
tion136,144−146. In recent studies, the introduction of polar-
ization information has proven that the input of polariz-
ation images into the network can improve the quality of
qualitative and quantitative evaluations. This section dis-
cusses the polarization end-to-end descattering network. Several improved methods focusing on this insuffi-
ciency have been proposed. For example, Huang et al. es-
timated the polarization difference image (PDI) of an ob-
ject signal using feasible region fitting to overcome the
limitations of the second assumption. Hu et al. estim-
ated the spatial distributions of the DoP of an object and In 2020, Hu et al. first employed a deep learning tech-
nique in polarimetric underwater imaging46, as shown in
Fig. 9(a), which is a typical end-to-end descattering
12 230042-12 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Zhang et al., which contained four pairs of networks
consisting of polarized and gray versions. Furthermore,
this method assumes that the intensity and polarization
images are information streams, two types of informa-
tion flows in their respective networks, and join together
at the end of the model. Furthermore, the addition of po-
larization information to gray information and feeding
these two parts into fused networks, called DENSE-U-
NET BLOCK, at the forefront of the network could
achieve better results than the front or end of the net-
work70. The experiments with different turbidity levels
performed better than the other methods and demon-
strated the excellent robustness of the proposed method. architecture. End-to-end descattering network The three dimensional inputs of different
linear polarization orientations with 0°, 45°, 90° are fed into
an end-to-end polarimetric dense network (PDN) rather
than intensity images, which contains three main com-
ponents: Shallow Feature Extraction (SFE) used to ex-
tract shallow polarization features, Residual Dense Block
(RDB) as the basic structure to connect with each other,
and concatenated by Dense Feature Fusion (DFF). The
third crucial component was utilized to fuse all the fea-
tures and output the descattering results. A dataset con-
taining abundant polarization image pairs was built using
a commercial DoFP camera, and a water tank filled with
milk was used to capture turbid and clear object signals. The same network structure, based only on intensity im-
ages, was trained to verify the significance of the polariz-
ation information. Compared with the intensity network
and existing methods, the polarization network demon-
strated higher values of image contrast (IC), measure of
enhancement (EME), PSNR, and SSIM, which indicate
higher image quality6,147, as shown in Fig. 9(b). In remote sensing, the scattering medium signific-
antly impacts on the results of object reconstruction,
even producing speckle patterns. Obtaining the corres-
pondence between the original object and the imaging
process is challenging and crucial. Li et al. combined an
object’s polarization information with a modified U-net-
based deep learning network (MU-DLN) to retrieve the
original object’s information influenced by the scattering Another end-to-end architecture was proposed by b1
b2
b3
b4
b5
a
b
S0
90°
0°
45°
Polarimetric-Net
3D input
d−1th
d−1th
1th
dth
dth
nth
Residual learning
SFE
RDB
RDB
DFF
Recovered image
(Output)
Single input
Intensity-Net
Conv. layer
ReLU
Concat. layer
Raw
Polarimetric-Net
He
Liang
Ground truth
EME=9.10
SSIM=0.77
EME=8.07
SSIM=0.66
EME=4.86
SSIM=0.36
EME=5.17
SSIM=0.39
EME=5.63
SSIM=0.31
EME=6.81
SSIM=0.31
Fig. 9 | The flow chart and results of the Hu et al. method, which is a typical end-to-end descattering architecture46. (a) The architecture
of proposed method. (b) Comparison of the enhanced images by different methods. Figure reproduced with permission from ref.46, Elsevier BV. 230042-13 d−1th
dth
Residual learning d−1th
1th
dth
nth
g
SFE
RDB
DFF
Recovered image
(Output) Recovered image Recovered image EME=5.17
SSIM=0.39 EME=5.17
SSIM=0.39 EME=6.81
SSIM=0.31 EME=8.07
SSIM=0.66 EME=8.07
SSIM=0.66 Fig. 9 | The flow chart and results of the Hu et al. method, which is a typical end-to-end descattering architecture46. (a) The architecture
of proposed method. Physical-model-integrated descattering network The physical-model-integrated descattering network in-
tegrates the descattering model into the network as the
backbone to guide the descattering process, which intro-
duces constraints compared to the physical-model-
guided descattering network. Therefore, the main task of
the network is to generate or refine specific parameters
before they are utilized to generate improved results. Furthermore, the physical model and its inverse process
can form a self-supervised closed loop to achieve im-
proved performance. Guided by a physical imaging model, Ren et al. trained
a lightweight dehazing CNN to rapidly process turbid
images, comparing it with conventional dehazing meth-
ods and introducing additional circular polarization in-
formation148. Furthermore, this is the first time circular
polarization information has been fed into a network. Two unknown parameters in the polarimetric descatter-
ing model resulted in an underdetermined function by
directly generating these parameters. Therefore, the pro-
posed method combines these two parameters into a
single formula to avoid an underdetermination problem
and minimize the reconstruction error. The new para-
meter K is expressed as:149 To further combine the physical formation model
with deep learning methods, some researchers have em-
bedded existing dehazing approaches into the proposed
pipeline. Zhou et al. proposed a robust polarization-
based dehazing architecture with a generalized physical
formation model that requires no specific clues to estim-
ate the required physical parameters or handcrafted pri-
ors48. Figure 10(a1) and 10(a2) show the network archi-
tecture and corresponding example with evaluation in-
dex(PSNR and multi-scale SSIM). The transmitted light
D (T in Fig. 10(a1)) and original scene radiance Lobject (R
in Fig. 10(a2)) can be calculated using the following
equations: K = (I −A∞)/t + A∞−1
I −1
. (13) (13) Lobject =KI −K + 1
The Eq. (12) can be rewritten as an
,
after which the descattering process is viable once the
parameter K is obtained. Tests were conducted in differ-
ent turbid environments to verify the feasibility of the
proposed method, and the results indicated the effective-
ness and high efficiency of the lightweight architecture. Subsequently, Ding et al. adopted a multi-polarization
fusion adversarial generative network to enhance turbid
images47. End-to-end descattering network (b) Comparison of the enhanced images by different methods. Figure reproduced with permission from ref.46, Elsevier BV. 230042-13 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 ˆA = I0 −I[1 −P]/2
PAcos2ϕA
,
(14) medium66. Data were acquired using a Monte Carlo sim-
ulation system and deep learning technology to learn a
physical model of the scattering process. The experi-
mental results show that the object’s information for the
Q-component can be reconstructed very well because of
the suppression of scattering light and highlighting of
ballistic light. Several fixed optical thickness environ-
ments are tested to reflect the superiority of the trained
MU-DLN. (14) ϕA
where the
is AoP of backscattering light computed by
the selected background region. They built the first color
polarization image datasets in the natural underwater en-
vironment, which selected the visually better enhanced
results among the results produced by several conven-
tional methods150−154. Compared with the underwater res-
ults by four supervised data-driven polarimetric meth-
ods mentioned above, the experimental results in labor-
atory simulated by the milk have huge improvement;
however, the natural results are improved to a lesser ex-
tent because the complicated environment would in-
crease the diversities of known or hidden parameters and
then result in the networks become more generalized
with higher robustness but lower performance in a spe-
cific example. ϕA
where the
is AoP of backscattering light computed by
the selected background region. They built the first color
polarization image datasets in the natural underwater en-
vironment, which selected the visually better enhanced
results among the results produced by several conven-
tional methods150−154. Compared with the underwater res-
ults by four supervised data-driven polarimetric meth-
ods mentioned above, the experimental results in labor-
atory simulated by the milk have huge improvement;
however, the natural results are improved to a lesser ex-
tent because the complicated environment would in-
crease the diversities of known or hidden parameters and
then result in the networks become more generalized
with higher robustness but lower performance in a spe-
cific example. Physical-model-guided descattering network The physical-model-guided descattering network is
trained to remove the scattering effect guided by the
physical model but does not participate in network train-
ing. Based on the physical model, the theoretical feasibil-
ity was proven before guiding the design of the network
architecture. Therefore, the middle stage of combining
the physical priors and models is instructive for further
fusing the physical model into the design of the network
pipeline. Physical-model-integrated descattering network Compared with the conventional model, the
proposed method introduces an angle of polarization to
calculate the backscattered light, expressed as: D = PI −IPA
PT −PA
and
Lobject =
DA∞
A∞−(I −D) ,
(15) (15) where PT and PA define the DoP of the transmitted light
and backscattered light, respectively, which are estim-
ated by the subnetworks. The symbols I and P are the 230042-14 230042-14 230042-14 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 g2
Estimated PA
and PT
b1
a1
g1
g3
g4
Refined T
Refined R
Calculated R
Calculated T
Estimated
infinite airlight
Precomputed haze
image and DoP
PSDNet-T2
PSDNet-T1
PSDNet-A
PSDNet-L
Estimated
target radiation
Orthogonal
scene image
Fused
transmission map
Estimated
scene airlight
Estimated
transmission map
I⊥
Composite haze
image
I||
I⊥
I||
b2
a2
Polarization
sub-images
Fig. 10 | The methods combining the physical formation model with deep learning methods and corresponding examples. (a1) Zhou et
al. method and (a2) the corresponding example. (b1) Shi et al. method and (b2) the corresponding examples. g2
Estimated PA
and PT
a1
g1
g3
g4
Refined T
Refined R
Calculated R
Calculated T
Estimated
infinite airlight
Precomputed haze
image and DoP
a2
Polarization
sub-images a2 Fig. 10 | The methods combining the physical formation model with deep learning methods and corresponding examples. (a1) Zhou et
al. method and (a2) the corresponding example. (b1) Shi et al. method and (b2) the corresponding examples. corresponding results, with assessment criteria(IC and
entropy-based no-reference image quality assessment
(ENIQA)) which consist of three subnetworks to com-
pute the object radiation, transmitted light, and backs-
cattered light. The pipeline processed a self-supervised
closed loop to optimize the network. The end-to-end
descattering network is part of the total pipeline, which
effectively reduces the scale of the network and enhances
performance. Additionally, a secondary product with an
accurate transmission map was produced, which may be
helpful for other computer vision tasks, such as 3D re-
construction. Several experiments demonstrated that the
proposed architecture can effectively improve the visibil-
ity of object details and is highly robust for the scene. haze image and its DoP, respectively, which can be calcu-
lated from the polarized images. The DoPs of the object
signal and scattered light can be generated by subnet-
work g1 before estimating the transmitted light using the
imaging model. The refined light transmitted by subnet-
work g2 is utilized to calculate the original scene radi-
ance, in which the saturated backscattered light is ob-
tained by subnetwork g3. Finally, the refined subnet-
work g4 was adopted to generate the refined results. The
raw polarization direction images join each subnetwork. The generation of the synthetic dataset was instruct-
ive. Clear images with depth and semantic segmentation
maps must be provided for the generation process. 230042-14 Specificly, α is a com-
pensation factor for the water absorption is set to 0.99 as
the subject was placed in a comparably shallow position
with pure water in the underwater environment, where
the influence on the absorption of natural light could be
ignored. η is a bias factor and ps° is degree of the scatter-
ing light, where the range of η is from 1 to 1/ ps°. The
measurement value of ps° is set to 0.8333 and η=1.13 to
meet the range of 1 ≤ η ≤ ps°. S∞ is the scattered light ra-
diation at an infinite distance estimated by the intensit-
ies of the brightest region of the no object. Lobject =
I −(A/ε)
1 −(A/εA∞) ,
(17) (17) where the ε is bias factor. Consequently, the object radi-
ance was optimized and recovered based on the under-
water image recovery process. The proposed method has
a much lower cost for preparing the training datasets and
demonstrates the capability of recovering underwater
images under different nonuniform optical fields. The
flow chart, proposed architecture of the Yang et al. meth-
od76, corresponding results, and comparisons with exist-
ing methods142,147,164,165 are shown in Fig. 12. The proposed methods not only overcome the acquisi-
tion of polarization characteristics of the environment
and object in the conventional process but also minim-
ize the dependency on datasets even when training on
only one image. Moreover, the mismatch between the
model and a real scene lacking environmental priors is
significantly reduced. Figure 11(a) and 11(b)49 show the
proposed architecture and a visual comparison among
the different descattering methods147,160−163 together with
statistical index of image contrast. The method repres-
ents a pioneering attempt in the realm of unsupervised
descattering imaging. However, its capacity for enhan-
cing imaging outcomes remains somewhat limited. Data-driven polarization descattering methods have
been gradually introduced into physical models to guide
network training, which resolves the limitations of tradi-
tional methods. Data-driven methods learn more com-
prehensive features and adjust them for more complex
media. Furthermore, it improves the training perform-
ance and provides another training process for a
self-supervised closed loop to optimize the network. 230042-14 The
Foggy Cityscapes-DBF dataset was eligible, and reason-
able values of the corresponding parameters were set to
generate the synthetic dataset155−157. Gaussian noise was
introduced to make them spatially variant and conform
to real-world scattering conditions to improve the ro-
bustness of the network158,159. R(·)
Because it is hard to capture the ground truth corres-
ponding to the object in the underwater environment,
unsupervised learning polarimetric underwater methods
were proposed. Zhu et al. synergistically make use of an
untrained network and polarimetric imaging formation
model to recover images from scattering in underwater
scenarios without requiring additional datasets49. There
are two stages during the network training. First, the raw
input images are input to the network
to generate Contrarily, Shi et al. processed a polarization-based
self-supervised dehazing network called PSDNet to elim-
inate the influence of haze on images72. Figure 10(b1)
and 10(b2) show the proposed network architecture and 230042-15 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Yang et al. trained a network to inpaint backscattered
light with different polarization orientations, which was
used to calculate the DoP and AoP of the background
light76. Furthermore, this is another unsupervised meth-
od that does not require clear ground truth. The primary
task of the proposed method is to calculate the complete
backscattered light. After removing the region of the ob-
ject in the captured polarized images using the GrabCut
algorithm, the incomplete image, randomly erasing a re-
gion, is input to the network to generate a mission re-
gion before using a filtering method that compares the
gray value of one pixel with those in other nearby pixels
and replacing the singular point with the averaged value
in a 7×7 square. Furthermore, a clear image is calculated
using the following modified function: M (·)
the optimized image. Furthermore, using the imaging
formation model
to recalculate the degraded im-
age. After the circular process, the loss of raw image and
calculated image is adopted to optimize the network: [R (·; w∗) , Θ∗] =
argmin
w,Θ
∥M{R(I(x, y); w), Θ} −I(x, y)∥2
2 ,
(16) [R (·; w∗) , Θ∗] = (16) where w is the weights, Θ is parameters of image forma-
tion model including {α, η, ps°, S∞}. These parameters are
estimated using a neural network, where the initial value
is selected by traditional methods. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 lmage acquisition
and preprocessing
Collected polarization
images I (x, y)
Recovery and
optimization of L (x, y)
Optimization and recovery
of L (x, y) by Eq. (10)
Calculate EME of
L (x, y)
Get incomplete A∞ (x, y) (Remove
the target object in I (x, y))
Obtain B (x, y) from Eq. (6)
Estimation of
A∞ (x, y) and B (x, y)
Use unsupervised learning to
obtain the complete A∞ (x, y)
Calculate stokes parameters
of A∞ (x, y) and filter them
Estimate DOP of B from
DOP of A∞ (x, y)
Estimate the intensity of the
polarized part of B (x, y) from
DoP of I (x, y)
128×128×1
128×128×1
4×4×512
4×4×512
4×4×512
4×4
Trans
4×4
Conv
4×4
Conv
1×1
Conv
1×1×4096
128×128×1
Conv
ReLU
ReLU
ReLU
ReLU
Upsample
block
×5
Upsample
block:
Feature
extraction
block×5
Feature
extraction
block:
Feature
extraction
block×5
Generative network
Discriminative network
3×3 same Conv
Max-pooling
ReLU
4×4 s=2 p=1
TransConv
ReLU
c1
c2
c3
c4
EME=0.4007
EME=0.3994
EME=0.3829
EME=1.3175
EME=1.2754
EME=1.3378
a
b
d
real
or
fake
Fig. 12 | Unsupervised underwater descattering methods and corresponding results. For the equations and variables, see the ref.76. (a)
The flow chart. (b) Proposed architecture of Yang et al. method. (c) The results of generated backscattering light; (c1) the captured polarized im-
age; (c2) the generated backscattering image of proposed trained network; (c3) the backscattering image after smooth filtering; (c4) the corres-
ponding ground truth. (d) Final descattering results and comparisons with other methods. Figure reproduced with permission from ref.76, Optical
Society of America Publishing AG. g
p
(
)
p
g lmage acquisition
and preprocessing
Collected polarization
images I (x, y)
Recovery and
optimization of L (x, y)
Optimization and recovery
of L (x, y) by Eq. (10)
Calculate EME of
L (x, y)
Get incomplete A∞ (x, y) (Remove
the target object in I (x, y))
Obtain B (x, y) from Eq. (6)
Estimation of
A∞ (x, y) and B (x, y)
Use unsupervised learning to
obtain the complete A∞ (x, y)
Calculate stokes parameters
of A∞ (x, y) and filter them
Estimate DOP of B from
DOP of A∞ (x, y)
Estimate the intensity of the
polarized part of B (x, y) from
DoP of I (x, y)
a c2 c1 c3 c4 c4 c3 Fig. Yang K et al. Opto-Electron Sci 3, 230042 (2024) 12 | Unsupervised underwater descattering methods and corresponding results. For the equations and variables, see the ref.76. (a)
The flow chart. (b) Proposed architecture of Yang et al. method. (c) The results of generated backscattering light; (c1) the captured polarized im-
age; (c2) the generated backscattering image of proposed trained network; (c3) the backscattering image after smooth filtering; (c4) the corres-
ponding ground truth. (d) Final descattering results and comparisons with other methods. Figure reproduced with permission from ref.76, Optical
Society of America Publishing AG. Additionally, the physical model in the network may
generate extra parameters that are probably helpful for
other tasks, such as depth maps, backgrounds, and inher-
ent coefficients of media. The acquisition of dataset is
key for network training in scattering media. The syn-
thetic and generated methods may solve the problem
despite remaining crucial and challenging in the future. Additionally, the physical model in the network may
generate extra parameters that are probably helpful for
other tasks, such as depth maps, backgrounds, and inher-
ent coefficients of media. The acquisition of dataset is
key for network training in scattering media. The syn-
thetic and generated methods may solve the problem
despite remaining crucial and challenging in the future. the DoP, AoP, and micro-surface zenith and azimuth. An intrinsic drawback of deriving a shape from polariza-
tion is the ambiguous estimation of surface orientation. The suitable arctangent function in the model results in a
multivalued azimuth, commonly known as azimuth am-
biguity. Cues
from
various
aspects,
such
as
geometry168−170,
spectrum171,172,
photometry173−176. However, relying only on a physical-based imaging mod-
el, recovering the shape with high accuracy remains chal-
lenging under nonlaboratory conditions. The excellent
nonlinear representation ability of deep neural networks
can narrow the gap between ideal and real-world condi-
tions. This section reviews the existing 3D shapes from
polarization methods combined with deep learning (DL). 230042-14 90°
0°
45°
Input (I0°, I45°,I90°)
Generated
measurement
Loss
θ*
Underwater
IFM
M (·, Θ)
Network (R (·; w))
w
w, θ
w*
arg min||M {R (I (x, y); w),Θ)−I (x, y)||2
2
Input
IFM
Ours
MIP
HE
RGHS
DCP
GC
RoWS
a
b
Contrast=0.257
Contrast=0.273
Contrast=0.354
Contrast=0.279
Contrast=0.286
Contrast=0.335
Contrast=0.263
Contrast=0.216
Contrast=0.252
Fig. 11 | Unsupervised underwater descattering method. (a) Architecture of Zhu et al. method49 and (b) visual comparison among different
de-scattering methods. Figure reproduced with permission from ref.49, Optical Society of America Publishing AG. 230042-16 MIP
Contrast=0.279 HE
RoWS
Contrast=0.286
Contrast=0.252 HE
Contrast=0.286 Ours
Contrast=0.354 IFM
Contrast=0.273 Generated
measurement HE
Contrast=0.286 MIP
Contrast=0.279 Ours
Contrast=0.354 MIP
GC
C
0
9
Contrast=0.216 HE HE
RoWS
Contrast=0.252 RoWS
Contrast=0.252 RoWS Fig. 11 | Unsupervised underwater descattering method. (a) Architecture of Zhu et al. method49 and (b) visual comparison among different
de-scattering methods. Figure reproduced with permission from ref.49, Optical Society of America Publishing AG. 230042-16 Principles of polarization 3D shape reconstruction where n denotes the refractive index, θ refers to the
zenith angle, assuming that ηi =1 because in most condi-
tions light is incident from air. The refractive index of
specular surface is denoted ηt = n. Because the azimuth
angle is perpendicular to the phase of the specular polar-
ization178, leading to the π/2 shift of azimuth angle. This
is another ambiguity problem regarding shape owing to
polarization. The surface shape changed the polarization states of the
incident illumination, providing the possibility of recov-
ering the shape from polarization. In polarization detec-
tion, polarization information can be obtained using a
camera and a rotated linear polarizer mounted in front
of it or a camera with a pixelated polarizer. The captured
image intensity varied sinusoidally. ˆn = n (1 + iκ)
Material object reconstruction is more complicated
than that of regular specular surfaces. The refraction in-
dex of metal is a complex number defined as
, where κ is the attenuation coefficient. And then Eq. (20) can be derived as: I
(
ϕpol
)
= Imax + Imin
2
+ Imax −Imin
2
cos
(
2ϕpol −2ϕ
)
,
(18) where ϕpol denotes the angle of polarizer axis relative to a
chosen reference orientation, ϕ denotes the azimuth
angle of micro-surface of an object, Imax and Imin refer to
the observed maximum and minimum intensity during
ϕpol from 0 to π. A whole 2π period of sinusoidal func-
tion results in a π ambiguity. For instance, if ϕ=ϕpol, the
maximum intensity is obtained. However, the minimum
intensity corresponds to two azimuth angles, i.e. ϕ±π/2. The π-ambiguity problem is the one of shape from polar-
ization. Psm =
2ntanθsinθ
tan2θsin2θ + |ˆn|
2 . (21) (21) Diffuse reflection originates from the light refracted by
the shallow surface of an object, in which it is partially
polarized owing to the irregular interactions between the
light and the interior particles. Therefore, the DoP of dif-
fuse reflection is determined by the Fresnel transmission
coefficients. The relationship between the DoP and the
Fresnel coefficients of diffuse reflection is defined as: The polarization state of the reflected light directly de-
pends on the reflection type occurring over the surface,
which is primarily specular or diffuse reflection, as
shown in Fig. 13. Pd = T∥−T⊥
T∥+ T⊥
=
R⊥−R∥
2 −R⊥−R∥
. (22) (22) Under these conditions, light is refracted to the air in
the object. Three-dimensional shape reconstruction By analyzing the interactions between light and surface
geometry, we can reconstruct the 3D shapes of
objects166,167, where polarization is crucial. Natural illu-
mination becomes partially polarized after reflection
from an object’s surface. Polarized reflection implies
shape information because the Fresnel equations relate 230042-17 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 ing conditions using only polarization images. n (u) = [nx (u) , ny (u) , nz (u)]
T In addition to the ambiguity problem, other limita-
tions to the shape owing to polarization exist. For ex-
ample, estimating the zenith angle requires an unknown
prior for the refractive index, which limits the recon-
struction of complex objects and natural scenes. Second,
when the zenith angle is close to zero, the influence of
noise increases because the DoP is small. Third, mixed
reflections were common in real-world scenarios. Moreover, achieving satisfactory reconstruction results
in complex scenarios by using the linear superposition of
a single physical model is challenging. Finally, the dis-
continuous depth is also a significant challenge for re-
covering the shape from the derived surface normal by
integration. Consequently, the introduction of other in-
formation is essential to avoid the problems mentioned
above and expand the application fields. = [sinϕ (u) sinθ (u) , cosϕ (u) sinθ (u) , cosθ (u)]
T ,
(24) where nx(u), ny(u), nz(u) are the normal vectors of ele-
ment surface u. The normal vector can be expressed us-
ing the surface gradient, as shown in Eq. (25) n (u) =
1
√
p(u)
2 + q(u)
2 + 1
[
−p(u)
2
−q(u)
2
1
]T ,
(25)
p (u) = ∂zx (u)
q (u) = ∂zy (u)
∇z (u) =
[
p (u)
q (u)
]T
where
,
,
i.e.,
. Finally, the surface shape
z(u) was reconstructed. (25) The shape of the polarization differs according to the
type of reflection. Compared to specular reflection, the
DoP and zenith angles for diffuse reflection are one-to-
one maps. However, diffuse reflection makes the recon-
struction process challenging because of the lower signal-
to-noise ratio and higher dependence of the DoP on the
refractive index. For specular reflection, the mirror-like
surface maintains a relatively uniform direction and
phase, thus avoiding the influence of random noise. However, specular reflection leads to more ambiguous
problems than diffuse reflection does. Moreover, it is
challenging to find an object when the viewing direction
exceeds the reflection direction. Table 2 compares the
advantages and disadvantages of the shape from polariz-
ation based on both specular and diffuse reflections. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Typical methods include a combination of heuristic
priors, such as the boundary and convexity of objects180,
shading181, and photometric stereo64, but noise is a major
limitation. This complicated calculation amplifies the
noise, leading to a degraded texture or profile in the re-
covered shapes. Data-driven imaging is powerful for 3D
imaging with a nonlinear modeling ability. Furthermore,
this primarily depends on the semantic information of
the image. Under the guidance of the physical model,
this brings new possibilities for shapes from polarization. The shape of the polarization depends on the estim-
ated azimuth and zenith angles. Ambiguity is a critical
challenge. Regarding the azimuth angle, two phase angles
with a π shift are derived from the period of the sinusoid
function. For specular reflection, the azimuth angle
would be retrieved with ± π/2 operation. However, the
two zenith angles of the ambiguous solution are determ-
ined for a given DoP in the specular reflection, which
cannot be excluded without other information. These
contributing factors result in high error rates and limita-
tions in the generalization to mixed materials and light- Principles of polarization 3D shape reconstruction Therefore ηt =1, i.e. the refraction index of air,
and ηi =n, i.e., the refraction index of diffuse surface. Eq. (23) can then be derived as: The Fresnel equations describe how incident light
changes when propagating in media with different re-
fractive indices. When specular reflection dominates, the
DoP of the specular reflection is calculated using the
Fresnel reflection coefficients: Pd =
(
n −1
n
)2
sin2θ
2 + 2n2 −
(
n + 1
n
)2
sin2θ + 4cosθ
√
n2 −sin2θ
. (23) Ps = R⊥−R∥
R⊥+ R∥
. (19) (19) Combined with the Fresnel function, it has the expres-
sion in Eq.(20)177: (23) ϕ
Given the azimuth angle and zenith angles θ, the
normal vector of shape surface at any point could be ex-
pressed as179: Ps =
2sin2θcosθ
√
n2 −sin2θ
n2 −sin2θ −n2sin2θ + 2sin4θ ,
(20) (20) lncident
light
Object
Surface
normal
a
b
Object
Microfacet
Air
Air
Partially
polarized
light
Incident
light
Surface
normal
Reflected
light
Reflected
light
light
Subsurface scattering
Unpolarized
Fig. 13 | Polarization of specular reflection and diffuse reflection. (a) Specular reflection. (b) Diffuse reflection. 230042-18 lncident
light
Object
Surface
normal
b
Air
Reflected
light
Subsurface scattering Surface
normal Surface
normal lncident
light Reflected
light Reflected
light Fig. 13 | Polarization of specular reflection and diffuse reflection. (a) Specular reflection. (b) Diffuse reflection. 230042-18 Data-driven shape from polarization in single
reflection In certain situations, the reflection can be purely specu-
lar or diffuse. For instance, in human face recognition or
clothed body reconstruction tasks, the skin, clothes, and
other human tissues are diffuse surfaces, and specular re-
flection is negligible. In transparent object reconstruc-
tions, specular reflection dominates. For 3D clothed human shape reconstruction with
clothing details, Zou et al. introduced polarization im-
ages and two ambiguous normal maps into the designed Table 2 | Comparisons of specular reflection and diffuse reflection. Advantages
Disadvantages
Specular reflection
● Higher signal-to-noise ratio
● Lesser influence of the refraction index
● Suffer from more ambiguity problem
● Lower viewing angle
● Lesser application fields
Diffuse reflection
● Monotony of degree of polarization about refraction index
● Wide viewing angle
● More application fields even for scene in wild
● More influence of noise
● Worse visual effects
● Stronger influence of refraction index
230042-19 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 human face reconstruction, Han et al. proposed a learn-
ing-based method for passive 3D face reconstruction
from polarization183, as shown in Fig. 15(a). Further-
more, it derives the ambiguous normal of each microfa-
cet over the face at the pixel level based on the polariza-
tion of the diffuse reflection. The CNN-based 3D morph-
able model (3DMM) generates a rough depth map of the
face based on a directly captured polarization image, and
is used to amend the ambiguous polarization normal and
further
reconstruct
an
accurate
3D
face
using
Frankot–Chellappa 3D surface restoration functions. Figure 15(b) illustrates the final results, including a male
face under indoor lighting, a male face under natural
outdoor illumination, and an indoor plaster statue. The
3D rendering features fit well with the original appear-
ance, and the lighting conditions had little influence. The
experiments also demonstrate the benefits of introdu-
cing deep learning into 3D polarization reconstruction. network65, as shown in Fig. 14. Specular reflection was
omitted because of the rough surface of the clothing. Owing to the azimuth ambiguity problem, two possible
maps were resolved and input into the network as phys-
ical priors. The two ambiguous normal maps, n1 and n2
are classified into three categories: n1, n2, and back-
ground. Each pixel point was classified as belonging to
one of these three categories and then merged into n3 us-
ing Eq. (26) with probabilities p0, p1 and p2. Data-driven shape from polarization in single
reflection 230042-20 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 each segmented area. Moreover, this problem can also be
solved by reconstructing each area separately; however,
the segmentation algorithm and stitching of the 3D
shape of an object are huge challenges. Neural networks
provide solutions to fuse explainable or inexplicable fea-
tures with mixed reflections, relying on their excellent
nonlinear representation ability. network to reconstruct 3D shape-transparent objects
from specular reflections81. However, transmitted light is
often a diffuse reflection. Therefore, separating the trans-
mitted light is critical. The AoLP features indicate
stronger background noise in areas with higher transmit-
tance. The closer it is to the center area, the stronger the
noise, as shown in Fig. 16. The physical prior confidence
concept is based on intrinsic faults in the AoLP maps of
transparent objects. The first method combines deep learning and polariz-
ation-reconstructed models; Ba et al. fed polarization im-
ages and ambiguous normal maps into the network and
trained the network to learn the effective inputs from
training data automatically64. The inputs were four im-
ages captured with a polarizer, and the ambiguous nor-
mal maps consisted of one diffuse and two specular am-
biguous maps. The proposed method achieved the low-
est test error on the tested data under the three lighting
conditions compared with conventional methods. ConfidenceK,m (i, j) =
∑
p∈Pi,j
|p −¯pi,j|
m
max
0⩽x⩽W
0⩽y⩽H
∑
p∈Px,y
|p −¯px,y|
m ,
(27) (27) ¯px,y
where pi,j represents the pixel values in the K×K neigh-
borhood of the point (x, y),
is the mean of the pixel
values in this area, m is the smoothing exponential term. H and W denote the height and width of the map, re-
spectively. This physics-based prior confidence map is
then input into the network as an attention map to guide
the fusion of the original-polar (DoLP and AoLP maps)
and physics-based prior (four ambiguous maps). The
proposed method achieves optimal performance and
provides a new perspective for further transparent
shapes from polarization research. Based on a polarimetric Bidirectional Reflectance Dis-
tribution Function (pBRDF) model and real polarization
scene rendering, Kondo et al. applied rendered polarized
images to train a network for an accurate surface normal
estimation71. A physics-based renderer was built to simu-
late the polarization behavior of the rays based on the
proposed pBRDF model for each material. Data-driven shape from polarization in single
reflection Furthermore,
it can correctly reproduce the polarization property, in-
cluding the inter-reflection effect, in real-life scenes. Therefore, the synthetic-colored image and simulated
polarization information, such as the phase and DoP,
were fed into the CNN to estimate the surface normal. The detailed process and reconstruction results are
shown in Fig. 17(a). Data-driven shape from polarization in single
reflection n3 = (1 −p0) ·
p1n1 + p2n2
∥p1n1 + p2n2∥2
. (26) (26) The final surface-normal prediction is refined using a
denoising network. The smoothed normal concatenates
the fused normal and raw polarization direction images
as the input to accurately estimate the surface normal. Subsequently, the skinned multi-person linear (SMPL)
representation182 and deformation stage were used to re-
construct the refined 3D human shape with clothing de-
tails rather than naked. The transparent objects exhibited typical specular re-
flections. Shao et al. proposed a multibranch fusion Regarding diffuse-reflection-dominated cases, such as Ambiguous
normal
Classify net
Fused normal
Single
polarization
images
Predicted normal
Deformation
ResNet 50
Joints
Detailed body shape
Fig. 14 | The proposed architecture of the Zou et al. method. Ambiguous
normal
Classify net
Fused normal
Single
polarization
images
Predicted normal
Deformation
ResNet 50
Joints
Detailed body shape
Fig. 14 | The proposed architecture of the Zou et al. method. Improved
3DMM net
3D depth map
Polarization-
based 3D
model
Dimensional
transformation
Rough depth
map
High-precision
3D face
a
b
I
Imax
Imin
Fig. 15 | Han et al. passive 3D polarization face reconstruction method. (a) Overall schematic of the proposed method. (b) 3D polarization
face reconstruction results. Figure reproduced with permission from ref.183, Multidisciplinary Digital Publishing Institute. 230042-20 Single
polarization
images Joints Joints Detailed body shape Detailed body shape Deformation Deformation Fig. 14 | The proposed architecture of the Zou et al. method. Improved
3DMM net
3D depth map
Polarization-
based 3D
model
Dimensional
transformation
Rough depth
map
High-precision
3D face
a
b
I
Imax
Imin
Fig. 15 | Han et al. passive 3D polarization face reconstruction method. (a) Overall schematic of the proposed method. (b) 3D polarization
face reconstruction results. Figure reproduced with permission from ref.183, Multidisciplinary Digital Publishing Institute. 230042-20 Improved
3DMM net
3D depth map
Polarization-
based 3D
model
Dimensional
transformation
Rough depth
map
High-precision
3D face
a
b
I
Imax
Imin
Fig. 15 | Han et al. passive 3D polarization face reconstruction method. (a) Overall schematic of the proposed method. (b) 3D polarization
face reconstruction results. Figure reproduced with permission from ref.183, Multidisciplinary Digital Publishing Institute. 230042-20 Improved
3DMM net High-precision
3D face High-precision
3D face Fig. 15 | Han et al. passive 3D polarization face reconstruction method. (a) Overall schematic of the proposed method. (b) 3D polarization
face reconstruction results. Figure reproduced with permission from ref.183, Multidisciplinary Digital Publishing Institute. Data-driven shape from polarization in mixed
reflections Mixed reflections stemming from the two primary con-
ditions were prevalent in the natural scenarios. First, the
reflectance of the surface determines the type of reflec-
tion: specular reflection or diffuse reflection. For materi-
als such as ceramics, plastics, and lacquers, specular re-
flection dominates the highlighted areas, whereas diffuse
reflection dominates the other areas. Second, objects
made of different materials create reflections that vary in The proposed pBRDF model is described by the angle
of incident light, plane, reflection angle, camera direc-
tion, and half-vector, which allows accurate transmis-
sion Mueller matrix modeling for arbitrary cameras and Mixed
region
Specular
dominated
region
AoLP map
Physics-based prior confidence map
Fig. 16 | The physical-based prior confidence map according to the differences of polarization characteristics between transparent ob-
ject and background. Physics-based prior confidence map Physics-based prior confidence map AoLP map Fig. 16 | The physical-based prior confidence map according to the differences of polarization characteristics between transparent ob-
ject and background 230042-21 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 lighting positions. Specular and diffuse reflection mod-
els were established separately. The Mueller matrix of
specular reflection considers the rotation matrix of light in the incident plane, Fresnel elements, delay metrics,
and the rotation matrix of light in the camera, as de-
scribed by Eq. (28), R−χl
0
R+χlγc + R×γlχccosδ
R×χcsinδ
−R+χlχc + R×γlγccosδ
R×γcsinδ
−R×γlsinδ
R×cosδ
,
(28) Ms
i,o =
R+
R−γl
R−γc
R+γlγc −R×χlχccosδ
−R−χc
−R+γlχc −R×χlγccosδ
0
R×χlsinδ (28) R+ = (R⊥+ R∥) /2
R−= (R⊥−R∥) /2
R× = √R⊥R∥
where
,
, and
are Fresnel reflection coefficients. The
other coefficients are elements of the rotation matrix. Accordingly, the Mueller matrix of diffuse reflection
contains a rotation matrix of light into the incident
plane, two Fresnel elements into and out of the surface, a
depolarization matrix, and a rotation matrix of light into
the camera, as denoted by Eq. (29), timization, all parameters can be calculated. Experi-
ments show well-rendered results close to the real ones
used to generate polarized images as synthetic datasets. This study guides the establishment of 3D reconstruc-
tion of polarized datasets and encourages exploration to
accurately transmit the interaction process between po-
larized light and objects, even in the entire scenario. Similarly, Deschaintre et al. Data-driven shape from polarization in mixed
reflections coupled polarimetric ima-
ging with a CNN to estimate the 3D shape and calcu-
lated the Spatially Varying Bidirectional Reflectance Dis-
tribution Function (SVBRDF) using single-view polari-
metric imaging under frontal flash illumination80, as
shown in Fig. 17(b). U-Net contains three branched de-
coders to generate the 3D shape: 1) surface normal and
depth maps, 2) spatially varying reflectance properties as
diffuse, and 3) specular albedo maps and specular rough-
ness maps. Next, it was fed by the flash image, normal-
ized diffuse color, and the Stokes map computed by the
polarization image results, which were plausible, and the
proposed method captured the real appearance of the in-
puts. However, as the lighting or object becomes gradu-
ally complex, for instance, multi-illumination, multiple
objects with blurred details, etc., the methods based on Md
i,o =
T+
o T+
i
T+
o T−
i βln
T+
o T−
i αln
0
T−
o T+
i βnc
T−
o T−
i βlnβnc
T−
o T−
i αlnβnc
0
−T−
o T+
i αnc −T−
o T−
i βlnαnc −T−
o T−
i αlnαnc 0
0
0
0
0
,
(29) Md
i,o =
T−
o T+
i βnc
T−
o T−
i βlnβnc
T−
o T−
i αlnβnc
0
−T−
o T+
i αnc −T−
o T−
i βlnαnc −T−
o T−
i αlnαnc 0
0
0
0
0
,
(29)
T+ = (T⊥+ T∥) /2
T−= (T∥−T⊥) /2
where
and
are
Fresnel transmission coefficients. The final normalized
Mueller matrix is the linear superposition of the reflec-
tion matrix and depolarization matrix mentioned above,
representing the diffraction and scattering characterist-
ics of the light inside the materials. The linear superposi-
tion process can effectively simulate the mixed reflec-
tions. A generalized Lambertian reflection distribution
function model was used to parameterize luminance and
linear combination coefficients. Through the final op- Polarization
characteristic
measurement
Polarization
BRDF
modeling
Physical based
polarization
rendering
Polarization
information
Surface
normal
normal/ diffuse/ roughness/ specular/ depth map
Computed
inputs
Captured
inputs
Direct light
0
0
10
10
20
20
−0.15
−1
−1
1
1
0
0
0
0.01
0.02
0.03
−0.10
−0.05
0
a
b
Fig. 17 | The 3D shape reconstruction methods based on the pBRDF. (a) Y Kondo et al. method and the reconstruction examples. (b) V Des-
chaintre et al. method and corresponding results. Reflection removal In summary, existing data-driven polarization 3D
shape reconstruction methods are always end-to-end
structures guided by a physical model. The basic struc-
ture is input to the raw polarization images and other
prior images, which are always ambiguous, to optimize
the network, and the refined normal image is generated. In addition, other prior images are crucial in the corres-
ponding methods for enhancing the reconstructed ef-
fects. Different inputs may also result in various network
architectural designs. Therefore, as shown in Fig. 18, the
inputs in the 3D shape reconstruction task are visually displayed. The data-driven dataset for the ground truths was ob-
tained in two ways. First, a Kinect depth camera is the
most commonly used equipment for capturing a coarse
depth map as the ground truth. Other operations may be
conducted to refine the captured depth map, such as de-
noising, exclusion of inaccurate values, and sparse point
cloud67. Second, a simulation method based on the BRDF
is adopted to generate the synthetic datasets. Plausible
results could be produced, and other features such as
roughness and depth maps80 could also be generated,
which may be helpful for other computer vision tasks. However, robustness will decrease in a complicated
environment. Mixed reflection dominated the outdoor scenario with
multiple objects with different refractive indices. Lei et al. constructed the first real-world SfP dataset for complex
scenes to train a network67. The proposed network input
includes three parts: 1) four raw captured polarization
images and 2) polarization feature images, including in-
tensity, DoP, and encoded AoP by sine and cosine opera-
tions. Encoded AoP solves the problem regarding raw
AoP maps being similar at two given polarization angles
with π difference; 3) The viewing encoding to account
for non-orthographic projection in scene-level SfP. The
introduction of viewing encoding effectively calibrates
the polarization parameters influenced by the spatially
varying viewing directions. Data-driven shape from polarization in mixed
reflections 230042-22 Polarization
information
Surface
normal Polarization
characteristic
measurement
−1
−1
1
1
0
0
0
0.01
0.02
0.03
a Polarization
BRDF
modeling
0
0
10
10
20
20
−0.15
−0.10
−0.05
0 Physical based
polarization
rendering
Direct light Polarization
BRDF
modeling Physical based
polarization
rendering Polarization
characteristic
measurement normal/ diffuse/ roughness/ specular/ depth map b normal/ diffuse/ roughness/ specular/ depth map normal/ diffuse/ roughness/ specula Captured
inputs Captured
inputs Computed
inputs Computed
inputs Fig. 17 | The 3D shape reconstruction methods based on the pBRDF. (a) Y Kondo et al. method and the reconstruction examples. (b) V Des-
chaintre et al. method and corresponding results. 230042-22 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 BRDF71,80 will suffer poor recovery, and accurate simula-
tion of mixed polarization remains an open challenge. Data-driven polarization reflection removal based on
synthetic datasets The reflected light is polarized, and polarization has
proven to be a feasible solution to this problem. Based on
the Fresnel functions, the number of unknowns was sim-
ilar to that of the input images. The reflection and trans-
mission components can be separated by viewing the
Brewster angle. Moreover, the closer to the Brewster
angle, the better the reflection removal performance. Despite the incident angle having to be known, obtain-
ing it in the real world is challenging. Additionally, the
robustness and generalization of existing polarization-
based methods cannot meet the requirements of real-
world high-quality imaging, considering the various
viewing angles, complex refractive indices, smoothness,
and local curvature of the surface. Synthetic datasets are commonly used to train networks
because they are hard-accessible real-world datasets. The
traditional method directly sums the candidate image as
a reflection and transmission using normalized weights. Real-world tests exhibit poor performance. Therefore,
based on the polarimetric imaging model, synthetic data-
sets of high quality and robustness were generated. T′
R′
By leveraging the properties of light polarization and
residual representation, Wieschollek et al. presented the
first deep learning approach to separate reflected and
transmitted components73, as shown in Fig. 19(a1). The
proposed network architecture uses three polarization
orientation images to calculate the parallel and perpen-
dicular components I∥, I⊥ as the input into the network. The output of network comprises the residual images
,
and the two single-channel weights ξ∥, ξ⊥. The final
estimates of the reflection and transmission can be com-
puted as follows: Deep learning methods based on CNN are excellent
for extracting hidden features. Furthermore, it enables
the prediction of potential prior information from cap-
tured images and demonstrates good performance in the
reflection removal task. Introducing polarization and
imaging models into the network can improve removal
performance and expand the diversity of datasets and ar-
chitectures. However, acquiring the ground truth data-
set is crucial. Thus, the generation of a synthetic dataset
based on the proposed method and refined real dataset
methods was proposed. Table 3 lists the elements that
must be considered in synthetic and real-world dataset
acquisition and their advantages and disadvantages. Ir = ξ⊥R′ + (1 −ξ⊥) I⊥and It = ξ∥T′ +
(
1 −ξ∥
)
I∥,
(30) ∥
(30) where Ir and It denote the estimation of reflected and
transmitted light, respectively, represented as predicted
transmission and reflection in Fig. 19(a1). Limitations of reflection removal based on
polarization Removal of reflection contamination is a challenging but
critical and frequently encountered task because it may
contaminate image quality. Several studies have been
conducted based on diverse physical and image charac-
teristics, but this remains an unsubstantial task14,184,185. Because the transmission image through the surface and
the image reflected by the surface are simultaneously
captured by a photographer, recovering two images from
a single-mixture image is a highly ill-posed problem. 135°
90°
45°
0°
Physical prior
input
Polar image
input
Diffuse
Specular 1
Specular 2
RGB
Phase
DoP
I45
IV
IH
I135
Normalized color
Flash
image
Stokes map
Computed inputs
Captured inputs
Iun
ϕe
ρ
Imin
Imax
DoLP
AoLP
Physics-based prior
input
Physics-based prior
confidence
Original polar
input
Polarization representation P
Viewing encoding V
Polarization images
Raw polarization
image
Calculate
Ambiguous normal
n1, n2
I0 Iπ/4
I3π/4
Iπ/2
a
d
e
f
g
b
c
Fig. 18 | The inputs of network in 3D shape reconstruction task. (a) The raw polarization images as well as the diffuse and specular normal
prior. (b) The raw polarization images and two diffuse ambiguous normal. (c) The DoFP image consists of four polarization sub-images, viewing
encoding, intensity, DoP image and encoded AoP image. (d) The chromatic intensity, phase and DoP image. (e) The raw polarization images,
flash image, normalized color and Stokes map. (f) The maximum and minimum polarization images. (g) The raw polarization images, physics-
based prior confidence, DoLP and AoLP. 230042 23 Calculate
Ambiguous normal
n1, n2
b 135°
90°
45°
0°
Physical prior
input
Polar image
input
Diffuse
Specular 1
Specular 2
a Iun
ϕe
ρ
Polarization representation P
Viewing encoding V
Polarization images
Raw polarization
image
I0 Iπ/4
I3π/4
Iπ/2
c Calculate I45
IV
IH
I135
Normalized color
Flash
image
Stokes map
Computed inputs
Captured inputs I45 d DoP RGB
Phase
DoP
d Phase RGB
Phase Fig. 18 | The inputs of network in 3D shape reconstruction task. (a) The raw polarization images as well as the diffuse and specular normal
prior. (b) The raw polarization images and two diffuse ambiguous normal. (c) The DoFP image consists of four polarization sub-images, viewing
encoding, intensity, DoP image and encoded AoP image. (d) The chromatic intensity, phase and DoP image. (e) The raw polarization images,
flash image, normalized color and Stokes map. (f) The maximum and minimum polarization images. (g) The raw polarization images, physics-
based prior confidence, DoLP and AoLP. Limitations of reflection removal based on
polarization 230042-23 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) Constraints are essential to solve this problem. Constraints are essential to solve this problem. Data-driven polarization reflection removal based on
synthetic datasets An accurate synthetic data generation pipeline is in-
troduced, including the simulation of realistic reflections,
such as high-dynamic-range scenes, nonstatic scenes,
and curved and nonideal surfaces, to enhance the robust-
ness of the proposed method, as shown in Fig. 19(a2). First, because the world consists of high-dynamic-range
elements, the light intensity naturally diminishes as it Therefore, artificial manipulations must be added to
generate synthetic images or collect real-world images. In this section, based on the acquired aspects of the
training datasets, we review the existing reflection re-
moval methods using synthetic and real-world datasets. Table 3 | The comparison of synthetic and real-world datasets. Synthetic dataset
Real-world dataset
● Elements should be
considered in acquisition of
datasets
● High dynamic range in the real-world. ● Disappearance or interruption of the reflection. ● Discontinuous scene of the reflection or
transmission. ● Dynamic scenes. ● Semi-reflective surface. ● Curved or irregular surface. ● Rotating of reflection. ● Acquisition of ground truth. ● Dynamic scenes. ● Misalignment of refraction and transmission light. ● Luminousness and thickness of glass. ● Image registration with or without glass. ● Overexposure areas. ● Advantages
● Abundant samples. ● Easily generation. ● Real. ● Complicated enough to cover the real-world
conditions. ● Disadvantages
● Illusory. ● Difficult to take fully account all conditions. ● Domain gap between synthesis and real-world data. ● Difficult capturing process. ● Limited samples. 230042 24 Table 3 | The comparison of synthetic and real-world datasets. ● Elements should be
considered in acquisition of
datasets Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 travels. Consequently, artificial adjustments match this
phenomenon in real-world environments. The proposed
method separately manipulates the dynamic range of the
transmitted and reflected input images using a random
factor. Second, for nonstatic scenes, such as cases where
a swaying tree branch occurs during capture, local and
nonrigid deformations are adopted by perturbing each
grid over a patch. Third, for curved and non-ideal sur-
faces, a parabola was utilized to simulate unconstrained
surface curvatures with four variable parameters. where ϕpol denotes the polarization angle and ϕ denotes
the azimuth angle. Data-driven polarization reflection removal based on
synthetic datasets The unpolarized and polarized im-
ages were then calculated using: Iunpol =ξ
2 · Ir + 2 −ξ
2
· It
Ipol = ζ
2 · It + 2 −ζ
2
· It ,
(32) (32) where Iunpol, Ipol, ξ and ζ denotes the unpolarized, polar-
ized images and weights for reflection and transmission,
respectively. Next, the reflection and transmission im-
ages can be computed as: Lyu et al. exploited the physical constraints from a pair
of unpolarized and polarized images to separate reflec-
tion and transmission74, as shown in Fig. 19(b). The coef-
ficients of the glass plane are predicted by the semireflec-
tor orientation module to compute the reflection and
transmission based on the proposed physical image
formation, denoted as: Ir =2 · (2 −ξ) · Ipol −(1 −ζ) · Iunpol
2ζ −ξ
,
It =2 · ζ · Iunpol −ξ · Ipol
2ζ −ξ
. (33) (33) Finally, to close the gap between the physical model
and real data, a refined module was adopted to improve
the initial estimation. Additionally, the proposed captur-
ing setup can potentially be integrated into smartphones ξ =R⊥+ R∥
ζ =R⊥cos2 (
ϕpol −ϕ
)
+ R∥sin2 (
ϕpol −ϕ
)
,
(31)
a1
Produce
image
Parallel/vertical
image a1
Produce
image a1
b
a2
Produce
image
Parallel/vertical
image
Residual images/
weights map
Predicted transmission &
reflection
Transmission & reflection/
polarization sub-images
Reflection
physics
Dealing with
dynamic scenes
Dynamic range
manipulation
Original
data
Unpolarized
image
Weight map
Weight map
Computed
reflection
Refined
reflection
Polarized
image
Computed
transmission
Refined
transmission
−1 +1 +1 +1
Ps
f′|xp
θ
Fig. 19 | The typical data-driven polarimetric methods of reflection removal. (a1) P Wieschollek et al. method. (a2) The image-based data
generation procedure of (a1). (b) Y Lyu et al. method. 230042-25 Residual images/
weights map Predicted transmission &
reflection Predicted transmission &
reflection Produce
image Parallel/vertical
image Residual images/
weights map g
weights map
reflection
Transmission & reflection/
polarization sub-images
Reflection
physics
−1 +1 +1 +1
Ps
f′|xp
θ
reflection
Transmission & reflection/
polarization sub-images weights map
Reflection
physics
−1 +1 +1 +1
Ps
f′|xp
θ
Transmission & reflection/
polarization sub-images b
Unpolarized
image
Weight map
Weight map
Computed
reflection
Refined
reflection
Polarized
image
Computed
transmission
Refined
transmission Unpolarized
image Polarized
image Refined
transmission Fig. 19 | The typical data-driven polarimetric methods of reflection removal. (a1) P Wieschollek et al. method. Data-driven polarization reflection removal based on
synthetic datasets (a2) The image-based data
generation procedure of (a1). (b) Y Lyu et al. method. al data-driven polarimetric methods of reflection removal. (a1) P Wieschollek et al. method. (a2) The image-based data
re of (a1). (b) Y Lyu et al. method. https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) without affecting the original photography quality or
achieving reliable results. The dataset was generated
based on the PLACE2186 dataset, in which two random
images were selected as the original reflection and trans-
mission images. Reflection is blurred by Gaussian
smoothing based on the assumption that people take
photos of the transmitted light. eliminate the misalignment between these two elements,
a loss of image similarity at the perceptual level, such as
perceptual loss and contextual loss, was designed. However, the intensity delay and color distortion persist. The collected reflection-free images are not perfectly
aligned with the input mixed images owing to glass re-
fraction. To avoid misalignment issues, Lei et al. used a
piece of black cloth to cover the back of the glass to block
all transmissions for clear reflection53. This dataset in-
cludes approximately 100 types of glass in the real world,
which guides the proposed method to handle different
types of reflections without introducing artifacts. The re-
flection removal network uses multi-polarization direc-
tion images as input. Furthermore, the calculated intens-
ity, degree, and angle of polarization, and the overexpos-
ure mask eliminating the overexposed areas, were com-
bined into the network. Two stages were adopted to es-
timate reflection and transmission. This design signific-
antly improves the performance of the proposed method
by a large margin. Pang et al. proposed a novel progressive polarization-
based reflection removal network (P2R2Net) to generate a
preliminary estimation of coarse transmission images be-
fore guiding the final reflection removal187. The input im-
ages for the network consisted of two parts: a reflection-
obstructed image and high-column features from a pre-
trained VGG19, which is a successful example of using
pre-trained features as prior information. Reflection-ob-
structed images are synthetic, based on physical func-
tion Eq. (33). The high dynamic range in the real-world,
considered as light intensity, is nonlinearly compressed
in the captured image through the power function of
gamma encoding73. Two independent parameters were
used to simulate diverse practical imaging environments. Data-driven polarization reflection removal based on
synthetic datasets Additionally, flat and parabolic surface models were ad-
opted to simulate curved surfaces, which can be calcu-
lated using the camera position (xc, yc) and incidence
point (xp, yp): In summary, reflection removal is crucial because ob-
taining analytical solutions to ill-posed problems is chal-
lenging. The introduction of polarization information
can guide reflection removal using Fresnel functions des-
pite the unknown incident angle. Thus, combining deep
learning to learn prior parameters is a feasible method. Acquiring datasets determines the effectiveness and ro-
bustness of the parameter estimation of the proposed
methods. In this study, synthetic and real-world meth-
ods are proposed. In the future, more comprehensive en-
vironments and complete theories must be developed to
solve reflection-removal tasks effectively. θ = arccos
xcyp −xpyc −xpyp
√x2
p + y2
p
√(
x2
p −x2
c
)
+
(
y2
p −y2
c
) ,
(34) (34) where the θ is the angle of incidence. Additionally, ran-
dom deformation, rotation, and wrapping expand the
scale of the dataset and improve the robustness when
synthesizing the reflected obstructed image. Target detection For target classification or detection, polarimetric data-
driven methods can improve efficiency and do not re-
quire manual extraction of image features compared to
traditional methods. However, existing methods use only
intensity information images, resulting in a reduction in
the accuracy rate for low-light environments or camou-
flaged targets188−193. The targets and backgrounds also dif-
fer in their polarization characteristics. Polarimetric ima-
ging can effectively reveal these differences and assist in
target detection194,195. Therefore, we can expect positive
results by introducing polarization into data-driven
target detection. Despite synthetic datasets not being easy to obtain, they
are often too ideal, and complex conditions cannot be
fully considered in real-world environments. Real-world
datasets are also crucial but challenging to obtain with
the influence of glass and misalignment issues. Real-world datasets are typically collected using re-
movable glass. Reflection-obstructed images were cap-
tured using a camera with glass in front of the detector. The ground truth of the transmission was captured after
the glass was removed. However, the difference between
transmission and refractive transmission cannot be ig-
nored. Intensity delay caused by attenuation and color
distortion caused by colored glass are also common. To Fan et al. first proposed the use of polarization com-
plementary to intensity-based information to improve 230042-26 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) car detection accuracy54. A feature-selection process was
performed to select the most informative polarization
feature. Final detection is based on a fusion rule that
takes the polarization-based model to confirm the color-
based one. Gao et al. presented a similar work51. Blin et
al. proved that polarimetric imaging is useful for target
detection in road scenes52. Sun et al. adopted three-di-
mensional convolutions to consider the relationship
among S0, S1, and S2 images to improve the detection
rate with limited polarimetric images59. Xie et al. used the
Stokes vector to obtain four different configurations of
polarization parameter image datasets: I, DoP, [I, DoP,
AoP], and [S0, S1, S2] and trained different polarization
image detection models, indicating that increased polar-
ization information fusion enabled more learned target
features and better target detection55. Tian et al. pro-
posed a human face anti-spoofing method for real-life
scenarios, which extracts and classifies the unique polar-
ized features of faces using a CNN and an SVM
together196. Experiments covering diverse face-spoofing
attacks (print, replay, and mask) under uncontrolled in-
door and outdoor conditions were conducted. Target detection Usmani et
al. proposed unified polarimetric target detection and
classification in degraded environments using 3D polari-
metric integral imaging data197. 3D polarimetric images
with deep neural networks can effectively detect and
classify polarimetric targets under different low-light
conditions and in the presence of occlusions. Shen et al. combined the advantages of polarimetric imaging and
deep learning for rapid target detection of artificial tar-
gets camouflaged in natural scenes198, as shown in Fig. 20. The color difference of each image is calculated to
prove the proposed method can highlight the camou-
flaged artificial targets to a greater extent. pathology. This section reviews the existing data-driven
biomedical imaging and pathological diagnosis methods
and applications. Next, we discuss the interpretation of
physical properties of the network layers based on the
distance-based learning classifier. Existing biomedical imaging methods Li et al. first presented a Mueller matrix imaging system
to classify morphologically similar algae using a CNN60. Because of the low contrast in the polarimetric signals of
algae based on previous measurements of the algal
Mueller matrix, performing classification without high-
precision instruments is challenging. The proposed
methods compare the performances of various stacks of
network layers to identify the number of convolution
layers. The classifier network was trained to extract fea-
tures from the Mueller matrix and achieved a classifica-
tion accuracy of 97%. Subsequently, they introduced a
distance metric learning method called the Siamese net-
work, which aimed to learn good distance metrics of
algal Mueller matrix images in low-dimensional feature
spaces61. Compared to the convolutional CNN method,
in the Siamese approach, data pairs are generated
stochastically as inputs to train the network to determ-
ine if they belong to the same category. The experiments
demonstrated that the coupling of Mueller matrix ima-
ging and CNN of the Siamese approach may be an effi-
cient solution for the automatic classification of morpho-
logically similar algae. Zhao et al. proposed a giant cell tumor bone detection
method using Mueller matrix polarization microscopic
imaging and a multi-parameter fusion network (MPFN)
that combines three extracted polarimetric features: deep
micro-Pol features, MMPD features, and MMT
features62, as shown in Fig. 21. Wang et al. and Zhou et
al. used polarized speckle images for in vivo skin cancer
detection85 and polarized hyperspectral images for head
and neck squamous cell carcinoma detection86. Yao et al. characterized
the
microstructures
of
endometrial
samples at the typical proliferative and secretory phases
using Müller matrix polar decomposition and a set of ro-
tation-invariant parameters and their corresponding an-
gular parameters87. In this study, polarimetric imaging
was combined with a digital pathology technique to
quantitatively study the microstructural features of
endometrial samples. Furthermore, the incorporation of
local image texture information through Local Binary
Pattern (LBP) analysis improves the characterization Biomedical imaging and pathological diagnosis Biomedical imaging and pathological diagnosis methods
based on Mueller matrix features, a typical polarization
feature, are emerging label-free and noninvasive tech-
niques suitable for characterizing the microstructures of
biological tissues with anisotropic properties. Recently,
results have been published based on Mueller matrix
imaging for digital pathology25,28,29,199−207. However,
achieving accurate pathological diagnosis by observing
and evaluating stained pathological sections for interns is
challenging. Furthermore, pathological diagnosis is a
classification problem; therefore, learning-based meth-
ods are crucial in achieving fast and accurate digital 230042-27 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Splitting original
polarization image
Highlighting locations
of camouflaged targets
Input
Output
H,S, V channel images
S0 stokes image
Is image
IE image
S0
Is
IE
Tyo's method
Wang's method
Our method
S1,S2 stokes images
I0, I45, I90, I135
Decomposing
S0 stokes image
Extracting
polarization signatures
Enhancing
S0 stokes image
if necessary
Calculating polarization
parameter Is image
Calculating
stokes images
DC=0.539
DC=0.445
DC=0.810
DC=0.599
DC=0.486
DC=0.849
DC=0.218
DC=0.248
DC=0.567
DC=0.620
DC=0.400
DC=0.452
a
b
a1
a2
a3
a4
a5
a6
b1
b2
b3
b4
b5
b6
c1
c2
c3
c4
c5
c6
d1
d2
d3
d4
d5
d6
Fig. 20 | Shen et al. method. (a) The flow chart of proposed method. (b) The detected results. Figure reproduced with permission from ref.198, In-
stitute of Electrical and Electronics Engineers. Splitting original
polarization image
Input
a Input Is S0 IE Fig. 20 | Shen et al. method. (a) The flow chart of proposed method. (b) The detected results. Figure reproduced with permission from ref.198, In-
stitute of Electrical and Electronics Engineers. MMT
MMPD
Mueller matrix
elements
Hand crafted
features
Detected
results
Fig. 21 | The architecture and results of multi-parameters fusion network (MPFN) and the corresponding results. Fig. 21 | The architecture and results of multi-parameters fusion network (MPFN) and the corresponding results. architecture and results of multi-parameters fusion network ability of the polarization parameter images. The experi-
ments demonstrated the feasibility of combining polari-
metric imaging with digital pathological techniques in
typical proliferative and secretory phases. main unclear. In data-driven polarimetric imaging, the
Muller matrix provides the most comprehensive inform-
ation representing the polarization information, and
most decomposition methods that provide raw funda-
mental parameters have been proposed. Thus, the
Mueller matrix is crucial in exploring the interpretation
of network layers. Yang K et al. Opto-Electron Sci 3, 230042 (2024) For example,
complex cervical precancerous samples exhibit polariza-
tion characteristics of various types of anisotropic super-
positions. The depolarization ability of precancerous cer-
vical samples changed with the development of lesions. In addition, changes in retardation and depolarization
occur during the propagation and scattering of patholo-
gical cervical samples at different stages. Therefore, the
proposed method has high sensitivity and precision for
the screening of cervical lesion pathological tissues, and
may bring physical interpretability to the CNN. D =
xT
1
xT
2... xT
N
N×M
,
X = Dω =
xT
1 · ω
xT
2 · ω
... xT
N · ω
N×1
,
L (ω) = d(PNormal (X) , PCIN1 (X) )
−1 ,
(35) D =
xT
1
xT
2... xT
N
N×M
,
X = Dω =
xT
1 · ω
xT
2 · ω
... xT
N · ω
N×1
,
L (ω) = d(PNormal (X) , PCIN1 (X) )
−1 ,
(35)
where xi is an M×1 vector representing PBPs elements. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 In ref.61, the authors calculated the Pearson correla-
tion coefficients between the elements of the algal
Mueller matrix and features extracted by the CNN from
f0 to f15. The experiments demonstrate that features f2, f3,
f6, and f7 are positively correlated with depolarization-re-
lated elements; however, f1, f4, f10, f12, and f13 are negat-
ively correlated. In addition, the fast-axis-orientation-de-
pendent periodic variations were preserved in f0, f5, f9,
and f15. Dong et al. proposed a data-driven polarimetric
imaging framework and constructed a dual-modality
machine-learning framework for the quantitative dia-
gnosis of cervical precancerous lesions63, as shown in Fig. 22. The U-net architecture was adopted to segment the
epithelium in digitized cervical hematoxylin-eosin-
stained images and mask the corresponding cervical
sample’s polarimetry basis parameters (PBPs), which
were decomposed based on the MMPD, MMT, and oth-
er Mueller matrix rotation-invariant parameters. Fur-
thermore, these masked parameters are processed by the
designed statistical distance-based learning classifier for
deriving a polarimetry feature parameter (PFP). The
classifier of the negative class and those of the CIN1(mild
dysplasia) samples can be expressed as: M and N are the number of PBPs and target pixels, re-
spectively. X is an N×1 vector, calculated as a linear pro-
jection of the input PBPs. PNormal(X) is the probability
distribution of X from Normal cervical pathological tis-
sues, whereas PCIN1(X) represents CIN1 tissues. ω rep-
resents weight coefficients of PBPs. L(ω) is the energy
distance between PNormal(X) and PCIN1(X) by energy dis-
tance function d. PFP can be represented a simplified lin-
ear combination of the PBPs, which is similar to the dis-
tribution of specific microstructural variations. The dif-
ferent weights indicate the significance of the elements of
the PFP feature. The results demonstrate the physical interpretability
of the polarimetry feature parameters. For example,
complex cervical precancerous samples exhibit polariza-
tion characteristics of various types of anisotropic super-
positions. The depolarization ability of precancerous cer-
vical samples changed with the development of lesions. In addition, changes in retardation and depolarization
occur during the propagation and scattering of patholo-
gical cervical samples at different stages. Therefore, the
proposed method has high sensitivity and precision for
the screening of cervical lesion pathological tissues, and
may bring physical interpretability to the CNN. The results demonstrate the physical interpretability
of the polarimetry feature parameters. Physical interpretation of network layers However, the physical properties of network layers re- 230042-28 Input and utilization of polarization information Input information is a crucial element in network train-
ing with polarimetric imaging and deep learning. However, various inputs and polarization parameters ex-
ist for different tasks. Three perspectives were con-
sidered as network inputs: original polarization images
(OPI), polarimetric parameter feature maps (PPFM), and
associated parameter maps (APM), as shown in Fig. 25. Shaunak et al. transformed the information in an aug-
mented dataset into a compact representation of polari-
metric synthetic aperture radar data to classify and seg-
ment urban areas82. The segmentation of road scenes is a
typical application in which water hazards, transparent
glass, and metallic surfaces are key challenges. Yang et al. proposed the prediction of polarization information
from monocular RGB images as a complement to RGB-
based pixel-wise semantic segmentation for applications
in real-world wearable assistive navigation systems69, as
shown in Fig. 23. Similarly, Zhang et al.56 and Blanchon
et al.83 used different architectures to achieve the same
goals: robust and accurate scene parsing of outdoor en-
vironments paves the way for autonomous navigation
and relationship inference. Focusing on transparent ob-
ject segmentation, the polarization textures of transpar-
ent objects provide extra but very different information
than the background. Therefore, a polarized CNN frame- Original polarization images are among the most
widely used inputs. OPI refers to images captured dir-
ectly using a DoFP, camera, Mueller Matrix Polarization
Microscope, or other equipment. Because of the rapid re-
sponse and comprehensive polarization information
captured in one shot, raw super-pixel images captured by
DoFP are common input42−45. Other common inputs are
polarization-oriented images, usually set to 0°, 45°, and
90°46−49,52. Furthermore, there are variants with the addi-
tion of 135°50,51 or circular polarization information46. Parallel and perpendicular polarization components
were used to train the dehazing network72. Polarization RGB image
RGB & depth
image
Semantic segmentation/
polarization difference image
Fig. 23 | Yang et al. method: proposed architecture and produced results of depth and segmented Semantic segmentation/
polarization difference image RGB & depth
image Fig. 23 | Yang et al. method: proposed architecture and produced results of depth and segmented results. a
b
Class
Mask
Bbox
lntensity & DoLP & AoLP
Segmentation mask
Ground truth
Ground truth
Intensity Mask
R-CNN
Intensity Mask
R-CNN
Polarization
Mask R-CNN
Polarization
Mask R-CNN
mAP=0.281
mAP=0.511
mAP=0.878
mAP=0.889
Fig. 24 | Transparent object segmentation. (a) The designed architecture. Semantic segmentation Semantic segmentation Segmentation is a popular topic for understanding the
scene in remote sensing and automatic navigation fields. According to learning from different types of massive
data, the data-driven segmentation method achieved where xi is an M×1 vector representing PBPs elements. where xi is an M×1 vector representing PBPs elements. a
b
c
d
g
f
e
500 μm
500 μm
500 μm
500
μm
500
μm
Fixed χ
Select ROI
Caculate PBPs
Moving γ
Registration between
the two images by
transformation T
m11 image
Segmented by Mask f
Mask M
Select target
pixels by
mapping
Mask M to
PBPs
Generate
Mask M
M=T·f
T=χ·inv(γ)
H & E image
U-net for
epithelium
segmentation
0 0.0150.030
DL
PL
qL
rL
δ
DL
D
PL qL rL
δ
Target pixels in PBPs
Input data to
designed classifier
Analyze PFP
PBPs
0.04
0.03
0.02
0.01
0
0.005
0.010
0.015
0.020
0.025
Probability
Sets of PBPs as input data
PFP's statistical features
as diagnostic indicators
Probility
x
Classification
Normal | CIN1 | CIN2 | CIN3
A statistical distance-based machine
learning classifier for deriving PFPs
ε(X, Y)=2E|X−Y|d−E|X−X′|d−E|Y−Y′|d
ε--Energy distance E|X|d<∞ E|Y|d<∞
Fig. 22 | Polarization-imaging-based ML framework for quantitative diagnosis of cervical precancerous lesions. Figure reproduced with
permission from ref.174, Institute of Electrical and Electronics Engineers. 230042-29 Fig. 22 | Polarization-imaging-based ML framework for quantitative diagnosis of cervical precancerous lesions. Figure reproduced with
permission from ref.174, Institute of Electrical and Electronics Engineers. 230042-29 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 work can be trained based on the intensity and polariza-
tion information57, which will be helpful for applications
in broad areas such as robotics, autonomous driving, and
face authentication, as shown in Fig. 24. The mean
average precision(mAP) is used to measure accuracy. good performance. However, intensity-based methods
always suffer from degradation in scenes with similar
colors, clutter, or reflective areas208−212. This choice is ori-
ented towards polarimetric imaging, which provides the
ability to distinguish and recover from changes in com-
plex scenes. Several approaches have been proposed to
achieve segmentation of remote sensing, road scenes,
and transparent objects via polarimetric imaging and
deep learning. Discussion Input and utilization of polarization information Datasets speckle images are another type of OPI66,85 captured by
the detector after scattering the media. Owing to the demand for different tasks, the number of
data-driven polarimetric imaging datasets has gradually
increased, as listed in Table 5. Seven types of datasets
were associated with the corresponding tasks described
in previous section. Three strategies for building the
datasets were considered. First, ground truths corres-
ponding to the inputs exist; therefore, the real ground
truth is captured directly to rectify the outputs. Second,
the transfer function of the imaging system was con-
sidered, and the generative process was simulated to gen-
erate the ground truth. In addition, comparing different
traditional methods and selecting the best results regard-
ing the ground truth is another method that combines
the advantages of existing methods. The polarimetric parameter feature maps were calcu-
lated using the OPI. Based on the Stokes representation,
[S0, S1, S2] is a common set widely used in polarization
network training52,55,58,59. Furthermore, the DoP and AoP
computed by the OPI or Stokes vector images are a type
of new material for network training52−57. In biomedical
diagnosis, Mueller matrix images are the most common
input to the network91,95, and Mueller matrix parameters
decomposed by Mueller matrix images can also be fed
into network training62,208. Table 4 lists the existing de-
composition elements of Mueller matrix methods. Associated parameter maps are images in which OPI,
and PPFM are combined with other information or pre-
processing for different tasks. Intensity information is
the most common complement based on the OPI and
PPFM53,55−57,66,70,74,196. Similarly, the spectrum86 and
phase71,75 are general additions to the polarization in-
formation. In 3D shape reconstruction tasks, there are
different complements such as zenith and azimuth angle
maps derived from specular and diffuse reflection64,65,
viewing encoding, encoded AoP67, normalized color80,
and physics-based prior confidence81 based on different
conditions. The raw images were interpolated using the
bicubic interpolation method in the demosaicing task68. An overexposure mask is used in the network input to
avoid overexposed areas during reflection removal53. The
scene segmentation network utilizes HSL color space
representation by incorporating a polarizing pseudo-col-
or image83. The overall datasets have a large gap between each
other in terms of number and size, even in the same task,
as shown in Table 5, which would not avoid the differ-
ence in the extraction of features by the CNN. Datasets Further-
more, researchers use self-collected training and test
datasets; however, evaluating and comparing different
methods is challenging. Therefore, authoritative datasets
must be built for this task. Input and utilization of polarization information (b) The segmented results of intensity Mask R-CNN and polarized
Mask R-CNN in several dataset. Fig. 24 | Transparent object segmentation. (a) The designed architecture. (b) The segmented results of intensity Mask R-CNN and polarized
Mask R-CNN in several dataset. 230042-30 230042-30 Yang K et al. Opto-Electron Sci 3, 230042 (2024)
https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Input and utilization of polarization information
polarizer
DoFP
DoP
DoLP
DoCP
AoP
AoLP
AoCP
Polarization
orientation
images
Circular
polarization
image
Dual-rotating
retarder
images
Polarization
speckle
images
Stokes
vectors
MMT
Rotation
invariant
parameters
MMPD
Convolutional
neural
network
Mueller matrix images
Preprocessing
Polarizing
pseudo-color
image
Physics-
based prior
confidence
Normalized
color
Polarimetry feature
parameters
Viewing
encoding
Encoded
AOP
Diffuse
reflection
Phase
Spectrum
Intensity
Specular
reflection
a
b
c
Fig. 25 | Input and utilization of polarization information. (a) Original polarization images (OPI). (b) Polarimetric parameter feature maps
(PPFM). (c) Associated parameters maps (APM). Rotation
invariant
parameters Convolutional
neural
network Fig. 25 | Input and utilization of polarization information. (a) Original polarization images (OPI). (b) Polarimetric parameter feature maps
(PPFM). (c) Associated parameters maps (APM). Loss function Loss functions are critical elements, and their selection is
crucial in guiding network training. Each loss function
has advantages and disadvantages. Therefore, a specific
loss function was adopted based on the given task and
imaging environment. Table 6 lists the latest quality-loss
functions for data-driven polarimetric imaging. The 230042-31 Yang K et al. Opto-Electron Sci 3, 230042 (2024)
https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Table 4 | Existing decomposition of Mueller matrix. Decomposition of Mueller matrix
Physical meanings
Mueller matrix polar decomposition (MMPD)101
M = MΔMRMD
δ = arccos
[
(MR(2, 2) + MR(3, 3))2+
(MR(3, 2) + MR(2, 3))2
] 1
2 −1
Linear retardation
D =
√
m2
12 + m2
13 + m2
14 (0 ⩽D ⩽1)
Diattenuation
Δ = 1 −|tr (MΔ −1)|
3
(0 ⩽Δ ⩽1)
Depolarization
θ = 0.5arctan
( r2
r1
)
Orientation of fast axis
ψ = 1
2 arctan
[ MR(3, 2) −MR(2, 3)
MR(2, 2) −MR(3, 3)
]
Optical rotation
Mueller matrix transformation (MMT)102
t1 =
√
(m22 −m33)2 + (m23 + m32)2
2
Anisotropy degree
t2 =
√
m2
21 + m2
31
2
b = m22 + m33
2
Polarizance
A = 2b · t1
b2 + t2
1
∈[0, 1]
Normalized anisotropy
t3 =
√
m2
42 + m2
43
2
Linear retardation
Rotation invariant parameters213−215
PL =
√
m2
21 + m2
31 ∈[0, 1]
Linear polarizance
PC = m41 ∈[−1, 1]
Circular polarizance
DL =
√
m2
12 + m2
13 ∈[0, 1]
Linear diattenuation
DC = m14 ∈[−1, 1]
Circular diattenuation
qL =
√
m2
42 + m2
43 ∈[0, 1]
Capability of transforming between linear and circular polarizations
rL =
√
m2
24 + m2
34 ∈[0, 1]
αP = 1
2 arctan
( m31
m21
)
Orientation of maximum transmittance
αD = 1
2 arctan
( m13
m12
)
αq = 1
2 arctan
(
−m42
m43
)
Orientation of fast axis
αr = 1
2arctan
(
−m24
m34
)
Mueller matrix anisotropy coefficients103
α =
√
1
Σ
[
(m01 + m10)2 + (m23 −m32)2]
Σ = 3m2
00 −
(
m2
11 + m2
22 + m2
33
)
+ m01m10 + m02m20 + m03m30
−(m23m32 + m13m31 + m12m21)
Horizontal linear anisotropy
β =
√
1
Σ
[
(m02 + m20)2 + (m13 −m31)2]
45° linear anisotropy
γ =
√
1
Σ
[
(m03 + m30)2 + (m12 −m21)2]
Circular anisotropy
230042-32 Table 4 | Existing decomposition of Mueller matrix. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Therefore, the measured information consists
of polarimetric representations, such as DoP(DoLP),
AoP(AoLP), Stokes vectors, Mueller matrix, spectrum,
HSV color space, and their perceptual representations
computed by a network model VGG. ˆy
where y is the ground truth, and is the output of the
network. Therefore, the measured information consists
of polarimetric representations, such as DoP(DoLP),
AoP(AoLP), Stokes vectors, Mueller matrix, spectrum,
HSV color space, and their perceptual representations
computed by a network model VGG. LSSIM = 1 −
2μyμˆy + c1
μ2
y + μ2
ˆy + c1
·
2σyˆy + c2
σ2
y + σ2
ˆy + c2
,
(39) (39) μ∗
σ∗
σyˆy
ˆy
where
and
denote the mean and standard devi-
ations of the image, respectively. is the cross-covari-
ance computed from the images of y and . where c1 and
c2 are constants. ii) The mean absolute error (MAE) is a widely used in-
dicator. Compared to the MSE, the MAE has less blurri-
ness and noise; however, it is more unstable. The utiliza-
tion of the MAE loss function was similar to that of MSE
because the range of AoLP is 0 to π that always maps in-
to 0–177. However, 0 and 1 indicate the same physical
meaning where the error is the largest. Therefore, the
HSV spatial display rule was introduced to design a
closer distance on the circle of the AoLP. The loss func-
tion is defined as follows: Yang K et al. Opto-Electron Sci 3, 230042 (2024) Mueller matrix transformation (MMT)102 Mueller matrix transformation (MMT)102 t1 =
√
(m22 −m33)2 + (m23 + m32)2
2
t2 =
√
m2
21 + m2
31
2
b = m22 + m33
2
A = 2b · t1
b2 + t2
1
∈[0, 1]
t3 =
√
m2
42 + m2
43
2 Capability of transforming between linear and circular polarizations Mueller matrix anisotropy coefficients103 230042-32 230042-32 Yang K et al. Opto-Electron Sci 3, 230042 (2024)
https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) Table 4 (Continued)
Decomposition of Mueller matrix
Physical meanings
Differential decomposition104
m =
16
∑
n=1
mn =
κi
κq + κ′
q
κu + κ′
u
κv + κ′
v
κq −κ′
q
κi −κ′
i,q
ηv + η′
v
ηu + η′
u
κu −κ′
u
−ηv + η′
v
κi −κ′
i,u
ηq + η′
q
κv −κ′
v
−ηu + η′
u
−ηq + η′
q
κi −κ′
i,v
Differential Mueller matrix for depolarizing anisotropic media
Symmetric decomposition105
M = MD2MR2MΔdMT
R1MD1
Symmetric decomposition of Mueller matrix
M = MR2M′
D2MΔdM′
D1MT
R1
M = MD2MR2MΔdM′
D1MT
R1
M = MR2M′
D2MΔdMT
R1MD1
Cloude decomposition106
mij = 1
2 Tr
(
Tcη4i+j
)
Cloude decomposition following is a detailed description of several functions
that differ from the intensity loss functions. is different from the other information forms, ensuring
that the CS loss becomes the most suitable indicator. The
loss function is defined as follows: i) Mean squared error (MSE) is the most widely used
indicator in deep learning, which standards the differ-
ence between the output images and the ground truth. In
data-driven polarimetric imaging, in addition to the dif-
ference in intensity, the polarization parameters also en-
sure the accuracy of the polarimetric representations. The loss can be expressed as: LCS = 1 −
y · ˆy
∥y∥2∥ˆy∥2
. (38) (38) iv) SSIM is a widely used indicator of end-to-end net-
works. SSIM focuses on the brightness, contrast, and
structural similarity between two images. The SSIM im-
proves as the value increases within the range of [0, 1],
opposite to the goal of minimizing the similarity. The
loss function is defined as follows: LMSE = ∥y −ˆy∥2 ,
(36) (36) ˆy
where y is the ground truth, and is the output of the
network. Future of data-driven polarimetric imaging The field of polarimetric imaging has been influenced by
deep learning, which has recently become one of the
most disruptive technologies. First, we analyze the trends
in data-driven polarimetric imaging, focusing on the ap-
plication of data-driven polarimetric imaging and re-
view existing research achievements. Furthermore, the
acquisition of high-accuracy polarization information is
the foundation for subsequent imaging and semantic
processing. Descattering, 3D shape reconstruction, re-
flection removal, biomedical imaging, pathological dia-
gnosis, target detection, and semantic segmentation are
crucial in the application of data-driven polarimetric
imaging. A comprehensive discussion is essential owing LMAE,AOLP = min
(
∥y −ˆy∥1, 1 −∥y −ˆy∥1
)
. (37) (37) iii) Cosine similarity (CS) is commonly used in the
surface-normal estimation of 3D reconstructions based
on polarimetric imaging. In the 3D polarimetric ima-
ging task, the surface normal map is calculated using
Fresnel's formula to generate the normal vectors, which iii) Cosine similarity (CS) is commonly used in the
surface-normal estimation of 3D reconstructions based
on polarimetric imaging. In the 3D polarimetric ima-
ging task, the surface normal map is calculated using
Fresnel's formula to generate the normal vectors, which 230042-33 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 to the input and utilization of polarization information,
datasets and loss functions
Strengths
Data-driven polarimetric imaging represents a new
Table 5 | Summary of data-driven polarimetric imaging datasets. Task
Data
Dataset
Paired or not
No. Future of data-driven polarimetric imaging of images
Size
Synthetic/Real
Section Restoration and enhancement of
accurate polarization information
2020
Hu42
Paired
300000
64×64
Real
2022
LLCP50
Paired
—
64×64
Real
2018
Zhang68
Paired
215
640×480
Synthetic
2019
Zeng43
Paired
76890
40×40
Synthetic
2021
Wu44
Paired
—
64×64
Synthetic
2020
Li45
Paired
137000
64×64
Synthetic
2022
Yang78
Paired
337
1961×2381
Real
2022
Liu77
Paired
140000
64×64
Real
2021
Ting79
Paired
42228
512×512
Real
2022
Si58
Paired
—
256×256
Real
Section Polarimetric descattering
2020
Hu46
Paired
103000
64×64
Real
2021
Zhang70
Paired
2000
256×256
Real
2021
Ren148
Paired
—
—
Real
2021
Zhou48
Paired
—
240×240
Synthetic
2022
Shi72
Paired
60000
960×576
Synthetic
2022
Ding47
Paired
700
512×512
Real+Synthetic
2022
Li66
Paired
5000
256×256
Real+Synthetic
Section Three-dimensional shape
reconstruction
2019
Ba64
Paired
300
256×256
Real
2020
SURREAL+PHSPD65
Paired
312915
—
Real
2020
Kondo71
Paired
—
—
Real+Synthetic
2021
Lei67
Paired
522
1224×1024
Real
2021
Deschaintre80
Paired
100000
512×512
Synthetic
2022
TransSfP81
Paired
936
512×512
Real+Synthetic
Section Reflection removal
2018
URD73
Paired
—
128×128
Synthetic
2019
Lyu74
Paired
—
256×256
Synthetic
2020
Lei53
Paired
100107
—
Real
Section Target detection
2018
Fan54
Paired
153
—
Real
2019
Blin52
Paired
2730
—
Real
2020
Sun59
Paired
—
96×96
Real
2020
CASIA-DOLP196
Unpaired
10697
224×224
Real
2021
USMANI197
Paired
240
—
Real
2022
Gao51
Paired
60
1224×1024
Real
Section Biomedical imaging and
pathological diagnosis
2017
Li60
Paired
10463
—
Real
2018
Li61
Paired
12162
—
Real
2020
Zhao62
Paired
100000
—
Real
2021
Zhou86
Paired
4500
40×40
Real
2021
Yao87
Paired
400
64×64
Real
2021
Dong63
Unpaired
49870
128×128
Real
Section Semantic segmentation
2018
Yang69
Paired
9736
320×240
Real
2019
POLABOT56
Paired
700
256×256
Real
2020
Kalra57
Paired
1000
—
Real Table 5 | Summary of data-driven polarimetric imaging datasets. Table 5 | Summary of data-driven polarimetric imaging datasets. to the input and utilization of polarization information,
datasets, and loss functions. to the input and utilization of polarization information,
datasets, and loss functions. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Table 6 | Summary of loss functions. Loss function
Applications
Frobenius matrix norm45,46,68
Section Restoration and enhancement of accurate polarization information, Section Polarimetric
descattering
Weighted mean squared error (WMSE)70
Section Polarimetric descattering
Preceptual loss53,70,77
Section Polarimetric descattering
Mean squared error
(MSE)46,48,49,61,65,71−74,76−78
Section Restoration and enhancement of accurate polarization information, Section Polarimetric
descattering, Section Three-dimensional shape reconstruction, Section Reflection removal
Mean absolute
Error(MAE)42−44,47,48,50,66,75,79,80,148
Section Restoration and enhancement of accurate polarization information, Section Polarimetric
descattering, Section Three-dimensional shape reconstruction
Negative pearson correlation coefficient
(NPCC)66
Section Polarimetric descattering
Cosine similarity (CS)64,65,67,81
Section Three-dimensional shape reconstruction
Perceptual normalized cross-correlation
(PNCC)53
Section Reflection removal
Structural similarity index (SSIM)43,79
Section Restoration and enhancement of accurate polarization information
Total variation (TV)58,75
Section Restoration and enhancement of accurate polarization information
Contrastive loss196
Section Target detection
Logistic regression87
Section Biomedical imaging and pathological diagnosis
Ridge loss87
Section Biomedical imaging and pathological diagnosis
Focal loss69
Section Semantic segmentation
Cross entropy loss (CEL)83
Section Semantic segmentation Table 6 | Summary of loss functions. representation domains. Applying the information rep-
resented by the physical model and the deep network
layers enables researchers to exploit the potential fea-
tures embedded in all information-transmitted paths. ric imaging network, researchers must weigh the costs
associated with data-driven polarimetric imaging and
conventional approaches, which include the establish-
ment of polarimetric imaging datasets, storage of data al-
ways four times that of traditional datasets, and imaging
systems. Because balancing these costs and benefits is not
exact, some uncertainties are considered in this process. During the imaging process, the light source, trans-
mitted media, imaging system, and image processing
method influence visual performance. Moreover, model-
ing these complex processes using physical functions or
traditional methods is challenging. Network layers are
high-order polynomials expressed by the convolution
neural network. Therefore, nonlinear representations of
the network layers may be promising for simulating the
process. A fundamental element of data-driven polarimetric
imaging is the availability of comprehensive datasets. Most data-driven polarimetric imaging methods have fo-
cused on supervised deep learning. However, based on
polarimetric imaging methods and imaging environ-
ments, the corresponding ground truth is more challen-
ging to capture than in an intensity-based network. Ad-
ditionally, different polarimetric imaging techniques in-
troduce various errors and influence the visual perform-
ance of the network. Yang K et al. Opto-Electron Sci 3, 230042 (2024) For example, the division of time al-
ways suffers from mismatching in dynamic scenes, and
the division of the focal plane has a mosaicking problem
in principle. Existing methods have established their
datasets to respond to specific tasks. However, ensuring
similar performance from other datasets is challenging;
moreover, the existing dataset is insufficient to cover all
conditions, and its generalization ability is insufficient. Real data changes over time, indicating increasing
volume and improper handling of methods. The introduction of deep learning into conventional
polarimetric imaging and pattern recognition generates
more accurate coefficients for the physical model, simu-
lating a complicated processing approach not modeled
by physical functions. However, introducing a polari-
metric imaging model into the deep network would add
physical constraints and polarization information to
guide the network training to achieve better perform-
ance compared with the intensity network. Therefore,
data-driven polarimetric imaging probably enables cap-
abilities that cannot be realized using traditional methods. Weaknesses Strengths Data-driven polarimetric imaging represents a new
paradigm that combines deep learning and traditional
physical properties, including altering the patterns of
physical properties to achieve better results and balan-
cing the physical model and information extraction
between the traditional physical and high-order nonlinear This section evaluates the future of data-driven polari-
metric imaging based on its strengths, weaknesses, and
opportunities. Furthermore, this approach is suitable for
developing instructive strategies for further studies that
combine deep learning and polarization information. 230042-34 The opportunities of methods The assistance of physical model: A synthetic dataset
solves the scarcity of datasets. An accurate physical mod-
el that simulates information transmission is crucial in
dataset generation. With an additional physical con-
straint on the CNN, fewer training data are required to
achieve a more generalized result than conventional
methods. In addition, to obtain a synthetic dataset, vari-
ous parameters covering different imaging conditions
are crucial. Thus, the development of conventional po-
larimetric imaging methods is suitable for designing net-
work architectures. Black boxes and their acceptance by applied research-
ers are inherent drawbacks of deep learning methods by
health professionals. Most researchers in practical ap-
plied fields are wary because deep learning theories have
not yet provided a complete and reasonable answer. In
addition, further development and optimization would
depend only on the performance of tasks without any
guidance from theories, which results in indeterminacy
in the study. Moreover, the legal implications of black
box functionality could be another challenge. For ex-
ample, who would be responsible if the results were in-
correct in pathological diagnosis or target detection? In
data-driven polarimetric imaging, the introduction of
polarization information may be helpful for the inter-
pretability of deep learning. Dong et al. attempted to use
a linear projection of input PBPs to interpret their signi-
ficance by learning the factors of each parameter. However, many studies have been conducted to achieve
these goals63. Unsupervised or semi-supervised learning216−218: Ob-
taining the ground truth for a large polarization dataset
is challenging. Therefore, unsupervised or semi-super-
vised learning is required to reduce the dependence on
the ground truth. However, image enhancement or im-
age processing is an end-to-end task; thus, existing learn-
ing methods without ground truth achieve poor per-
formance. A more comprehensive physical model must
be established, and more effective loss functions de-
signed to guide pipelines. In addition, the middle para-
meter may be generated without the ground truth, which
is also a feasible way to improve the performance. Transfer learning219,220: Transfer learning allows the
optimized parameters for one dataset to train a new net-
work as initialization values for another dataset, which is
a feasible approach for reducing the dependence on data-
sets in data-driven polarimetric imaging because the
learned features can be promptly transferred from a
trained network to a new network for another task. Weaknesses To achieve a better performance by training a polarimet- The loss function, which is crucial in guiding the The loss function, which is crucial in guiding the 230042-35 230042-35 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) training of the network, is always similar to the intensity-
based methods used in data-driven polarimetric imaging. General operators replace intensity images with polariza-
tion parameters. However, the intensity and polarization
information have entirely different optical properties. The intensity image describes the reflectivity and trans-
missivity of the object; however, the polarization image
describes the texture details, material properties, shape,
shading, and roughness. These differences determine the
disparate designs of loss functions. However, there are
insignificant loss functions that focus on polarization
parameters. ging or training theories to guide the optimization pro-
cess. In addition, three broad application areas: descat-
tering imaging, even high-scattering media, detection of
camouflage, spoofing targets, and enhancement and fu-
sion of information assess the potential of data-driven
polarimetric imaging in future applications. The opportunities of methods The
fine-tuning technique is a typical method used in trans-
fer learning, which is faster and easier than training a
network from scratch. Therefore, the extracted features
were similar to the shallow layers; moreover, the shallow
layers in a trained network can be copied to the new net-
work for another task to reduce the cost of training time. The combination and utilization of polarization in-
formation in deep learning are still in their infancy. In
most existing methods, polarization parameter images
are the only approach that uses polarization information
as the input into the network. The extraction of polariza-
tion features relies on the automatic processing of net-
work layers, which remains a challenge to utilize polariz-
ation information despite the unlimited opportunity for
improvement yet to be explored. Opportunities Based on the weaknesses of data-driven polarimetric
imaging, solutions have been proposed to address these
gaps. Furthermore, many novel training methods and
physical models exist, such as unsupervised or semi-su-
pervised training, transfer learning, and computational
imaging. Therefore, it must be combined with other ima- Multi-dimensional learning221,222: The introduction of
polarization information that displays different physical
properties into a traditional intensity network can
provide more constraints and information sources to 230042-36 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 For descattering imaging in high-scattering media,
such as clouds, water, haze, smoke, smog, fog, mist in the
air, soil particles, algae, and mineral salt, in underwater
scenes, there are potential opportunities for data-driven
polarimetric imaging. However, further development of
physical models has amplified this ability. However, tra-
ditional model functions are challenging to handle in
complex imaging environments and always use simple
assumptions to simulate real parameters. The introduc-
tion of deep learning can model complicated conditions
in nonlinear cases using convolutional neural layers. Fu-
ture opportunities will arise from the development of
more accurate parameters for forming improved ima-
ging functions generated by deep learning. promote network inference. Similarly, the phase, spec-
trum, and other physical properties can be embedded in-
to a network to enhance performance. Phase is a repres-
entative generation of the change in light, and the spec-
trum describes the characteristics of the wavelength of
light. These properties would fill the gap in which a
single domain cannot fully represent the light domain for
all physical properties. Federated learning223: To provide a practical training
set for deep learning in polarimetric imaging applica-
tions, obtaining various available datasets from different
institutes or corporations may be a possible solution. There are several datasets for different tasks, as shown in
Table 5; however, the datasets collected by different
groups are not uniform because it is difficult to guaran-
tee a similar performance from other datasets. Therefore,
different datasets were beneficial, increasing the di-
versity of the collected samples. In addition, different
imaging systems, detectors, environments, and observa-
tion directions are challenging to simulate using existing
physical functions, which can improve the generaliza-
tion ability of the network and avoid overfitting. There-
fore, this is instructive for building and optimizing the
evaluation criterion, focusing on polarization images. Spoofing targets are another opportunity for camou-
flage detection. Opportunities Target detection is widely applied in po-
larimetric imaging because the polarization information
can describe the material of an object, which is suitable
for camouflaging and spoofing targets of the same color
that the intensity information cannot distinguish. Next,
more comprehensive extraction of special features by the
neural network may further improve the success rate of
target detection. The material surface, texture, and contrast are the
main characteristics described by polarization informa-
tion for enhancing and fusing information. Polarization
parameters are observable in low- or hard-light environ-
ments because they are unaffected by intensity. Con-
sequently, the fusion of polarization and other images
can extend the feature domain of an object. The network
generates a fusion based on data-driven polarization fu-
sion, enhancing performance by extracting more fea-
tures and providing more information on imaging ob-
jects or scenes compared with artificial coefficients. Moreover, complementary features from various do-
mains are advantageous for other computer vision tasks,
such as object detection. Emergence of metasurface and metalens224−227: The util-
ization of lenses and metasurfaces allows for tailored
control over light with specific polarization states,
achieved through deliberate design. This deliberate con-
trol enables superior capture, separation, and analysis of
polarized light signals, thereby significantly enhancing
the sensitivity and accuracy of acquiring polarization
datasets. These advancements not only amplify the po-
tential of data-driven polarimetric imaging but also com-
plement the capabilities of deep learning methodologies,
promising refined insights and higher precision in polar-
ization imaging applications. References Fry ES, Voss KJ. Measurement of the Mueller matrix for phyto-
plankton. Limnol Oceanogr 30, 1322–1326 (1985). 22. Ronchi V. Barocas V. The Nature of Light: An Historical Sur-
vey (Harvard University Press, Cambridge, 1970). 1. Ronchi V. Barocas V. The Nature of Light: An Historical Sur-
vey (Harvard University Press, Cambridge, 1970). 1. Svensen Ø, Stamnes JJ, Kildemo M et al. Mueller matrix
measurements of algae with different shape and size distribu-
tions. Appl Opt 50, 5149–5157 (2011). 23. Huard S. Polarization of Light (Wiley, Chichester, 1997). 2. Huard S. Polarization of Light (Wiley, Chichester, 1997). 2. Schechner YY, Karpel N. Recovery of underwater visibility and
structure by polarization analysis. IEEE J Oceanic Eng 30,
570–587 (2005). 3. Wang WF, Lim LG, Srivastava S et al. Investigation on the po-
tential of Mueller matrix imaging for digital staining. J. Bio-
photonics 9, 364–375 (2016). 24. Li XB, Hu HF, Zhao L et al. Polarimetric image recovery meth-
od combining histogram stretching for underwater imaging. Sci
Rep 8, 12430 (2018). 4. Li XB, Hu HF, Zhao L et al. Polarimetric image recovery meth-
od combining histogram stretching for underwater imaging. Sci
Rep 8, 12430 (2018). 4. Du E, He HH, Zeng N et al. Mueller matrix polarimetry for dif-
ferentiating characteristic features of cancerous tissues. J Bio-
med Opt 19, 076013 (2014). 25. Liu TG, Guan ZJ, Li XB et al. Polarimetric underwater image
recovery for color image with crosstalk compensation. Opt
Laser Eng 124, 105833 (2020). 5. Liu TG, Guan ZJ, Li XB et al. Polarimetric underwater image
recovery for color image with crosstalk compensation. Opt
Laser Eng 124, 105833 (2020). 5. Le DL, Huynh TN, Nguyen DT et al. Characterization of
healthy and nonmelanoma-induced mouse utilizing the Stokes-
Mueller decomposition. J Biomed Opt 23, 125003 (2018). 26. Liang J, Ren LY, Qu ES et al. Method for enhancing visibility of
hazy images based on polarimetric imaging. Photonics Res 2,
38–44 (2014). 6. Liang J, Ren LY, Qu ES et al. Method for enhancing visibility of
hazy images based on polarimetric imaging. Photonics Res 2,
38–44 (2014). 6. Pierangelo A, Manhas S, Benali A et al. Multispectral Mueller
polarimetric imaging detecting residual cancer and cancer re-
gression after neoadjuvant treatment for colorectal carcino-
mas. J Biomed Opt 18, 046014 (2013). 27. Liu F, Han PL, Wei Y et al. Conclusion In the future, the optimization of methods will aim for
better visual performance in more widely applied fields. For polarimetric imaging, the methods that depend on
polarization properties include descattering imaging,
high-scattering media, detection of camouflage, spoof-
ing targets, and enhancement and fusion of information. Deep learning, nonlinear representation ability, and po-
tential feature extraction improve the accuracy of estim-
ating parameters and feasible transmitted functions com-
pared to conventional methods. This review provides an overview of recent efforts to
summarize data-driven polarimetric imaging based on
seven classifications and discusses them comprehens-
ively from three perspectives. Based on the application
fields, the classifications consist of polarimetric descat-
tering, 3D shape reconstruction, reflection removal, res-
toration, enhancement of polarization information, tar-
get detection, biomedical imaging and pathological dia-
gnosis, and semantic segmentation. Subsequently, we 230042-37 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Bronstein AM, Bronstein MM, Zibulevsky M et al. Sparse ICA
for blind separation of transmitted and reflected images. Int J
Imag Syst Technol 15, 84–91 (2005). 15. synthetically analyze the input, datasets, and loss func-
tion, which are crucial in data-driven polarimetric ima-
ging, listing the existing datasets and loss functions with
an evaluation of their advantages and disadvantages. In
conclusion, deep-learning-based polarimetric imaging
introduces polarization information into the convolu-
tional neural network to achieve better performance than
traditional intensity imaging, bringing physical inter-
pretability to CNN through physical models. Through
research on existing data-driven polarimetric imaging,
the study of the corresponding fields can be improved to
a higher level, enabling them to enhance high-level visu-
al tasks. Forssell G. Test and analysis of the detectability of personnel
mines in a realistic minefield by polarization in the infrared LW
region. Proc SPIE 5415, 187–195 (2004). 16. Forssell G. Passive IR polarization measurements applied to
covered surface landmines. Proc SPIE 5089, 547–557 (2003). 17. Cremer F, de Jong W, Schutte K. Infrared polarization meas-
urements and modelling applied to surface laid anti-personnel
landmines. Opt Eng 41, 1021–1032 (2002). 18. Aron Y, Gronau Y. Polarization in the LWIR: a method to im-
prove target aquisition. Proc SPIE 5783, 653–661 (2005). 19. Ratliff BM, Lemaster DA, Mack RT et al. Detection and track-
ing of RC model aircraft in LWIR microgrid polarimeter data. Proc SPIE 8160, 816002 (2011). 20. Voss KJ, Fry ES. Measurement of the Mueller matrix for ocean
water. Appl Opt 23, 4427–4439 (1984). 21. References Deeply seeing through highly tur-
bid water by active polarization imaging. Opt Lett 43,
4903–4906 (2018). 7. Shukla P, Pradhan A. Mueller decomposition images for cer-
vical tissue: potential for discriminating normal and dysplastic
states. Opt Express 17, 1600–1609 (2009). 28. Liu F, Wei Y, Han PL et al. Polarization-based exploration for
clear underwater vision in natural illumination. Opt Express 27,
3629–3641 (2019). 8. Pierangelo A, Nazac A, Benali A et al. Polarimetric imaging of
uterine cervix: a case study. Opt Express 21, 14120–14130
(2013). 29. Wei Y, Han PL, Liu F et al. Enhancement of underwater vision
by fully exploiting the polarization information from the Stokes
vector. Opt Express 29, 22275–22287 (2021). 9. Egan WG. Polarization in remote sensing. Proc SPIE 0891
(1988). https://doi.org/10.1117/12.944289
30. Li X, Liu F, Han PL et al. Near-infrared monocular 3D compu-
tational polarization imaging of surfaces exhibiting nonuniform
reflectance. Opt Express 29, 15616–15630 (2021). 10. David G, Thomas B, Dupart Y et al. UV polarization lidar for re-
mote sensing new particles formation in the atmosphere. Opt
Express 22, A1009–A1022 (2014). 31. Han PL, Cai YD, Liu F et al. Computational polarization 3D:
new solution for monocular shape recovery in natural condi-
tions. Opt Laser Eng 151, 106925 (2022). 11. Carotenuto V, de Maio A, Clemente C et al. Invariant rules for
multipolarization SAR change detection. IEEE Trans Geosci
Remote Sens 53, 3294–3311 (2015). 32. Cui ZP, Gu JW, Shi BX et al. Polarimetric multi-view stereo. In
Proceedings of 2017 IEEE Conference on Computer Vision
and Pattern Recognition (IEEE, 2017);
http://doi.org/10.1109/CVPR.2017.47. 12. Nagdimunov L, Kolokolova L, Mackowski D. Characterization
and remote sensing of biological particles using circular polar-
ization. J Quant Spectrosc Radiat Transfer 131, 59–65 (2013). 33. Kadambi A, Taamazyan V, Shi BX et al. Depth sensing using
geometrically constrained polarization normals. Int J Comput
Vis 125, 34–51 (2017). 13. Wang F, Ainouz S, Lian CF et al. Multimodality semantic seg-
mentation based on polarization and color images. Neurocom-
puting 253, 193–200 (2017). 34. Kong N, Tai YW, Shin JS. A physically-based approach to re-
flection separation: from physical modeling to constrained op-
timization. IEEE Trans Pattern Anal Mach Intell 36, 209–221
(2014). 14. Xiang KT, Yang KL, Wang KW. Polarization-driven semantic
segmentation via efficient attention-bridged fusion. Opt Ex-
press 29, 4802–4820 (2021). 35. Ni J, Zhang F, Ma F et al. Random region matting for the high-
36. References Ni J, Zhang F, Ma F et al. Random region matting for the high-
36. 230042-38 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 resolution PolSAR image semantic segmentation. IEEE J Sel
Top Appl Earth Obs Remote Sens 14, 3040–3051 (2021). resolution PolSAR image semantic segmentation. IEEE J Sel
Top Appl Earth Obs Remote Sens 14, 3040–3051 (2021). detection. In Proceedings of the 2018 25th IEEE International
Conference on Image Processing 3069–3073 (IEEE, 2018);
https://doi.org/10.1109/ICIP.2018.8451397. Li ZS, Sun JS, Fan Y et al. Deep learning assisted variational
Hilbert quantitative phase imaging. Opto-Electron Sci 2,
220023 (2023). 37. Xie RC, Zu HY, Xue Y et al. Target detection method for polar-
ization imaging based on convolutional neural network. Proc
SPIE 11455, 114557Z (2020). 55. Wang YYD, Wang H, Gu M. High performance “non-local” gen-
eric face reconstruction model using the lightweight Speckle-
Transformer (SpT) UNet. Opto-Electron Adv 6, 220049 (2023). 38. Zhang Y, Morel O, Blanchon M et al. Exploration of deep learn-
ing-based multimodal fusion for semantic road scene segment-
ation. In Proceedings of the 14th International Joint Confer-
ence on Computer Vision, Imaging and Computer Graphics
Theory and Applications 336–343 (SciTePress, 2019);
https://doi.org/10.5220/0007360403360343. 56. Guo YM, Zhong LB, Min L et al. Adaptive optics based on ma-
chine learning: a review. Opto-Electron Adv 5, 200082 (2022). 39. Li YX, Qian JM, Feng SJ et al. Deep-learning-enabled dual-fre-
quency composite fringe projection profilometry for single-shot
absolute 3D shape measurement. Opto-Electron Adv 5,
210021 (2022). 40. Kalra A, Taamazyan V, Rao SK et al. Deep polarization cues
for transparent object segmentation. In Proceedings of 2020
IEEE/CVF Conference on Computer Vision and Pattern Re-
cognition 8599–8608 (IEEE, 2020);
https://doi.org/10.1109/CVPR42600.2020.00863. 57. Chen YX, Zhang FY, Dang ZB et al. Chiral detection of bio-
molecules based on reinforcement learning. Opto-Electron Sci
2, 220019 (2023). 41. Hu HF, Lin Y, Li XB et al. IPLNet: a neural network for intens-
ity-polarization imaging in low light. Opt Lett 45, 6162–6165
(2020). 42. Si L, Huang TY, Wang XJ et al. Deep learning-based polariza-
tion feature retrieval from a single stokes vector. Proc SPIE
11963, 1196307 (2022). 58. Sun R, Sun XB, Chen FN et al. Polarimetric imaging detection
using a convolutional neural network with three-dimensional
and two-dimensional convolutional layers. Appl Opt 59,
151–155 (2020). 59. Zeng XL, Luo Y, Zhao XJ et al. References An end-to-end fully-convolu-
tional neural network for division of focal plane sensors to re-
construct S0, DoLP, and AoP. Opt Express 27, 8566–8577
(2019). 43. Wu RY, Zhao YQ, Li N et al. Real-time division-of-focal-plane
polarization imaging system with progressive networks. arXiv:
2110.13823 (2021). 44. Li XP, Liao R, Zhou JL et al. Classification of morphologically
similar algae and cyanobacteria using Mueller matrix imaging
and convolutional neural networks. Appl Opt 56, 6520–6530
(2017). 60. https://doi.org/10.48550/arXiv.2110.13823 Li XP, Liao R, Ma H et al. Polarimetric learning: a Siamese ap-
proach to learning distance metrics of algal Mueller matrix im-
ages. Appl Opt 57, 3829–3837 (2018). 61. Li XB, Li HY, Lin Y et al. Learning-based denoising for polari-
metric images. Opt Express 28, 16309–16321 (2020). 45. Hu HF, Zhang YB, Li XB et al. Polarimetric underwater image
recovery via deep learning. Opt Laser Eng 133, 106152
(2020). 46. Zhao YQ, Reda M, Feng K et al. Detecting giant cell tumor of
bone lesions using Mueller matrix polarization microscopic
imaging and multi-parameters fusion network. IEEE Sens J 20,
7208–7215 (2020). 62. Ding XY, Wang YF, Fu XP. Multi-polarization fusion generat-
ive adversarial networks for clear underwater imaging. Opt
Laser Eng 152, 106971 (2022). 47. Dong Y, Wan JC, Wang XJ et al. A polarization-imaging-based
machine learning framework for quantitative pathological dia-
gnosis of cervical precancerous lesions. IEEE Trans Med Ima-
ging 40, 3728–3738 (2021). 63. Zhou C, Teng MG, Han YF et al. Learning to dehaze with po-
larization. In Proceedings of the 35th Conference on Neural In-
formation Processing Systems (NeurIPS, 2021). 48. Zhu YM, Zeng TJ, Liu KW et al. Full scene underwater ima-
ging with polarization and an untrained network. Opt Express
29, 41865–41881 (2021). 49. Ba YH, Gilbert A, Wang F et al. Deep shape from polarization. In Proceedings of the 16th European Conference on Com-
puter Vision 554–571 (Springer, 2020);
https://doi.org/10.1007/978-3-030-58586-0_33. 64. Xu XY, Wan MG, Ge JY et al. ColorPolarNet: residual dense
network-based chromatic intensity-polarization imaging in low-
light environment. IEEE Trans Instrum Meas 71, 5025210
(2022). 50. Zou SH, Zuo ZX, Qian YM et al. 3D human shape reconstruc-
tion from a polarization image. In Proceedings of the 16th
European Conference on Computer Vision 351–368 (Springer,
2020); https://doi.org/10.1007/978-3-030-58568-6_21
65. Gao SH, Cao Y, Zhang WJ et al. Learning feature fusion for
target detection based on polarimetric imaging. Appl Opt 61,
D15–D21 (2022). 51. References Front Phys 9, 789232 (2022). 72. Jellison GE. Four-channel polarimeter for time-resolved ellip-
sometry. Opt Lett 12, 766–768 (1987). 89. Wieschollek P, Gallo O, Gu JW et al. Separating reflection and
transmission images in the wild. In Proceedings of the 15th
European Conference on Computer Vision 90–105 (Springer,
2018); https://doi.org/10.1007/978-3-030-01261-8_6. 73. Compain E, Drevillon B. Broadband division-of-amplitude po-
larimeter based on uncoated prisms. Appl Opt 37, 5938–5944
(1998). 90. Lyu YW, Cui ZP, Li S et al. Reflection separation using a pair
of unpolarized and polarized images. In Proceedings of the
33rd International Conference on Neural Information Pro-
cessing Systems 1304 (Curran Associates Inc. , 2019);
https://doi.org/10.5555/3454287.3455591. 74. Ju HJ, Ren LY, Liang J et al. A Mueller matrix measurement
technique based on a division-of-aperture polarimetric camera. Proc SPIE 10839, 108391F (2019). 91. Pezzaniti JL, Chenault DB. A division of aperture MWIR ima-
ging polarimeter. Proc SPIE 5888, 58880V (2005). 92. Liu TR, de Haan K, Bai BJ et al. Deep learning-based holo-
graphic polarization microscopy. ACS Photonics 7, 3023–3034
(2020). 75. Gao SK, Gruev V. Bilinear and bicubic interpolation methods
for division of focal plane polarimeters. Opt Express 19,
26161–26173 (2011). 93. Yang SL, Qu BW, Liu GS et al. Unsupervised learning polari-
metric underwater image recovery under nonuniform optical
fields. Appl Opt 60, 8198–8205 (2021). 76. York T, Gruev V. Calibration method for division of focal plane
polarimeters in the optical and near-infrared regime. Proc SPIE
8012, 80120H (2011). 94. Liu HD, Zhang YZ, Cheng ZZ et al. Attention-based neural net-
work for polarimetric image denoising. Opt Lett 47, 2726–2729
(2022). 77. Hsu WL, Davis J, Balakrishnan K et al. Polarization micro-
scope using a near infrared full-Stokes imaging polarimeter. Opt Express 23, 4357–4368 (2015). 95. Yang XJ, Zhao QH, Huang TY et al. Deep learning for denois-
ing in a Mueller matrix microscope. Opt Express 13,
3535–3551 (2022). 78. Goldstein DH. Polarized Light 3rd ed (CRC Press, Boca Raton,
2017). 96. Wu XS, Zhang H, Hu XP et al. HDR reconstruction based on
the polarization camera. IEEE Robotics Autom Lett 5,
5113–5119 (2020). 79. Li XB, Liu TG, Huang BJ et al. Optimal distribution of integra-
tion time for intensity measurements in stokes polarimetry. Opt
Express 23, 27690–27699 (2015). 97. Deschaintre V, Lin YM, Ghosh A. Deep polarization imaging
for 3D shape and SVBRDF acquisition. References Li DK, Lin B, Wang XY et al. High-performance polarization re-
mote sensing with the modified U-Net based deep-learning
network. IEEE Trans Geosci Remote Sens 60, 5621110
(2022). 66. Blin R, Ainouz S, Canu S et al. Road scenes analysis in ad-
verse weather conditions by polarization-encoded images and
adapted deep learning. In Proceedings of 2019 IEEE Intelli-
gent Transportation Systems Conference 27–32 (IEEE, 2019);
https://doi.org/10.1109/ITSC.2019.8916853. 52. Lei CY, Qi CY, Xie JX et al. Shape from polarization for com-
plex scenes in the wild. In Proceedings of 2022 IEEE/CVF
Conference on Computer Vision and Pattern Recognition
12622–12631 (IEEE, 2021);
https://doi.org/10.1109/CVPR52688.2022.01230. 67. Lei CY, Huang XH, Zhang MD et al. Polarized reflection re-
moval with perfect alignment in the wild. In Proceedings of
2020 IEEE/CVF Conference on Computer Vision and Pattern
Recognition 1747–1755 (IEEE, 2020);
https://doi.org/10.1109/CVPR42600.2020.00182. 53. Zhang JC, Shao JB, Luo HB et al. Learning a convolutional
demosaicing network for microgrid polarimeter imagery. Opt
Lett 43, 4534–4537 (2018). 68. Yang KL, Bergasa LM, Romera E et al. Predicting polarization
69. Fan W, Ainouz S, Meriaudeau F et al. Polarization-based car
54. 69. 230042-39 230042-39 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) beyond semantics for wearable robotics. In Proceedings of the
2018 IEEE-RAS 18th International Conference on Humanoid
Robots 96–103 (IEEE, 2018);
https://doi.org/10.1109/HUMANOIDS.2018.8625005. Wang YH, Louie DC, Cai JY et al. Deep learning enhances po-
larization speckle for in vivo skin cancer detection. Opt Laser
Technol 140, 107006 (2021). 85. Zhou XM, Ma L, Brown W et al. Automatic detection of head
and neck squamous cell carcinoma on pathologic slides using
polarized hyperspectral imaging and machine learning. Proc
SPIE 11603, 116030Q (2021). 86. Zhang R, Gui XY, Cheng HY et al. Underwater image recov-
ery utilizing polarimetric imaging based on neural networks. Appl Opt 60, 8419–8425 (2021). 70. Yao Y, Zuo M, Dong Y et al. Polarization imaging feature char-
acterization of different endometrium phases by machine
learning. OSA Continuum 4, 1776–1791 (2021). 87. Kondo Y, Ono T, Sun LG et al. Accurate polarimetric BRDF for
real polarization scene rendering. In Proceedings of the 16th
European Conference on Computer Vision 220–236 (Springer,
2020); https://doi.org/10.1007/978-3-030-58529-7_14. 71. Azzam RMA. Division-of-amplitude photopolarimeter (DOAP)
for the simultaneous measurement of all four Stokes paramet-
ers of light. Opt Acta Int J Opt 29, 685–689 (1982). 88. Shi YJ, Guo EL, Bai LF et al. Polarization-based haze removal
using self-supervised network. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 1109–1118 (2009). 1109–1118 (2009). Zhang JC, Shao JB, Chen JL et al. Polarization image fusion
with self-learned fusion strategy. Pattern Recognit 118, 108045
(2021). 127. Cloude SR. Group theory and polarisation algebra. Optik 75,
26–36 (1985). 106. Lin TY, Dollár P, Girshick R et al. Feature pyramid networks for
object detection. In Proceedings of 2017 IEEE Conference on
Computer Vision and Pattern Recognition 936–944 (IEEE,
2017); http://doi.org/10.1109/CVPR.2017.106. 128. Carnicer A, Javidi B. Polarimetric 3D integral imaging in
photon-starved conditions. Opt Express 23, 6408–6417 (2015). 107. Chen C, Chen QF, Xu J et al. Learning to see in the dark. In
Proceedings of 2018 IEEE/CVF Conference on Computer Vis-
ion and Pattern Recognition 3291–3300 (IEEE, 2018);
https://doi.org/10.1109/CVPR.2018.00347. 108. Song YB, Ma C, Gong LJ et al. CREST: convolutional residual
learning for visual tracking. In Proceedings of 2017 IEEE Inter-
national Conference on Computer Vision 2574–2583 (IEEE,
2017); https://doi.org/10.1109/iccv.2017.279. 129. https://doi.org/10.1109/CVPR.2018.00347. Tibbs AB, Daly IM, Roberts NW et al. Denoising imaging polar-
imetry by adapted BM3D method. J Opt Soc Am A 35,
690–701 (2018). 109. Xu Y, Wen J, Fei LK, Zhang Z. Review of video and image de-
fogging algorithms and related studies on image restoration
and enhancement. IEEE Access 4, 165–188 (2015). 130. Gao SK, Gruev V. Gradient-based interpolation method for di-
vision-of-focal-plane polarimeters. Opt Express 21, 1137–1151
(2013). 110. Guo Y, Liu RW, Lu YX et al. Haze visibility enhancement for
promoting traffic situational awareness in vision-enabled intelli-
gent
transportation. IEEE
Trans
Veh
Technol
72,
15421–15435 (2023). 131. Zhang JC, Luo HB, Hui B et al. Image interpolation for division
of focal plane polarimeters with intensity correlation. Opt Ex-
press 24, 20799–20807 (2016). 111. Zhang JC, Luo HB, Liang RG et al. Sparse representation-
based demosaicing method for microgrid polarimeter imagery. Opt Lett 43, 3265–3268 (2018). 112. Nguyen K, Nguyen P, Bui DC et al. Analysis of the influence of
de-hazing methods on vehicle detection in aerial images. Int J
Adv Comput Sci Appl 13, 846–856 (2022). 132. Ratliff BM, LaCasse CF, Tyo JC. Interpolation strategies for re-
ducing IFOV artifacts in microgrid polarimeter imagery. Opt Ex-
press 17, 9112–9125 (2009). 113. Liu JP, Wang SJ, Wang X et al. A review of remote sensing
image dehazing. Sensors 21, 3926 (2021). 133. Karavarsamis S, Gkika I, Gkitsas V et al. Yang K et al. Opto-Electron Sci 3, 230042 (2024) A survey of deep
learning-based image restoration methods for enhancing situ-
ational awareness at disaster sites: the cases of rain, snow
and haze. Sensors 22, 4707 (2022). 134. Wen SJ, Zheng YQ, Lu F et al. Convolutional demosaicing net-
work for joint chromatic and polarimetric imagery. Opt Lett 44,
5646–5649 (2019). 114. Song YF, Nakath D, She MK et al. Optical imaging and image
restoration techniques for deep ocean mapping: a compre-
hensive survey. PFG J Photogramm Remote Sens Geoinf Sci
90, 243–267 (2022). 135. Sargent GC, Ratliff BM, Asari VK. Conditional generative ad-
versarial network demosaicing strategy for division of focal
plane polarimeters. Opt Express 28, 38419–38443 (2020). 115. Sun YY, Zhang JC, Liang RG. Color polarization demosaick-
ing by a convolutional neural network. Opt Lett 46, 4338–4341
(2021). 116. Islam J, Xia YY, Sattar J. Fast underwater image enhance-
ment for improved visual perception. IEEE Robot Autom Lett 5,
3227–3234 (2020). 136. Pistellato M, Bergamasco F, Fatima T et al. Deep demosa-
icing for polarimetric filter array cameras. IEEE Trans Image
Process 31, 2017–2026 (2022). 117. Negi A, Chauhan P, Kumar K et al. Face mask detection clas-
sifier and model pruning with keras-surgeon. In Proceedings of
the 2020 5th IEEE International Conference on Recent Ad-
vances and Innovations in Engineering 1–6 (IEEE, 2020);
http://doi.org/10.1109/ICRAIE51050.2020.9358337. 137. Zhang JC, Chen JL, Yu HW et al. Polarization image demosa-
icking via nonlocal sparse tensor factorization. IEEE Trans
Geosci Remote Sens 60, 5607210 (2021). 118. Hagen N, Otani Y. Stokes polarimeter performance: general
noise model and analysis. Appl Opt 57, 4283–4296 (2018). 119. Narasimhan SG, Nayar SK. Vision and the atmosphere. Int J
Comput Vis 48, 233–254 (2002). 138. Schechner YY, Narasimhan SG, Nayar SK. Polarization-based
Vision through haze. Appl Opt 42, 511–525 (2003). 139. Li XB, Hu HF, Liu TG et al. Optimal distribution of integration
time for intensity measurements in degree of linear polariza-
tion polarimetry. Opt Express 24, 7191–7200 (2016). 120. Akkaynak D, Treibitz T. A revised underwater image formation
model. In Proceedings of 2018 IEEE/CVF Conference on
Computer Vision and Pattern Recognition 6723–6732 (IEEE,
2018); http://doi.org/10.1109/CVPR.2018.00703. 140. Lewis JJ, O’Callaghan RJ, Nikolov SG et al. Pixel- and region-
based image fusion with complex wavelets. Inf Fusion 8,
119–130 (2007). 121. Akkaynak D, Treibitz T. Sea-Thru: a method for removing wa-
ter from underwater images. References In Proceedings of 2021
IEEE/CVF Conference on Computer Vision and Pattern Re-
cognition 15562–15571 (IEEE, 2021);
https://doi.org/10.1109/CVPR46437.2021.01531. 80. Mueller H. On the theory of scattering of light. Proc Roy Soc A
Math Phys Eng Sci 166, 425–449 (1938). 98. Liu F, Zhang SC, Han PL et al. Depolarization index from
Mueller matrix descatters imaging in turbid water. Chin Opt
Lett 20, 022601 (2022). 99. Shao MQ, Xia CK, Yang ZD et al. Transparent shape from a
single view polarization image. arXiv: 2204.06331 (2023)
https://doi.org/10.48550/arXiv.2204.0633. 81. Ortega-Quijano N, Haj-Ibrahim B, García-Caurel E et al. Ex-
perimental validation of Mueller matrix differential decomposi-
tion. Opt Express 20, 1151–1163 (2012). 100. Lu SY, Chipman RA. Interpretation of Mueller matrices based
on polar decomposition. J Opt Soc Am A 13, 1106–1113
(1996). 101. De S, Bruzzone L, Bhattacharya A et al. A novel technique
based on deep learning and a synthetic target database for
classification of urban areas in PolSAR data. IEEE J Sel Top
Appl Earth Obs Remote Sens 11, 154–170 (2018). 82. He HH, Zeng N, Du E et al. A possible quantitative Mueller
matrix transformation technique for anisotropic scattering me-
dia/Eine mögliche quantitative Müller-Matrix-Transformations-
Technik für anisotrope streuende Medien. Photonics Lasers
Med 2, 129–137 (2013). 102. Blanchon M, Morel O, Seulin R et al. Outdoor scenes pixel-
wise semantic segmentation using polarimetry and fully convo-
lutional network. In Proceedings of the 14th International Joint
Conference on Computer Vision, Imaging and Computer
Graphics Theory and Applications 328–335 (SciTePress,
2019); https://doi.org/10.5220/0007360203280335. 83. Arteaga O, Garcia-Caurel E, Ossikovski R. Anisotropy coeffi-
cients of a Mueller matrix. J Opt Soc Am A 28, 548–553
(2011). 103. Akkaynak D, Treibitz T, Shlesinger T et al. What is the space
of attenuation coefficients in underwater computer vision? In
Proceedings of 2017 IEEE Conference on Computer Vision
and Pattern Recognition 568–577 (IEEE, 2017);
http://doi.org/10.1109/CVPR.2017.68. 84. Ortega-Quijano N, Arce-Diego JL. Mueller matrix differential
decomposition. Opt Lett 36, 1942–1944 (2011). 104. Ossikovski R. Analysis of depolarizing Mueller matrices
through a symmetric decomposition. J Opt Soc Am A 26,
105. 230042-40 230042-40 http://doi.org/10.1109/OCEANS.2010.5664428. IEEE International Conference on Robotics and Automation
7159–7165 (IEEE, 2018);
http://doi.org/10.1109/ICRA.2018.8460552. IEEE International Conference on Robotics and Automation
7159–7165 (IEEE, 2018);
http://doi.org/10.1109/ICRA.2018.8460552. Huang DM, Wang Y, Song W et al. Shallow-water image en-
hancement using relative global histogram stretching based on
adaptive parameter acquisition. In Proceedings of the 24th In-
ternational Conference on Multimedia Modeling 453–465
(Springer, 2018);
https://doi.org/10.1007/978-3-319-73603-7_37. 161. http://doi.org/10.1109/ICRA.2018.8460552. Anwar S, Li CY, Porikli F. Deep underwater image enhance-
ment, arXiv: 1807.03528 (2018). https://doi.org/10.48550/arX-
iv.1807.03528
145. Wang N, Zhou YB, Han FL et al. UWGAN: underwater GAN for
real-world underwater color restoration and dehazing. arXiv:
1912.10269 (2019). https://doi.org/10.48550/arXiv.1912.10269
146. Huang SC, Cheng FC, Chiu YS. Efficient contrast enhance-
ment using adaptive gamma correction with weighting distribu-
tion. IEEE Trans Image Process 22, 1032–1041 (2013). 162. He KM, Sun J, Tang XO. Single image haze removal using
dark channel prior. IEEE Trans Pattern Anal Mach Intell 33,
2341–2353 (2011). 147. Chao L, Wang M. Removal of water scattering. In Proceed-
ings of the 2010 2nd International Conference on Computer
Engineering and Technology V2-35–V2-39 (IEEE, 2010);
https://doi.org/10.1109/ICCET.2010.5485339. 163. Ren QM, Xiang YF, Wang GC et al. The underwater polariza-
tion dehazing imaging with a lightweight convolutional neural
network. Optik 251, 168381 (2022). 148. Liang J, Ren LY, Ju HJ et al. Visibility enhancement of hazy
images based on a universal polarimetric imaging method. J
Appl Phys 116, 173107 (2014). 164. Li BY, Peng XL, Wang ZY et al. AOD-Net: all-in-one dehazing
network. In Proceedings of 2017 IEEE International Confer-
ence on Computer Vision 4780–4788 (IEEE, 2017);
http://doi.org/10.1109/ICCV.2017.511. 149. Hitam MS, Awalludin EA, Yussof WNJHW et al. Mixture con-
trast limited adaptive histogram equalization for underwater im-
age enhancement. In Proceedings of 2013 International Con-
ference on Computer Applications Technology 1–5 (IEEE,
2013); http://doi.org/10.1109/ICCAT.2013.6522017. 165. Peng YT, Cao KM, Cosman PC. Generalization of the dark
channel prior for single image restoration. IEEE Trans Image
Process 27, 2856–2868 (2018). 150. Fan R, Wang HL, Cai PD et al. SNE-RoadSeg: incorporating
surface normal information into semantic segmentation for ac-
curate freespace detection. In Proceedings of the 16th
European Conference on Computer Vision 340–356 (Springer,
2020); https://doi.org/10.1007/978-3-030-58577-8_21. 166. Fu XP, Liang Z, Ding XY et al. Image descattering and absorp-
tion compensation in underwater polarimetric imaging. Opt
Laser Eng 132, 106115 (2020). 151. Drews PLJ, Nascimento ER, Botelho SSC et al. Underwater
depth estimation and image restoration based on single im-
ages. http://doi.org/10.1109/OCEANS.2010.5664428. IEEE Comput Graph Appl 36, 24–35 (2016). 152. Kazhdan M, Bolitho M, Hoppe H. Poisson surface reconstruc-
tion. In Proceedings of the Fourth Eurographics Symposium on
Geometry ProcessING 61–70 (2006);
https://dl acm org/doi/10 5555/1281957 1281965
167. Fu XY, Zhuang PX, Huang Y et al. A retinex-based enhancing
approach for single underwater image. In Proceedings of 2014
IEEE
International
Conference
on
Image
Processing
4572–4576 (IEEE, 2014);
http://doi.org/10.1109/ICIP.2014.7025927. 153. https://dl.acm.org/doi/10.5555/1281957.1281965. Qi XJ, Liao RJ, Liu ZZ et al. Geonet: geometric neural network
for joint depth and surface normal estimation. In Proceedings
of 2018 IEEE/CVF Conference on Computer Vision and Pat-
tern Recognition 283–291 (IEEE, 2018);
http://doi.org/10.1109/CVPR.2018.00037. 168. Fu XY, Fan ZW, Ling M et al. Two-step approach for single un-
derwater image enhancement. In Proceedings of 2017 Interna-
tional Symposium on Intelligent Signal Processing and Com-
munication Systems 789–794 (IEEE, 2017);
http://doi.org/10.1109/ISPACS.2017.8266583. 154. Huang JW, Zhou YC, Funkhouser T, Guibas L. FrameNet:
learning local canonical frames of 3D surfaces from a single
RGB image. In Proceedings of 2019 IEEE/CVF International
Conference on Computer Vision 8637–8646 (IEEE, 2019);
http://doi org/10 1109/ICCV 2019 00873
169. Li BY, Ren WQ, Fu DP et al. Benchmarking single-image de-
hazing and beyond. IEEE Trans Image Process 28, 492–505
(2019). 155. Wang R, Geraghty D, Matzen K et al. VPLNet: deep single
view normal estimation with vanishing points and lines. In Pro-
ceedings of 2020 IEEE/CVF Conference on Computer Vision
and Pattern Recognition 686–695 (IEEE, 2020);
http://doi.org/10.1109/CVPR42600.2020.00077. 170. Sakaridis C, Dai DX, Van Gool L. Semantic foggy scene un-
derstanding with synthetic data. Int J Comput Vis 126,
973–992 (2018). 156. Sakaridis C, Dai DX, Van Gool L. Model adaptation with syn-
thetic and real data for semantic dense foggy scene under-
standing. In Proceedings of the 15th European Conference on
Computer Vision (Springer, 2018);
https://doi.org/10.1007/978-3-030-01261-8_42. 157. Bansal A, Chen XL, Russell B et al. PixelNet: towards a gener-
al pixel-level architecture. arXiv: 1609.06694 (2016). https://arxiv.org/abs/1609.06694
171. Zhang YF, Ding L, Sharma G. HazeRD: an outdoor scene
dataset and benchmark for single image dehazing. In Proceed-
ings of 2017 IEEE International Conference on Image Pro-
cessing 3205–3209 (IEEE, 2017);
http://doi.org/10.1109/ICIP.2017.8296874. 158. Li B, Shen CH, Dai YC et al. Depth and surface normal estima-
tion from monocular images using regression on deep fea-
tures and hierarchical CRFs. In Proceedings of 2015 IEEE
Conference on Computer Vision and Pattern Recognition
1119–1127 (IEEE, 2015);
http://doi.org/10.1109/CVPR.2015.7298715. 172. Yang K et al. Opto-Electron Sci 3, 230042 (2024) In Proceedings of 2019 IEEE/CVF
Conference on Computer Vision and Pattern Recognition
1682–1691 (IEEE, 2019);
http://doi.org/10.1109/CVPR.2019.00178. 141. Nencini F, Garzelli A, Baronti S et al. Remote sensing image
fusion using the curvelet transform. Inf Fusion 8, 143–156
(2007). 122. Li ST, Kang XD, Hu JW. Image fusion with guided filtering. IEEE Trans Image Process 22, 2864–2875 (2013). 123. Hu HF, Zhao L, Li XB et al. Underwater image recovery under
the nonuniform optical field based on polarimetric imaging. IEEE Photonics J 10, 6900309 (2018). 142. Liu Y, Liu SP, Wang ZF. A general framework for image fusion
based on multi-scale transform and sparse representation. Inf
Fusion 24, 147–164 (2015). 124. Li ST, Kang XD, Fang LY et al. Pixel-level image fusion: a sur-
vey of the state of the art. Inf Fusion 33, 100–112 (2017). 125. Wei Y, Han PL, Liu F et al. Polarization descattering imaging: a
solution for nonuniform polarization characteristics of a target
surface. Chin Opt Lett 19, 111101 (2021). 143. Zhang JC, Shao JB, Chen JL et al. PFNet: an unsupervised
deep network for polarization image fusion. Opt Lett 45,
1507–1510 (2020). 126. Fabbri C, Islam J, Sattar J. Enhancing underwater imagery us-
ing generative adversarial networks. In Proceedings of 2018
144. Fabbri C, Islam J, Sattar J. Enhancing underwater imagery us-
ing generative adversarial networks. In Proceedings of 2018
144. 230042-41 230042-41 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) 2021); http://doi.org/10.1109/CVPR46437.2021.00074 SMPL: a skinned multi-
person linear model. ACM Trans Graph 34, 248 (2015). 182. Han PL, Li X, Liu F et al. Accurate passive 3D polarization face
reconstruction under complex conditions assisted with deep
learning. Photonics 9, 924 (2022). 183. Shen YX, Huang RR, He HH et al. Comparative study of the
influence of imaging resolution on linear retardance paramet-
ers derived from the Mueller matrix. Biomed Opt Express 12,
211–225 (2021). 200. Levin A, Weiss Y. User assisted separation of reflections from
a single image using a sparsity prior. IEEE Trans Pattern Anal
Mach Intell 29, 1647–1654 (2007). 184. Sun T, Liu T, He HH et al. Distinguishing anisotropy orienta-
tions originated from scattering and birefringence of turbid me-
dia using Mueller matrix derived parameters. Opt Lett 43,
4092–4095 (2018). 201. Li Y, Brown MS. Exploiting reflection change for automatic re-
flection removal. In Proceedings of 2013 IEEE International
Conference on Computer Vision 2432–2439 (IEEE, 2013);
https://doi.org/10.1109/ICCV.2013.302. 185. Rehbinder J, Haddad H, Deby S et al. Ex vivo Mueller polari-
metric imaging of the uterine cervix: a first statistical evalu-
ation. J Biomed Opt 21, 071113 (2016). 202. Zhou BL, Lapedriza A, Khosla A et al. Places: a 10 million im-
age database for scene recognition. IEEE Trans Pattern Anal
Mach Intell 40, 1452–1464 (2018). 186. Chue-Sang J, Bai YQ, Stoff S et al. Use of combined polariza-
tion-sensitive optical coherence tomography and Mueller mat-
rix imaging for the polarimetric characterization of excised bio-
logical tissue. J Biomed Opt 21, 071109 (2016). 203. Pang YX, Yuan MK, Fu Q et al. Progressive polarization based
reflection removal via realistic training data generation. Pat-
tern Recognit 124, 108497 (2022). 187. Novikova T, Pierangelo A, Manhas S et al. The origins of po-
larimetric image contrast between healthy and cancerous hu-
man colon tissue. Appl Phys Lett 102, 241103 (2013). 204. Girshick R, Donahue J, Darrell T et al. Rich feature hierarch-
ies for accurate object detection and semantic segmentation. In Proceedings of 2014 IEEE Conference on Computer Vision
and Pattern Recognition 580–587 (IEEE, 2014);
https://doi.org/10.1109/CVPR.2014.81. 188. Pierangelo A, Manhas S, Benali A et al. Ex vivo photometric
and polarimetric multilayer characterization of human healthy
colon by multispectral Mueller imaging. J Biomed Opt 17,
066009 (2012). 205. Girshick R. Fast R-CNN. In Proceedings of 2015 IEEE Interna-
tional Conference on Computer Vision 1440–1448 (IEEE,
2015); https://doi.org/10.1109/ICCV.2015.169. 189. 2021); http://doi.org/10.1109/CVPR46437.2021.00074 Liu W, Anguelov D, Erhan D et al. SSD: single shot multibox
detector. In Proceedings of the 14th European Conference on
Computer Vision 21–37 (Springer, 2016);
https://doi.org/10.1007/978-3-319-46448-0_2. 192. Zhu DZ, Smith WAP. Depth from a polarisation + RGB stereo
pair. In Proceedings of 2019 IEEE/CVF Conference on Com-
puter Vision and Pattern Recognition 7578–7587 (IEEE, 2019);
http://doi.org/10.1109/CVPR.2019.00777. 175. Law H, Deng J. CornerNet: detecting objects as paired keypo-
ints. In Proceedings of the 15th European Conference on
Computer Vision 765–781 (Springer 2018);
https://doi.org/10.1007/978-3-030-01264-9_45
193. Ding YQ, Ji Y, Zhou MY et al. Polarimetric helmholtz stereop-
sis. In Proceedings of 2021 IEEE/CVF International Confer-
ence on Computer Vision 5017–5026 (IEEE, 2021);
http://doi.org/10.1109/ICCV48922.2021.00499. 176. Wolff LB. Polarization-based material classification from spec-
ular reflection. IEEE Trans Pattern Anal Mach Intell 12,
1059–1071 (1990). 194. Atkinson GA, Hancock ER. Recovery of surface orientation
from diffuse polarization. IEEE Trans Image Process 15,
1653–1664 (2006). 177. Wolff LB. Surface orientation from polarization images. Proc
SPIE 850, 110–121 (1988). 195. Robles-Kelly A, Huynh CP. Imaging Spectroscopy for Scene
Analysis (Springer, London, 2013). 178. Tian Y, Zhang KB, Wang LY et al. Face anti-spoofing by learn-
ing polarization cues in a real-world scenario. In Proceedings
of the 4th International Conference on Advances in Image Pro-
cessing 129–137 (ACM, 2020);
https://doi.org/10.1145/3441250.3441254. 196. Frankot RT, Chellappa R. A method for enforcing integrability
in shape from shading algorithms. IEEE Trans Pattern Anal
Mach Intell 10, 439–451 (1988). 179. Miyazaki D, Tan RT, Hara K et al. Polarization-based inverse
rendering from a single view. In Proceedings of the Ninth IEEE
International Conference on Computer Vision 982–987 (IEEE,
2003); https://doi.org/10.1109/ICCV.2003.1238455. 180. Usmani K, Krishnan G, O'Connor T et al. Deep learning polari-
metric three-dimensional integral imaging object recognition in
adverse
environmental
conditions. Opt
Express
29,
12215–12228 (2021). 197. Mahmoud AH, El-Melegy MT, Farag AA. Direct method for
shape recovery from polarization and shading. In Proceedings
of the 2012 19th IEEE International Conference on Image Pro-
cessing 1769–1772 (IEEE, 2012);
https://doi org/10 1109/ICIP 2012 6467223
181. Shen Y, Lin WF, Wang ZF et al. Rapid detection of camou-
flaged artificial target based on polarization imaging and deep
learning. IEEE Photonics J 13, 7800309 (2021). 198. Liu T, Lu M, Chen BG et al. Distinguishing structural features
between Crohn’s disease and gastrointestinal luminal tubercu-
losis using Mueller matrix derived parameters. J. Biophotonics
12, e201900151 (2019). 199. https://doi.org/10.1109/ICIP.2012.6467223. Loper M, Mahmood N, Romero J et al. http://doi.org/10.1109/OCEANS.2010.5664428. Zhao SY, Zhang L, Huang SY et al. Dehazing evaluation: real-
world benchmark datasets, criteria, and baselines. IEEE Trans
Image Process 29, 6947–6962 (2020). 159. Atkinson GA. Polarisation photometric stereo. Comput Vis Im-
age Underst 160, 158–167 (2017). 173. Carlevaris-Bianco N, Mohan A, Eustice RM. Initial results in
underwater single image dehazing. In Proceedings of
OCEANS 2010 MTS/IEEE SEATTLE 1-8 (IEEE, 2010);
160. Fukao Y, Kawahara R, Nobuhara S et al. Polarimetric normal
stereo. In Proceedings of 2021 IEEE/CVF Conference on
Computer Vision and Pattern Recognition 682–690 (IEEE,
174. 230042-42 230042-42 230042-42 Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 2021); http://doi.org/10.1109/CVPR46437.2021.00074 2021); http://doi.org/10.1109/CVPR46437.2021.00074 Author contributions K Yang, F Liu and SY Liang contributed equally to this work and drafted the
manuscript. M Xiang contributed to the part on restoration and enhance-
ment of accurate polarization information. PL Han contributed on the part
on polarimetric descattering and three-dimensional shape reconstruction. JP Liu contributed to the part on reflection removal. X Dong contributed to
the part on target detection and semantic segmentation. Y Wei contributed
to the part on biomedical imaging and pathological diagnosis. BJ Wang, K
Shimizu, XP Shao provided resource support and supervised the project. All
authors read, corrected and approved the manuscript. Dy JG, Brodley CE. Feature selection for unsupervised learn-
ing. J Mach Learn Res 5, 845–889 (2004). 217. Dike HU, Zhou YM, Deveerasetty KK et al. Unsupervised
learning based on artificial neural network: a review. In Pro-
ceedings of 2018 IEEE International Conference on Cyborg
and Bionic Systems 322–327 (IEEE, 2018);
https://doi.org/10.1109/CBS.2018.8612259. 218. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Pan SJ, Tsang IW, Kwok JT et al. Domain adaptation via
transfer component analysis. IEEE Trans Neural Netw 22,
199–210 (2011). 220. Liu YJ, Jourabloo A, Liu XM. Learning deep models for face
anti-spoofing: binary or auxiliary supervision. In Proceedings of
2018 IEEE/CVF Conference on Computer Vision and Pattern
Recognition 389–398 (IEEE, 2018);
https://doi org/10 1109/CVPR 2018 00048
209. Stevens SY, Delgado C, Krajcik JS. Developing a hypothetical
multi-dimensional learning progression for the nature of matter. J Res Sci Teach 47, 687–715 (2010). 221. Xin M, Kundu S. Multi-task learning with high-dimensional
noisy images. J Am Stat Assoc (2021). 222. Wang T, He XM, Barnes N. Glass object localization by joint
inference of boundary and depth. In Proceedings of the 21st
International Conference on Pattern Recognition 3783–3786
(IEEE, 2012). 210. Li T, Sahu AK, Talwalkar A et al. Federated learning: chal-
lenges, methods, and future directions. IEEE Signal Process
Mag 37, 50–60 (2020). 223. Xu YC, Nagahara H, Shimada A et al. TransCut: transparent
object segmentation from a light-field image. In Proceedings of
2015 IEEE International Conference on Computer Vision
3442–3450 (IEEE, 2015);
https://doi.org/10.1109/ICCV.2015.393. 211. Rubin NA, D’Aversa G, Chevalier P et al. Matrix Fourier optics
enables a compact full-Stokes polarization camera. Science
365, eaax1839 (2019). 224. Zhang YX, Pu MB, Jin JJ et al. Crosstalk-free achromatic full
Stokes imaging polarimetry metasurface enabled by polariza-
tion-dependent phase optimization. Opto-Electron Adv 5,
220058 (2022). 225. Zhang SF, Wang XB, Liu AJ et al. A dataset and benchmark
for large-scale multi-modal face anti-spoofing. In Proceedings
of 2019 IEEE/CVF Conference on Computer Vision and Pat-
tern Recognition 919–928 (IEEE, 2019);
https://doi.org/10.1109/CVPR.2019.00101. 212. Tang DL, Shao ZL, Xie X et al. Flat multifunctional liquid crys-
tal elements through multi-dimensional information multiplex-
ing. Opto-Electron Adv 6, 220063 (2023). 226. Sun MH, He HH, Zeng N et al. Characterizing the microstruc-
tures of biological tissues using Mueller matrix and trans-
formed polarization parameters. Biomed Opt Express 5,
4223–4234 (2014). 213. Chen J, Wang DP, Si GY et al. Planar peristrophic multiplex-
ing metasurfaces. Opto-Electron Adv 6, 220141 (2023). 227. Acknowledgements Li PC, Lv DH, He HH et al. Separating azimuthal orientation
dependence in polarization measurements of anisotropic me-
dia. Opt Express 26, 3791–3800 (2018). 214. We are grateful for the financial support from the National Natural Science
Foundation of China (Nos. 62205259, 62075175, 61975254, 62375212,
62005203 and 62105254), the Open Research Fund of CAS Key Laboratory
of Space Precision Measurement Technology (No. B022420004), and the
Fundamental Research Funds for the Central Universities (No. ZYTS23125). Gil JJ. Invariant quantities of a Mueller matrix under rotation
and retarder transformations. J Opt Soc Am A 33, 52–58
(2016). 215. Le QV. Building high-level features using large scale unsuper-
vised learning. In Proceedings of 2013 IEEE International Con-
ference on Acoustics, Speech and Signal Processing
8595–8598 (IEEE, 2013);
https://doi.org/10.1109/ICASSP.2013.6639343. 216. 2021); http://doi.org/10.1109/CVPR46437.2021.00074 Dubreuil M, Babilotte P, Martin L et al. Mueller matrix polari-
metry for improved liver fibrosis diagnosis. Opt Lett 37,
1061–1063 (2012). 206. Ren SQ, He KM, Girshick R et al. Faster R-CNN: towards real-
time object detection with region proposal networks. IEEE
Trans Pattern Anal Mach Intell 39, 1137–1149 (2017). 190. Wang WF, Lim LG, Srivastava S et al. Roles of linear and cir-
cular polarization properties and effect of wavelength choice
on differentiation between ex vivo normal and cancerous gast-
ric samples. J Biomed Opt 19, 046020 (2014). 207. Redmon J, Divvala S, Girshick R et al. You only look once: uni-
fied, real-time object detection. In Proceedings of 2016 IEEE
Conference on Computer Vision and Pattern Recognition
779–788 (IEEE, 2016);
191. Chen GH, Wang JY, Zhang AJ. Transparent object detection
and location based on RGB-D camera. J Phys Conf Ser 1183,
208. 230042-43 230042-43 Competing interests Pan SJ, Yang Q. A survey on transfer learning. IEEE Trans
Knowl Data Eng 22, 1345–1359 (2010). 219. The authors declare no competing financial interests. Scan for Article PDF 230042-44
|
https://openalex.org/W3100757695
|
https://findresearcher.sdu.dk/ws/files/124858619/Baryogenesis_in_the_two_doublet_and_inert_singlet_extension_of_the_Standard_Model.pdf
|
English
| null |
Baryogenesis in the two doublet and inert singlet extension of the Standard Model
|
Journal of Cosmology and Astroparticle Physics
| 2,016
|
cc-by
| 14,268
|
Download date: 24. Oct. 2024 aryogenesis in the two doublet and inert singlet extension of the Standard Mode Citation for pulished version (APA):
Alanne, T., Kainulainen, K., Tuominen, K., & Vaskonen, V. (2016). Baryogenesis in the two doublet and inert
singlet extension of the Standard Model. Journal of Cosmology and Astroparticle Physics, 2016(8), Article 57. https://doi.org/10.1088/1475-7516/2016/08/057 Citation for pulished version (APA):
Alanne, T., Kainulainen, K., Tuominen, K., & Vaskonen, V. (2016). Baryogenesis in the two doublet and inert
singlet extension of the Standard Model. Journal of Cosmology and Astroparticle Physics, 2016(8), Article 57. https://doi.org/10.1088/1475-7516/2016/08/057 Go to publication entry in University of Southern Denmark's Research Portal Terms of use
This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: University of Southern Denmark Home
Search
Collections
Journals
About
Contact us
My IOPscience Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated these terms apply: • You may download this work for personal use only. y
p
y
• You may not further distribute the material or use it for any profit-making activity or commercial
Y
f
l di t ib t
th
URL id
tif i
thi
i y
p
y
You may not further distribute the material or use it fo y
p
y
• You may not further distribute the material or use it for any profit-makin at this document breaches copyright please contact us providing details and we will investigate your claim. enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright ple
Please direct all enquiries to puresupport@bib.sdu.dk Download date: 24. Oct. 2024 ournal of Cosmology and Astroparticle Physics
An IOP and SISSA journal
J ournal of Cosmology and Astroparticle Physics
An IOP and SISSA journal
J Article funded by SCOAP3. Content from this work may be used
under the terms of the Creative Commons Attribution 3.0 License.
Any further distribution of this work must maintain attribution to the author(s)
and the title of the work, journal citation and DOI. Article funded by SCOAP3. Content from this work may be used
under the terms of the Creative Commons Attribution 3.0 License.
Any further distribution of this work must maintain attribution to the author(s)
and the title of the work, journal citation and DOI. Baryogenesis in the two doublet and inert singlet extension of the Standard Model This content has been downloaded from IOPscience. Please scroll down to see the full text. Download details:
IP Address: 130.226.87.174
This content was downloaded on 26/05/2017 at 09:49
Please note that terms and conditions apply. View the table of contents for this issue, or go to the journal homepage for more
JCAP08(2016)057
(http://iopscience.iop.org/1475-7516/2016/08/057)
You may also be interested in:
A strong electroweak phase transition from the inflaton field
Tommi Tenkanen, Kimmo Tuominen and Ville Vaskonen
Scalar representations in the light of electroweak phase transition and cold dark matter
phenomenology
Shehu S. AbdusSalam and Talal Ahmed Chowdhury
Detectable gravitational waves from very strong phase transitions in the general NMSSM
Stephan J. Huber, Thomas Konstandin, Germano Nardini et al. A model for dark matter, naturalness and a complete gauge unification
Kimmo Kainulainen, Kimmo Tuominen and Jussi Virkajärvi
Signatures from scalar dark matter with a vector-like quark mediator
Federica Giacchino, Alejandro Ibarra, Laura Lopez Honorez et al. Asymmetric dark matter models and the LHC diphoton excess
Mads T. Frandsen and Ian M. Shoemaker
Lower bound on the electroweak wall velocity from hydrodynamic instability
Ariel Mégevand, Federico Agustín Membiela and Alejandro D. Sánchez
Fermi Bubbles under Dark Matter Scrutiny Part II: Particle Physics Analysis
Wei-Chih Huang, Alfredo Urbano and Wei Xue
The electroweak phase transition in the Inert Doublet Model
Nikita Blinov, Stefano Profumo and Tim Stefaniak A model for dark matter, naturalness and a complete gauge unification
Kimmo Kainulainen, Kimmo Tuominen and Jussi Virkajärvi Signatures from scalar dark matter with a vector-like quark mediator
Federica Giacchino, Alejandro Ibarra, Laura Lopez Honorez et al. Lower bound on the electroweak wall velocity from hydrodynamic instability
Ariel Mégevand, Federico Agustín Membiela and Alejandro D. Sánchez Fermi Bubbles under Dark Matter Scrutiny Part II: Particle Physics Analysis
Wei-Chih Huang, Alfredo Urbano and Wei Xue The electroweak phase transition in the Inert Doublet Model
Nikita Blinov, Stefano Profumo and Tim Stefaniak Baryogenesis in the two doublet and
inert singlet extension of the Standard
Model JCAP08(2016)057 Tommi Alanne,a Kimmo Kainulainen,b,d Kimmo Tuominenc,d and
Ville Vaskonenb,d Keywords: baryon asymmetry, cosmological phase transitions ArXiv ePrint: 1607.03303 doi:10.1088/1475-7516/2016/08/057 doi:10.1088/1475-7516/2016/08/057 Contents
1
Introduction
1
2
The model
2
2.1
Reparametrization invariance and tree-level vacuum stability
4
2.2
Spontaneus symmetry breaking
6
2.3
Finite-temperature potential
8
3
Results
8
3.1
Theoretical and experimental constraints
9
3.2
Dark matter abundance and direct detection limits
9
3.3
Electroweak phase transition
11
3.4
Electron EDM constraint
12
3.5
Baryogenesis
13
3.6
Bubble nucleation
15
4
Conclusions and outlook
17
A Diagonalization of mass matrices
19
B 1-loop beta functions for scalar couplings
20
C Singlet scalar annihilation cross sections
21 2
4
6
8
8
9
9
11
12
13
15
17
19
20
21 JCAP08(2016)057 C Singlet scalar annihilation cross sections Tommi Alanne,a Kimmo Kainulainen,b,d Kimmo Tuominenc,d and
Ville Vaskonenb,d aCP3-Origins, University of Southern Denmark,
Campusvej 55, DK-5230 Odense M, Denmark
bDepartment of Physics, University of Jyv¨askyl¨a,
P.O. Box 35 (YFL), FI-40014 Jyv¨askyl¨a, Finland
cDepartment of Physics, University of Helsinki,
P.O. Box 64, FI-00014 Helsinki, Finland
dHelsinki Institute of Physics, University of Helsinki,
P.O. Box 64, FI-00014 Helsinki, Finland
E-mail: alanne@cp3.sdu.dk, kimmo.kainulainen@jyu.fi,
kimmo.i.tuominen@helsinki.fi, ville.vaskonen@jyu.fi aCP3-Origins, University of Southern Denmark,
Campusvej 55, DK-5230 Odense M, Denmark
bDepartment of Physics, University of Jyv¨askyl¨a,
P.O. Box 35 (YFL), FI-40014 Jyv¨askyl¨a, Finland
cDepartment of Physics, University of Helsinki,
P.O. Box 64, FI-00014 Helsinki, Finland
dHelsinki Institute of Physics, University of Helsinki,
P.O. Box 64, FI-00014 Helsinki, Finland
E-mail: alanne@cp3.sdu.dk, kimmo.kainulainen@jyu.fi,
kimmo.i.tuominen@helsinki.fi, ville.vaskonen@jyu.fi aCP3-Origins, University of Southern Denmark,
Campusvej 55, DK-5230 Odense M, Denmark
bDepartment of Physics, University of Jyv¨askyl¨a,
P.O. Box 35 (YFL), FI-40014 Jyv¨askyl¨a, Finland
cDepartment of Physics, University of Helsinki,
P.O. Box 64, FI-00014 Helsinki, Finland
dHelsinki Institute of Physics, University of Helsinki,
P.O. Box 64, FI-00014 Helsinki, Finland
E-mail: alanne@cp3.sdu.dk, kimmo.kainulainen@jyu.fi,
kimmo.i.tuominen@helsinki.fi, ville.vaskonen@jyu.fi E-mail: alanne@cp3.sdu.dk, kimmo.kainulainen@jyu.fi,
kimmo.i.tuominen@helsinki.fi, ville.vaskonen@jyu.fi Received July 13, 2016
Accepted August 21, 2016
Published August 25, 2016 Abstract. We investigate an extension of the Standard Model containing two Higgs doublets
and a singlet scalar field (2HDSM). We show that the model can have a strongly first-order
phase transition and give rise to the observed baryon asymmetry of the Universe, consistent
with all experimental constraints. In particular, the constraints from the electron and neutron
electric dipole moments are less constraining here than in pure two-Higgs-doublet model
(2HDM). The two-step, first-order transition in 2HDSM, induced by the singlet field, may
lead to strong supercooling and low nucleation temperatures in comparison with the critical
temperature, Tn ≪Tc, which can significantly alter the usual phase-transition pattern in
2HD models with Tn ≈Tc. Furthermore, the singlet field can be the dark matter particle. However, in models with a strong first-order transition its abundance is typically but a
thousandth of the observed dark matter abundance. 1
Introduction The matter-antimatter asymmetry in the universe presents one of the major quests for particle
cosmology. Due to cosmic inflation, such asymmetry cannot be an initial condition for
the thermal history of the universe, but calls for a dynamical explanation. The Standard
Model (SM) of elementary particle interactions fails in providing a successful mechanism for
baryogenesis, and one must look at different extensions of the SM. In this paper we address
these issues in the context of a 2HDSM featuring an extended scalar sector with two gauged
Higgs doublets and an extra singlet. Generation of the matter-antimatter asymmetry in connection with the electroweak
phase transition, i.e. electroweak baryogenesis, is a particularly appealing scenario due to the
possibility of connecting it with the collider experiments. Generic 2HDMs have been studied
earlier in connection with the electroweak baryogenesis problem [1–9]. They provide both
a new source of CP violation arising from complex parameters in the 2HDM potential and
a strong first-order phase transition arising from the one-loop effective potential. However,
observational constraints are placing stringent limits also on 2HDMs [8]. Here we show that
these constraints are alleviated when the model is further extended by a real scalar singlet
field. A generic feature of 2HDM, also inherited by the 2HDSM, is the danger of generating
large flavour changing neutral currents. To avoid these, one has to constrain the Higgs-
fermion couplings in one way or the other. Here we choose to work in the context of universal – 1 – Yukawa alignment, which may be argued for by a requirement that the whole Lagrangian is
invariant under the group GL(2,C) of linear reparametrization transformations in the doublet
space. We also use the reparametrization invariance to develop an elegant way explore the
vacuum stability and the phase-transition pattern in the model. In the 2HDM context large CP violation requires that scalar couplings have large com-
plex phases and strong transition requires that couplings are large in magnitude. When
combined, these requirements tend to give too large electron and neutron electric dipole mo-
ments (EDMs). We will show that the presence of the additional scalar allows for a strong
two-step electroweak phase transition, which does not rely on large radiative corrections to
the effective potential. This alleviates the burden on the scalar self-couplings and significantly
increases the phase space consistent with EDM constraints in the 2HDSM. 1
Introduction JCAP08(2016)057 The singlet scalar can also be a dark matter (DM) candidate when a discrete Z2 symme-
try is imposed to stablize it. However, we will find that a strong first-order phase transition
is not consistent with a dominant singlet scalar DM particle. The problem is that a strong
two-step transition requires a large coupling between the singlet and doublet sectors and this
implies so large annihilation rate for the DM that its relic abundance becomes too small to
account for the full observed DM density. This conclusion is generic for all models of this type. We observe that two-step transitions may also give rise to too strong transitions. It is
possible that fields get trapped in the metastable minimum so that electroweak symmetry
remains unbroken. Also, the latent heat released in the transition may be so large that the
transition walls necessarily become supersonic. However, we find also parameters for which
walls may be subsonic, consistent with the electroweak baryogenesis scenario. Overall, we
are able to find models that satisfy all observational and experimental constraints and can
also give rise to a successful electroweak baryogenesis, accompanied by a subleading DM in
the 2HDSM context. The structure of the paper is as follows: in section 2 we introduce the model and
discuss the most general GL(2,C)-reparametrization invariant 2HDSM Lagrangian including
Yukawa couplings. Here we also develop methods to study the vacuum stability and the
phase-transition patterns in the theory. In section 3 we first go through the experimental
constraints on the model and evaluate the DM relic abundance and the DM search limits on
model parameters. We then evaluate the strength of the transition and compute the baryon
asymmetry created in the electroweak phase transition. The section is concluded by a study
of bubble nucleation in the 2HDSM and in the singlet extension of the SM. In section 4 we
conclude and outline some directions for future research. 2
The model We start from the most general two-Higgs-doublet and inert-singlet extension of the SM with
the scalar field Lagrangian: Lscalar = Zij(DµHi)†DµHj + 1
2(∂µS)2 −V (H1, H2, S) ,
(2.1) (2.1) – 2 – where Zij is an arbitrary Hermitian 2 × 2 matrix and the most general potential is given by V (H1, H2, S) = −m2
1|H1|2 −m2
2|H2|2 −
m2
12H†
2H1 + h.c.
−1
2m2
SS2
+ λ1|H1|4 + λ2|H2|4 + λ3|H1|2|H2|2 + λ4(H†
1H2)(H†
2H1)
+
λ5(H†
2H1)2 + λ6|H1|2(H†
2H1) + λ7|H2|2(H†
2H1) + h.c.
+ 1
4λSS4+ 1
2λS1S2|H1|2+ 1
2λS2S2|H2|2+
1
2λS12S2H†
2H1+h.c.
. (2.2) (2.2) JCAP08(2016)057 Both doublets Hi are assumed to be gauged under SU(2)L × U(1)Y , while the scalar S
is a singlet under all SM gauge interactions. The singlet S is a crucial ingredient in the
model because it will disentangle the source of a strongly first-order transition from that of
sufficiently strong CP violation. The Lagrangian (2.1) is invariant under a reparametrization transformation Φ →Φ′ ≡
PΦ (and a simultaneous rescaling of S), where P is an element of the general linear group
GL(2,C), and Φ is the Higgs hyperdoublet: Φ ≡(H1, H2)T . (2.3) (2.3) GL(2,C) is the semidirect product of special linear transformations SL(2,C) and multiplica-
tive group of dilatations C×. We can always use the dilatation and a hyperbolic SL(2,C)
transformation to bring the kinetic term into the canonical form, Zij →diag(1, 1), i.e. Zij(DµHi)†DµHj
→
|DµH1|2 + |DµH2|2 . The resulting Lagrangian is still invariant under elliptic SL(2,C) transformations, i.e. the
usual SU(2) rotations of the doublets. The resulting Lagrangian is still invariant under elliptic SL(2,C) transformations, i.e. the
usual SU(2) rotations of the doublets. A generic 2HDM gives rise to unacceptably large flavour-changing neutral currents
(FCNCs) and the presence of a singlet does not change the situation. One way to avoid
FCNCs is the Yukawa alignment [10], which assumes that both doublets couple to fermions
with the same matrix structure (since S is a singlet under SM gauge interactions its couplings
to charged SM fermions are excluded): (2.4) LYukawa = yuCi
u ¯QL ˜HiuR + ydCi
d ¯QLHidR + yℓCi
ℓ¯LLHieR + h.c,
(2.4) where ˜H2 ≡iσ2H∗
2. Here ya are flavour matrices independent of the doublet index, and Ca
i
are doublet-index dependent complex numbers. 1This actually involves a rotation and a redefining of the scale of ya matrices. 2
The model In general the alignment may be different
in different fermion sectors: Ca
i ̸= Cb
i . However, for simplicity, we choose to work in the
special case of universal Yukawa alignment, where Ca
i ≡Ci. In this case we can, without a
further loss of generality, choose the basis where only the H2 field couples to fermions. This
corresponds to setting C1 = 0 and C2 = 1,1 so that: (2.5) LYukawa = yu ¯QL ˜H2uR + yd ¯QLH2dR + yℓ¯LLH2eR + h.c . (2.5) – 3 – The choice of basis leading to (2.5) can be effected by an SU(2) rotation of Φ, and it exhausts
our remaining freedom to perform elliptic SL(2,C)-reparametrization transformations after
diagonalizing the kinetic term.2 Let us stress that while the Yukawa sector (2.5) appears to be of type-I 2HDM, we did
not impose any discrete symmetry to derive it. This is why we have kept the λ6 and λ7
terms in the scalar potential. Note that renormalization does not change the form of the
theory; while it both re-introduces a kinetic mixing between doublets and a coupling of H1
to fermions, these changes can be countered by another GL(2,C) transformation. Also, we
point out that the universal Yukawa alignment can be argued for based on reparametrization
invariance: only in the context of universal alignment is the complete Lagrangian including
the 2HDM and Yukawa sectors invariant under GL(2,C) transformations. JCAP08(2016)057 Interestingly, the universal alignment structure arises as a low-energy effective theory in
models of dynamical electroweak symmetry breaking [11–18]. In the bosonic technicolor [11–
13], the ultraviolet theory contains a new gauge theory responsible for dynamically breaking
the electroweak symmetry and an elementary scalar doublet H, which communicates the
symmetry breaking to the SM fields through its renormalizable Yukawa couplings. At low
energies the strong technicolor dynamics is described in terms of an effective Lagrangian for a
composite Higgs doublet, which couples with the elementary one through a Lagrangian of the
form (2.1), including the non-trivial kinetic mixing. Only the elementary scalar couples to SM
fermions, which naturally introduces Yukawa alignment. Moreover, when the kinetic mixing
is removed by a non-unitary transformation, the Yukawa Lagrangian becomes naturally of the
universally Yukawa-aligned form with Ca
i = Ci. After a final SU(2) rotation, the model has a
diagonal kinetic term, type-I Yukawa sector (2.5), and the most general potential of eq. (2.2). 2Note that in the case of general Yukawa alignment, where Ca
i ̸= Cb
i , one could still use the SU(2) rotation
to set Cu
1 = 0, so that up-type quarks couple only to H2. Most of our subsequent analysis would hold also
for this scenario, because it is mostly sensitive only to the large top-quark coupling. The only exception is
the electron EDM, for which our analysis covers only a part of the full phase space available in the context of
general alignment.
†
† g
g
3Clearly r0 = P
i |Hi|2 ≥0. Also rµrµ = 4(|H1|2|H2|2 −(H†
1H2)(H†
2H1)) ≥0 , by Schwartz inequality. 2.1
Reparametrization invariance and tree-level vacuum stability In this notation
one can rewrite the Higgs potential (2.2) in a very compact form: V = −1
2m2
SS2 −1
2M2
µrµ + 1
4rµλµνrν + 1
4λSµrµS2 + 1
4λSS4 ,
(2.7) (2.7) where we defined mass and coupling four-vectors where we defined mass and coupling four-vectors M2
µ ≡
m2
1 + m2
2, 2m2
12R, −2m2
12I, m2
1 −m2
2
,
λSµ ≡(λS1 + λS2, 2λS12R, −2λS12I, λS1 −λS2)
(2.8) (2.8) and a symmetric coupling tensor JCAP08(2016)057 λµν ≡
λ1 + λ2 + λ3
λ6R + λ7R
−λ6I + λ7I
λ1 −λ2
λ6R + λ7R
λ4 + 2λ5R
−2λ5I
λ6R −λ7R
−λ6I + λ7I
−2λ5I
λ4 −2λ5R
−λ6I −λ7I
λ1 −λ2
λ6R −λ7R
−λ6I −λ7I
λ1 + λ2 −λ3
,
(2.9) (2.9) where the subscripts R and I refer to the real and imaginary parts of the couplings, respec-
tively. The reparametrization invariance is manifest in eq. (2.7), because V depends only on
Lorentz-invariant products of vectors and tensors. This form is particularly suitable for a
study of the vacuum stability and phase-transition patterns of the model. First consider the direction S = 0 in the potential (2.7). Here the term rµλµνrν must
be bounded from below. In [19] it was shown that this is the case precisely when λµν is
positive definite in the future light cone. That is, all stable potentials can be written as
λµν ≡ΛµαλD
αβΛβν, where Λµα is an SO(1,3)+ transformation and λD
αβ = diag(λD
00, −λD
11, −λD
22, −λD
33),
with
λD
00 > 0
and
λD
00 > λD
ii . (2.10) (2.10) Note that the four parameters in λD
αβ together with the six parameters in Λβν add up to the
ten real degrees of freedom in the most general 2HDM potential. Second, if we set rµ = 0
(H1 = H2 = 0), we see that we must have λS > 0 . (2.11) (2.11) Finally, we have to consider the directions where both S and Hi are nonzero. First, if the
vector λSµ of couplings which mix S and Hi lies in the future light cone, λSµ ∈LC+, i.e. λSµλµ
S = 4
λS1λS2 −|λS12|2
> 0
and
λ0
S = λS1 + λS2 > 0 ,
(2.12) (2.12) then the mixing term 1
4λSµrµS2 in the potential, eq. 2.1
Reparametrization invariance and tree-level vacuum stability The original Lagrangian with the most general potential, kinetic term, and the universally
aligned Yukawa sector has 27 real parameters (not counting the parameters entering the
Yukawa-flavour-mixing matrices). We removed the four arbitrary parameters from kinetic
terms and three from the complex Yukawa coefficients Ci by the use of the GL(2,C) invariance
of the theory. This still leaves us with 15 real couplings and five real mass parameters in the
model potential V (H1, H2, S). Our next task is to find out which sets of these parameters
correspond to physically viable models with a stable potential. We can use the reparametrization invariance to our advantage in constructing the stable
potentials. To this end, it is convenient to rephrase the invariance in terms of Lorentz in-
variance of the potential, written in terms of bilinears formed from hyperdoublets. Following
the analysis of ref. [19–21], we define rµ ≡Φ†σµΦ ,
where
σµ = (1, σi) . (2.6) (2.6) The bilinear four-vector rµ is positive definite.3 That is, rµ vectors span the future light
cone, LC+, of a Minkowski space. Thus, in bilinear representation the elliptic and hyperbolic
SL(2,C) basis transformations of fields Φ →Φ′ ≡PΦ correspond to proper orthochronous The bilinear four-vector rµ is positive definite.3 That is, rµ vectors span the future light
cone, LC+, of a Minkowski space. Thus, in bilinear representation the elliptic and hyperbolic
SL(2,C) basis transformations of fields Φ →Φ′ ≡PΦ correspond to proper orthochronous 2Note that in the case of general Yukawa alignment, where Ca
i ̸= Cb
i , one could still use the SU(2) rotation
to set Cu
1 = 0, so that up-type quarks couple only to H2. Most of our subsequent analysis would hold also
for this scenario, because it is mostly sensitive only to the large top-quark coupling. The only exception is
the electron EDM, for which our analysis covers only a part of the full phase space available in the context of
general alignment. g
3Clearly r0 = P
i |Hi|2 ≥0. Also rµrµ = 4(|H1|2|H2|2 −(H†
1H2)(H†
2H1)) ≥0 , by Schwartz inequality. – 4 – Lorentz transformations rµ →r′µ = (ΛP)µ
νrν, where (ΛP)µ
ν ∈SO(1, 3)+. In this notation
one can rewrite the Higgs potential (2.2) in a very compact form: Lorentz transformations rµ →r′µ = (ΛP)µ
νrν, where (ΛP)µ
ν ∈SO(1, 3)+. where Λµν ∈SO(1, 3)+. Let us comment on the role of the kinetic and Yukawa terms in the above construction
of the potential. We implicitly assumed that kinetic term becomes diagonal, and the Yukawa
term becomes of type-I form in the final frame, after the Lorentz transformation. Thus, they
necessarily must be nontrivial in the original, diagonal frame. Indeed, the six degrees of
freedom “missing” in the diagonal potential are in this frame evenly divided between the Ci
coefficients in the Yukawa Lagrangian and the mixing parameters in the kinetic term, which
in the bilinear notation can be written as Kµ(DαΦ)†σµ(DαΦ) . (2.16) (2.16) Here Kµ is some positive definite, but otherwise arbitrary four-vector of unit length (here
α refers to the usual space-time indices and µ to potential indices).4 It is amusing to see
that exactly a Lorentz boost (a hyperbolic SL(2,C) transformation on fields) is needed to
bring an arbitrary Kµ into the canonical form: Kµ →(1; 0, 0, 0), after which the kinetic term
is manifestly invariant under Lorentz rotations (elliptic SL(2,C) transformations on fields). These boosts and rotations into the canonical frame activate the whole SO(1, 3)+ group
discussed above and thus create all physically viable Lagrangians with bounded potentials. 4In the most general case Kµ has four free parameters, but one parameter is here assumed to be removed
by a dilatation, used to bring the length of Kµ to unity. 2.1
Reparametrization invariance and tree-level vacuum stability (2.7), is always positive, and no new
conditions arise. However, if λSµ /∈LC+, there are always directions rµ ∈LC+ along which
the product λSµrµ is negative. If we in such cases rewrite the quartic part of the potential as Vquartic = 1
4rµ
λµν −
1
4λS
λSµλSν
rν + 1
4λS
S2 + λSµrµ
2λS
2
,
(2.13) Vquartic = 1
4rµ
λµν −
1
4λS
λSµλSν
rν + 1
4λS
S2 + λSµrµ
2λS
2
,
(2.13) (2.13) we see that the potential is most negative as a function of S along direction 2λSS2 = −λSµrµ. In this subspace, the potential reduces to the form 4Vquartic = rµλS
µνrν, with a new coupling
matrix: λS
µν ≡λµν −
1
4λS
λSµλSν . (2.14) (2.14) – 5 – – 5 – The matrix λS
µν is not in general diagonalizable by an SO(1, 3)+ rotation, and it may have
also complex eigenvalues. Unfortunately we cannot restrict its properties like we did for λµν,
because λS
µν does not need not be positive definite in the entire future light cone, but only in
the subset of LC+ where λSµrµ is negative. Instead of covering the full range of possibilities,
we will require a sufficient (but not necessary) condition that λS
µν is positive definite in the
future light cone whenever λSµ /∈LC+. We now have the recipe to construct the space of stable potentials: we first choose a
λD
αα which satisfies eqs. (2.10). Then we generate a vector λD
Sµ and check if it satisfies the
positivity constraint (2.12), or if the matrix λS
µν in eq. (2.14) is positive definite in the subset
of LC+ where λSµrµ < 0. Having found an acceptable set, we generate a random Lorentz
transformation and define JCAP08(2016)057 λµν ≡ΛµαλD
αβΛβν
and
λµ
S ≡Λµν(λD
S)ν ,
(2.15) (2.15) 2.2
Spontaneus symmetry breaking Since we are interested in the cases where the singlet scalar S is a DM candidate, we restrict
our considerations to the cases where the potential is unbroken in S direction and hence
Z2 symmetric at low temperatures. However, to enhance the strength of the latter phase
transition, we also need the S symmetry to be broken at high temperatures before the
symmetry breaks in the doublet direction. To this end, we must have negative quadratic
term in the S direction in the potential (m2
S > 0). This implies that there may be other
minima away from the ⟨S⟩= 0 vacuum, and we must check that none of these minima is the
global one at zero temperature. The extremization conditions are: ∂V
∂S = SζS = 0 ,
where
ζS ≡−m2
S + λSS2 + 1
2λSµrµ ,
(2.17) (2.17) 4In the most general case Kµ has four free parameters, but one parameter is here assumed to be removed
by a dilatation, used to bring the length of Kµ to unity. – 6 – and and ∂V
∂H†
i
= σµ
ijHjζµ = 0 ,
where
ζµ = −1
2
M2
µ −1
2λSµS2
+ 1
2λµνrν . (2.18) ∂V
∂H†
i
= σµ
ijHjζµ = 0 ,
where
ζµ = −1
2
M2
µ −1
2λSµS2
+ 1
2λµνrν . (2.18) (2.18) Eqs. (2.18) are complex, so we have five equations relating vacuum fields to the parameters of
the theory. Our goal is to have an unbroken singlet and a broken neutral doublet vacuum at
zero temperature. In [19] it was shown that the neutral and charged vacua cannot coexist in
the pure 2HDM case. When S = 0, our potential reduces to the pure 2HDM case, and so the
above statement applies here as well. Therefore, if we find a neutral vacuum, we know it is
the global one in the doublet space and the charged extremum may at best be a saddle point. Moreover, even with S ̸= 0, the doublet-symmetry-breaking pattern is formally similar to
the pure 2HDM case, only with an effective mass parameter JCAP08(2016)057 −m2
µ ≡−M2
µ + 1
2λSµS2. (2.19) (2.19) This implies that also any neutral minimum with S breaking is the lowest one in the doublet
space. 2.2
Spontaneus symmetry breaking The most general neutral vacuum in 2HDM field space is given by ⟨H1⟩=
1
√
2
0
v1eiθ1
,
and
⟨H2⟩=
1
√
2
0
v2eiθ2
. (2.20) (2.20) Of course the local SU(2)-gauge invariance guarantees that only the relative phase θ ≡θ2−θ1
is physical, and one could rotate for example θ1 →0. Explicitly we find that this corresponds
to to rµ
0 = (v2
1 + v2
2, 2v1v2 cos θ, 2v1v2 sin θ, v2
1 −v2
2) ,
(2.21) (2.21) so that r2
0 = 0 as it should for a neutral vacuum [19]. This construction actually only ensures
that the special point (2.20) is an extremum of the potential. To check that this extremum
is a also a minimum, one needs to compute second derivative matrices of the potential
corresponding to scalar field masses and require that there are no negative eigenvalues. For
the mass of the physical excitation in the singlet direction, this corresponds to requiring that d2V
dS2
vev
= −m2
S + 1
2λSµrµ
0 ≡M2
S > 0 ,
(2.22) (2.22) where the left hand side is evaluated at the extremum ⟨S⟩= 0 and ⟨Hi⟩̸= 0 as defined
in eq. (2.20). These requirements on the spectrum allow one to set the Lagrangian mass
parameters in terms of physical masses and vacuum expectation values (vevs) of the fields. However, it still remains to check that the vacuum with S = 0 is the global minimum for any
given set of parameters. It is straightforward to show that the value of the potential at the
desired ⟨S⟩= 0, ⟨Hi⟩̸= 0 vacuum is V (⟨S⟩= 0, ⟨Hi⟩̸= 0) = −1
4rµ
0 λµνrν
0 . (2.23) (2.23) In the direction Hi = 0, the potential has at least a directional minimum at ⟨S⟩2 = m2
S/2λS,
and the value of the potential in this directional minimum is V (⟨S⟩̸= 0, ⟨Hi⟩= 0) = −1
4λS
M2
S −1
2λSµrµ
0
2
. (2.24) (2.24) – 7 – We impose the condition that the minimum in eq. (2.23) is below that in eq. (2.24). Finally,
there is an extremum where both ⟨S⟩̸= 0 and ⟨Hi⟩̸= 0, but this is a local maximum. 2.3
Finite-temperature potential JCAP08(2016)057 The final ingredient we need for our analysis is the effective potential at finite temperature. Here we only consider the leading corrections to the potential, which bring about the sym-
metry restoration at high temperatures. In the high-temperature limit, these corrections are
accounted for by the thermal masses: m2
a(T) = −m2
a + ca
T 2
12 ,
(2.25) (2.25) (2.25) where (a = 1, 2, 12, S): c1 = cSM + 6λ1 + 2λ3 + λ4 + 1
2λS1
c2 = cSM + 6λ2 + 2λ3 + λ4 + 1
2λS2
c12 = cSM + 3λ6R + λ7R + 1
2λS12R −i
3λ6I −λ7I + 1
2λS12I
cS = 3λS + 2(λS1 + λS2) ,
(2.26) (2.26) and and cSM = 9
4g2
L + 3
4g2
Y + 3y2
t
(2.27) (2.27) is the common SM contribution from the SU(2)L and U(1)Y gauge fields and the top quark. is the common SM contribution from the SU(2)L and U(1)Y gauge fields and the top quark. 2.2
Spontaneus symmetry breaking In addition to the massive scalar S, the physical spectrum contains the usual states
arising from the two Higgs doublets: the three neutral scalars h0, H0 and A0, and two charged
scalars H±. The diagonalization of the mass matrices is presented in detail in appendix A. The lightest neutral scalar state h0 is identified with the 125 GeV Higgs particle observed
LHC, while the masses of the heavier neutral and charged scalar states are constrained to lie
above the current limits. 3.1
Theoretical and experimental constraints The couplings between scalar fields tend to run strongly, which typically leads to a relatively
low cut-offscale for the validity of the effective theory. To be specific, we demand that our
model is consistent up to Λ = 1.5 TeV, i.e. we check that the vacuum is stable and that all
couplings remain perturbative5 up to Λ. The 1-loop renormalization-group equations needed
for this calculation are summarized in appendix B. Moreover, at zero temperature we impose |⟨H1⟩|2 + |⟨H2⟩|2 = v2
0,
⟨S⟩= 0
(3.1) (3.1) and check that this choice gives the global minimum of the potential as detailed in previous
section. JCAP08(2016)057 Second, we address the current experimental constraints. We only accept points that
give a light scalar of mass mh0 = 125 GeV, and for which the heavy scalars satisfy the mass
limits from direct searches at LEP on charged particles, mH+ > 500 GeV [22], and at LHC
on heavy neutral scalars, mH0, mA0 > 600 GeV [23]. For consistency, we also constrain the
heavy scalar masses to be below the cut-offΛ = 1.5 TeV. We also take into account the
electroweak precision data using the S and T parameters [24]. The necessary formulae for
computing S and T can be extracted from [25], where the oblique parameters have been
calculated for models with extra doublets and extra singlets. The current values of S and T
parameters are S = 0.00 ± 0.08, T = 0.05 ± 0.07 with a correlation factor ρ = 0.90 [26]. We
accept points that lie within the 2σ region in the (S, T) plane. Moreover we take into account constraints on the Higgs boson couplings to SM particles
using the signal-strength data from the LHC and Tevatron experiments [27–29]. First, we
impose the constraint from the invisible width of the Higgs boson from LHC at 2σ level: RI =
ΓI
ΓI + ΓSM, tot
< 0.020,
(3.2) (3.2) which here implies Γh0→SS ≲1.0 MeV. Second, we allow for modifications to the Higgs
couplings and parametrize the deviations from the SM with parameters af and aV , Leff= aV
2M2
W
v0
h0W +W −+ M2
Z
v0
h0ZZ
−af
X mψ
v0
h0ψψ . 3.1
Theoretical and experimental constraints (3.3) (3.3) We accept points which are within 2σ from the best fit values aV = 0.993 ,
af = 0.968 ,
(3.4) (3.4) which we obtain by performing a χ2 fit to the Higgs boson signal strength data. We have
neglected the imaginary part of af and the h0H+H−coupling in the fit, since we have checked
that these couplings are very small.6 which we obtain by performing a χ2 fit to the Higgs boson signal strength data. We have
neglected the imaginary part of af and the h0H+H−coupling in the fit, since we have checked
that these couplings are very small.6 3
Results We now test our model against experimental constraints and for the consistency of its cos-
mological predictions. We scan the parameter space by solving m2
1, m2
2 and m2
12 from the
vacuum conditions and create Monte Carlo chains in the coupling constant space by the al-
gorithm described in the previous section. We first subject the models to various theoretical
and experimental constraints. For models that pass these constraints, we compute the DM
abundance and the strength of the phase transition. We find that the model can provide
either the observed DM abundance or a strong transition, but not both simultaneously. For
the points providing a strong transition, we compute the prediction for the baryon-to-entropy
ratio ηB. We show that the parameter space where ηB is sufficiently large is strongly con-
strained but not excluded by the current experimental limit on the electric dipole moment of
the electron. Finally, we compute the nucleation temperatures Tn and show that typically,
and in particular for strong transitions, Tn is much smaller than the critical temperature Tc. – 8 – p
p
g
g
µ ≫
;
5
V
6As a comparison, we have performed the χ2 fit of af and aV with maximum values of the imaginary part of
af and h0H+H−coupling for out data points, and we find that the best-fit value does not change significantly. 5For perturbativity we use the upper limit (4π)2 on all scalar couplings. Over large portion of our data
points the couplings will remain small also at larger scales µ ≫Λ; the value 1.5 TeV is chosen for concreteness.
6
2 5For perturbativity we use the upper limit (4π)2 on all scalar couplings. Over large portion of our data
points the couplings will remain small also at larger scales µ ≫Λ; the value 1.5 TeV is chosen for concreteness.
6As a comparison, we have performed the χ2 fit of af and aV with maximum values of the imaginary part of
af and h0H+H−coupling for out data points, and we find that the best-fit value does not change significantly. 3.2
Dark matter abundance and direct detection limits We compute the relic abundance of the S bosons for all models passing the experimental
constraints described above. We assume that S is a thermal relic, i.e. we assume that at 5For perturbativity we use the upper limit (4π)2 on all scalar couplings. Over large portion of our data
points the couplings will remain small also at larger scales µ ≫Λ; the value 1.5 TeV is chosen for concreteness. 6As a comparison, we have performed the χ2 fit of af and aV with maximum values of the imaginary part of 5For perturbativity we use the upper limit (4π)2 on all scalar couplings. Over large portion of our data
points the couplings will remain small also at larger scales µ ≫Λ; the value 1.5 TeV is chosen for concreteness. 6
2 5For perturbativity we use the upper limit (4π)2 on all scalar couplings. Over large portion of our data
points the couplings will remain small also at larger scales µ ≫Λ; the value 1.5 TeV is chosen for concreteness. 6As a comparison, we have performed the χ2 fit of af and aV with maximum values of the imaginary part of
af and h0H+H−coupling for out data points, and we find that the best-fit value does not change significantly. – 9 – least some the portal couplings λS1, λS2 and λS12 are sufficiently large (larger than about
10−7 [30]). We then apply the standard freeze-out formalism [31], employing the accurate
approximation scheme introduced in [32]. The relevant annihilation channels of our WIMP
candidate are SS →h0h0, H0H0, A0A0, h0H0, h0A0, H0A0, H+H−, W +W −, ZZ, ¯ff. (3.5) (3.5) Cross-sections for all these processes are given in a very compact form in appendix C. To
treat cases where S is a subdominant DM candidate, we define the ratio [33] frel = ΩSh2
0.12 ,
(3.6) JCAP08(2016)057 (3.6) which expresses how large fraction of the observed DM abundance is in form of S bosons. All
annihilation channels are directly proportional to the couplings between the S boson and the
Higgs fields, although the precise dependence is rather complicated (see appendix C). Since
the relic abundance is roughly proportional to the inverse of the annihilation cross section,
large (small) couplings corresponds to a small (large) relic abundance. Direct search limits for S follow from the bound on the spin-independent cross section
for S scattering offnucleons. 3.2
Dark matter abundance and direct detection limits Shown is the LUX bound on a set of models passing all experimental constraints described
in section 3.1 and for which frel ≥0.01. 3.2
Dark matter abundance and direct detection limits It is given by σSI = λ2
efff2
N
4π
µm2
Nv2
0
m4
hM2
S
,
(3.7) (3.7) where mN = 0.939 GeV is the nucleon mass, µ = mNMS/(mN + MS) is the reduced mass
of the nucleon-scalar system, and fN ≈0.30 [32] gives the strength of the Higgs-nucleon
coupling: gh0N ¯
N = fNmN/v0. Finally, the effective SSh0 coupling is given by where mN = 0.939 GeV is the nucleon mass, µ = mNMS/(mN + MS) is the reduced mass
of the nucleon-scalar system, and fN ≈0.30 [32] gives the strength of the Higgs-nucleon
coupling: gh0N ¯
N = fNmN/v0. Finally, the effective SSh0 coupling is given by eff≡1
2
h RN44 cos β sin θ −RN42 cos β cos θ + RN41 sin β
λS12I
+
RN42 cos β sin θ −RN44 cos β cos θ −RN43 sin β
λS12R
+ cos β
RN43 cos θ + RN41 sin θ
λS1 −sin βRN44λS2
i
,
(3.8) (3.8) where β is the vacuum mixing angle and θ the phase between the doublet vevs, and RNij
are the components of the 4 × 4-mixing matrix between the neutral scalar fields given in
eq. (A.8). Currently the most stringent limit for σSI come from the LUX experiment [34]. However,
in the case of a subdominant DM, the LUX bound is not directly related to σSI shown in
eq. (3.7). Instead, assuming that all DM components cluster similarly, the actual signal
strength from S bosons is suppressed by the fraction frel. The relevant quantity to compare
with the LUX limit then is σeff= frelσSI. (3.9) (3.9) We show the effect of this limit on the models passing all experimental bounds on figure 1. Note that the scatter in σeffas a function of mS is much larger here than in the singlet
extension of the SM [33]. Note how also the models with relatively low abundance are
constrained by the LUX data. – 10 – – 10 – XENON100
LUX
100
200
300
400
-46.0
-45.5
-45.0
-44.5
-44.0
-43.5
-43.0
MS (GeV)
log10(σeff/cm2)
log10(frel)
-2.0
-1.5
-1.0
-0.5
0
Figure 1. Shown is the LUX bound on a set of models passing all experimental constraints described
in section 3.1 and for which frel ≥0.01. XENON100
LUX
100
200
300
400
-46.0
-45.5
-45.0
-44.5
-44.0
-43.5
-43.0
MS (GeV)
log10(σeff/cm2) JCAP08(2016)057 Figure 1. 3.3
Electroweak phase transition One of the necessary conditions for a successful baryogenesis scenario is the departure from
thermal equilibrium. In EWBG this is provided by a strong first-order phase transition. In
pure 2HDMs, with only leading thermal corrections (i.e. with thermal masses), the phase
transition is of second order. While full one-loop effects may induce a first-order transition,
it still tends to be rather weak [8, 9]. In a model with a singlet scalar, a strong transition
may take place with just the leading corrections (2.25), given a specific two-step symmetry-
breaking pattern [35]. This means that, when passing from high towards low temperatures, a
minimum of the potential is generated first along the singlet direction. At lower temperature,
then, the potential develops a minimum where the doublets have non-zero vevs while the
singlet symmetry is restored, (⟨h1⟩, ⟨h2⟩; ⟨s⟩) = (0, 0; 0) →(0, 0; w(T)) →(v1(T), v2(T); 0),
ensuing the actual electroweak phase transition. This pattern of minima should of course
develop in such a way that the true ground state at T = 0 is given by eq. (3.1). Including the thermal mass corrections of eq. (2.25) to the potential, we find the tran-
sition temperatures. In particular the electroweak transition temperature, Tc, where the two
minima are degenerate is determined by V (0, 0, wc, Tc) = V (v1c, v2c, 0, Tc),
(3.10) (3.10) where wc = w(Tc) and vic = vi(Tc). To determine when the transition is strong enough, we
require, as usual, that vc/Tc ≥1 ,
(3.11) vc/Tc ≥1 , (3.11) where vc =
p
v2
1c + v2
2c. It turns out not to be possible to have simultaneously strong tran-
sition and dominant DM candidate in the model with frel ≈1. This is because having a
strong transition requires that at least some of the mixing couplings λSi are large. However,
as we noted above, large mixing introduces large annihilation cross sections for S bosons and
hence small relic abundances. Qualitatively the behaviour is the same as in the pure singlet
model [32, 33]: a strong first-order EWPT and large frel ≈1 are realized in different portions
of the parameter space. where vc =
p
v2
1c + v2
2c. It turns out not to be possible to have simultaneously strong tran-
sition and dominant DM candidate in the model with frel ≈1. 3.3
Electroweak phase transition This is because having a
strong transition requires that at least some of the mixing couplings λSi are large. However,
as we noted above, large mixing introduces large annihilation cross sections for S bosons and
hence small relic abundances. Qualitatively the behaviour is the same as in the pure singlet
model [32, 33]: a strong first-order EWPT and large frel ≈1 are realized in different portions
of the parameter space. – 11 – 100
150
200
250
300
350
400
0.5
1
2
5
MS(GeV)
λeff
10-5
10-4
0.001
0.010
0.100
1
100
150
200
250
300
350
400
frel
MS(GeV)
Figure 2. Scanned data points which give a strong first-order EWPT. Yellow points are excluded by
direct DM searches. 10-5
10-4
0.001
0.010
0.100
1
100
150
200
250
300
350
400
frel
MS(GeV) 100
150
200
250
300
350
400
0.5
1
2
5
MS(GeV)
λeff JCAP08(2016)057 Figure 2. Scanned data points which give a strong first-order EWPT. Yellow points are excluded by
direct DM searches. Left panel of figure 2 shows the correlation between the DM particle mass MS and the
effective coupling λefffor models with strong EWPT. Accepted values of λeffincrease as a
function of MS because both quantities are linearly proportional to the mixing couplings λSi. Right panel of figure 2 shows the correlation between MS and the relative relic abundance
frel for the same set. Note that for MS between the Higgs resonance and MW , both a strong
transition and a relatively large DM abundance, frel ≈0.15 could be obtained, but this region
is now excluded by LUX. The fact that LUX most strongly constrains models with small λeffis because frel is
roughly inversely proportional to the square of λeff, and this to large extent cancels the direct
λeffdependence in σSI. Larger mass region beyond the Higgs resonance is less constrained
because the DM-number density falls with increasing mass. 3.4
Electron EDM constraint The non-observation of electric dipole moments (EDMs) of electrons, neutrons and atoms
gives stringent bounds on CP-violating interactions in multi-Higgs models. As shown in [36],
currently the most stringent bound for 2HDMs arises from the electron EDM, for which the
ACME experiment gives an upper limit |de| < 8.7 × 10−29ecm ,
(3.12) (3.12) with 90% confidence level [37]. We calculate de for the points which give a strong first-order
EWPT using the results from ref. [38], where Barr-Zee type contributions to fermionic EDM
were calculated in 2HDM. These results are directly applicable here as well, because the
singlet scalar S does not directly couple to gauge fields. In figure 3, we show the distribution
of models passing all previous cuts as a function of de and the neutral scalar mixing matrix
element RN42, which expresses the projection of h0 to complex part of the second doublet: RN42 ≡⟨H0
2I|h0⟩≡sin ∆CP . (3.13) (3.13) sin ∆CP is given in terms of the various mixing angles in eq. (A.8). The red region is excluded
by the electron EDM constraint. Small de naturally correlates with small sin ∆CP, because
the size of sin ∆CP is proportional to the size of the CP-violating mixing in the model. – 12 – 0.00
0.02
0.04
0.06
0.08
-3
-2
-1
0
1
2
|sinΔCP|
log10(de/8.7×10-29ecm) 0.00
0.02
0.04
0.06
0.08
-3
-2
-1
0
1
2
|sinΔCP|
log10(de/8.7×10-29ecm)
Figure 3. Scatter plot of all models with strong enough EWPT as a function of the mixing parameter
sin ∆CP and the electron EDM de. The red region is excluded by the eEDM limit. JCAP08(2016)057 Figure 3. Scatter plot of all models with strong enough EWPT as a function of the mixing parameter
sin ∆CP and the electron EDM de. The red region is excluded by the eEDM limit. 3.5
Baryogenesis The actual baryogenesis mechanism in our model relies on CP-violating interactions of the
top quark with the expanding phase-transition walls. The CP violation comes directly from
the spatial evolution of the complex phases of the Higgs field H2, which renders the top mass
a complex-valued function of the spatial coordinate across the wall. The first step for us is
then to work out the evolution of the scalar fields over the bubble wall. We shall approximate the true phase-transition-wall profile in the usual way, by the
stationary path that extremizes the Euclidean one-dimensional action Z
dz
|DzH1| + |DzH2| + 1
2|∂zS| + V + . . .
,
(3.14) (3.14) between two degenerate minima at critical temperature T = Tc, for which the condition (3.10)
holds. The covariant derivatives involve the classical Zµ field: Dµ = ∂µ −ig/(2 cos θW)Zµ. We write the neutral components of the doublets as hjeiϕj and observe that the effective
potential can depend only on the relative phase ϕ ≡ϕ1 −ϕ2. Following ref. [8], we work in
the gauge Zµ = 0, whereby we need to account for four fields: h1, h2, S and ϕ, while solving
the path. The relevant reduced action is S1 =
Z
dz
X
i
1
2(∂zhi)2 + 1
2(∂zS)2 + 1
2
h2
1h2
2
h2
1 + h2
2
(∂zϕ)2 + V (h1, h2, S, ϕ, Tc)
! . (3.15) (3.15) The invariance of the potential under the change of the total phase ϕ1 + ϕ2 implies a
conservation law, which in the Zµ = 0 gauge allows us to work out the phase ϕ2 in terms of
the relative phase ϕ [8]: The invariance of the potential under the change of the total phase ϕ1 + ϕ2 implies a
conservation law, which in the Zµ = 0 gauge allows us to work out the phase ϕ2 in terms of
the relative phase ϕ [8]:
2 ∂zϕ2 = −
h2
1
h2
1 + h2
2
∂zϕ . (3.16) (3.16) The complex, spatially-varying top mass can now be constructed from the phase ϕ2(x) and
the modulus h2(z): The complex, spatially-varying top mass can now be constructed from the phase ϕ2(x) and
the modulus h2(z): mt(z) = yt
√
2h2(z)eiϕ2(z). (3.17) (3.17) In fact, one does not need to solve for the top phase, since only its derivative, given by
eq. 3.5
Baryogenesis (3.16), appears in the source term for the diffusion equations for chemical potentials: In fact, one does not need to solve for the top phase, since only its derivative, given by
eq. (3.16), appears in the source term for the diffusion equations for chemical potentials: St = ξw
K8,t(x2
t ϕ′
2)′ −K9,tx2
t x2′
t ϕ′
2
. (3.18) (3.18) – 13 – 0.00
0.02
0.04
0.06
0.08
-5
-4
-3
-2
-1
0
|sinΔCP|
log10(ηB/8.7×10-11)
-2.0
-1.5
-1.0
-0.5
0.0
-5
-4
-3
-2
-1
0
log10(de/8.7×10-29ecm)
log10(ηB/8.7×10-11)
Figure 4. Left: shown is the correlation between the baryon-to-entropy ratio ηB and the mixing
matrix element sin ∆CP. Red dots correspond to models for which Tn cannot be found (in the thin-
wall approximation). Right: the correlation between ηB and de. The red region is excluded by the
eEDM limit and the black line shows the observed baryon-to-entropy ratio. 0.00
0.02
0.04
0.06
0.08
-5
-4
-3
-2
-1
0
|sinΔCP|
log10(ηB/8.7×10-11) -2.0
-1.5
-1.0
-0.5
0.0
-5
-4
-3
-2
-1
0
log10(de/8.7×10-29ecm)
log10(ηB/8.7×10-11) JCAP08(2016)057 Figure 4. Left: shown is the correlation between the baryon-to-entropy ratio ηB and the mixing
matrix element sin ∆CP. Red dots correspond to models for which Tn cannot be found (in the thin-
wall approximation). Right: the correlation between ηB and de. The red region is excluded by the
eEDM limit and the black line shows the observed baryon-to-entropy ratio. Here ξw is the wall velocity, primes denote ∂zT and Kn,t are dimensionless functions of
xt ≡|mt|/T arising from phase-space averaging of certain kinematic variables defined in [39]. Here ξw is the wall velocity, primes denote ∂zT and Kn,t are dimensionless functions of
xt ≡|mt|/T arising from phase-space averaging of certain kinematic variables defined in [39]. Given the source, one can calculate chemical potentials µj(z) for top, bottom, anti-top
and Higgs by solving a set of transport equations defined in [7] Finally the baryon to entropy Here ξw is the wall velocity, primes denote ∂zT and Kn,t are dimensionless functions of
xt ≡|mt|/T arising from phase-space averaging of certain kinematic variables defined in [39]. Gi
th
l
l t
h
i
l
t
ti l
( ) f
t
b tt
ti t Given the source, one can calculate chemical potentials µj(z) for top, bottom, anti-top
and Higgs by solving a set of transport equations defined in [7]. 3.5
Baryogenesis Finally the baryon-to-entropy
ratio ηB ≡nB/s is given by ηB =
405
4π2ξwg∗Tc
Z ∞
0
dz Γsph(z)µBL(z)e−45Γsph(z)z/4ξw. (3.19) (3.19) We take ξw = 0.1 for the wall velocity and g∗= 106.75 for the number of degrees of freedom
in the plasma. The left-chiral baryon chemical potential is We take ξw = 0.1 for the wall velocity and g∗= 106.75 for the number of degrees of freedom
in the plasma. The left-chiral baryon chemical potential is µBL = 1
2(1 + 4K1,t)µt + 1
2(1 + 4K1,b)µb −2K1,tcµtc. (3.20) (3.20) For the sphaleron rate we use a formula interpolating between the symmetric and the broken
phase [8],
6
40 ( )/T Γsph(z) = min(10−6Tc, 2.4Tce−40v(z)/Tc),
(3.21) (3.21) where v(z)2 = h1(z)2 + h2(z)2. where v(z)2 = h1(z)2 + h2(z)2. In the left panel of figure 4 we show how the baryon-to-entropy ratio relative to the
observed value ηobs
B
= 8.7 × 10−11 [40] correlates with the CP-violation-sensitive parameter
sin ∆CP defined in (3.13). Shown are only the points which survive the eEDM bound. As
expected, the size of sin ∆CP correlates with the size of ηB. This trend is similar to the
correlation between sin ∆CP and de shown in figure 3. However, a large ηB does not always
imply a large de, as is clear from the right panel of figure 4, where we show the correlation
between de and ηB, again for points that pass the EDM bound. Apparently, while both
quantities are sensitive to the CP-violating parameters in the model, they can be sensitive
to different linear combinations of them, so that large ηB may be obtained simultaneously
with a small enough de. Figure 5 shows the distributions of various physical parameters in our parametric scan. Orange colour refers to models that pass all experimental cuts described in sections 3.1
and 3.2, and give a strong EWPT, blue to models that in addition satisfy EDM constraint and
green to models which also give large baryon-to-entropy ratio ηB/ηobs
B
∈[0.5, 2]. These plots – 14 – Figure 5. Shown are the frequency distributions of the vevs of the scalar fields and the critical
temperature as well as scatter plots for the masses of the new heavy scalar particles in our parameter
scan. For details see the text. JCAP08(2016)057 Figure 5. 3.5
Baryogenesis Shown are the frequency distributions of the vevs of the scalar fields and the critical
temperature as well as scatter plots for the masses of the new heavy scalar particles in our parameter
scan. For details see the text. must be interpreted with care, since our scans were partly tuned by hand. Nevertheless, we
see that none of the vevs can be very large and in particular both v1 and v2 need to be nonzero. Also the critical temperature is bounded from above: Tc ≲100 GeV. Finally for the models
with large ηB, the new scalar masses are in general bound from above: mH, mA0, mH± ≲
1.4 TeV and mS ≲400 GeV, which is encouraging from the point of view of experimental
verifiability of the model. 3.6
Bubble nucleation So far we have implicitly assumed that the bubble nucleation takes place at a temperature not
too different from the critical temperature. This is typically the case in models where the first-
order phase transition is effected by cubic corrections to potential from infrared modes, which
leads to rather mild supercooling and small latent heat release. Here the situation is different,
because the barrier between the degenerate minima is essentially due to a tree-level term. Thus a stronger supercooling and more latent heat release may be expected, or even a possibil-
ity of a formation of a metastable vacuum where the electroweak breaking never takes place. We study the nucleation problem in the thin-wall limit [41]. The bubble nucleation rate
is given by
/ is given by Γ ∼T 4
S3(T)
2πT
3/2
exp
−S3(T)
T
,
(3.22) (3.22) where S3(T) is the three-dimensional action for an O(3)-symmetric bubble. In the thin-wall
limit, it is given by
3 S3(T) = 16π
3
σ3
∆V (T)2 ,
(3.23) (3.23) where ∆V (T) is the potential energy difference between the electroweak-symmetric and
electroweak-broken minima and σ is the surface tension, σ =
Z
dφ
√
2V ,
(3.24) (3.24) integrated along the path from the symmetric to the broken minimum at temperature T = Tc. The bubble nucleation temperature Tn is defined as the temperature at which creating at – 15 – 0.4
0.5
0.6
0.7
0.8
0.9
-5
-4
-3
-2
-1
0
Tn/Tc
log10(ηB/8.7×10-11)
0.4
0.5
0.6
0.7
0.8
0.9
-3
-2
-1
0
1
Tn/Tc
log10(α/αmax)
Figure 6. Left: shown is the baryon asymmetry ηB as a function of the ratio of the nucleation and
critical temperatures Tn/Tc. Right: shown is the bound for finding a deflagration wall. Only models
below the line α/αmax = 1 are allowed. For yellow points the nucleation temperatures were rescaled
by κ = 0.7. See text for details. We used αmax = 0.38, corresponding to ξw ≈0.1. 0.4
0.5
0.6
0.7
0.8
0.9
-5
-4
-3
-2
-1
0
Tn/Tc
log10(ηB/8.7×10-11) 0.4
0.5
0.6
0.7
0.8
0.9
-3
-2
-1
0
1
Tn/Tc
log10(α/αmax) JCAP08(2016)057 Figure 6. Left: shown is the baryon asymmetry ηB as a function of the ratio of the nucleation and
critical temperatures Tn/Tc. Right: shown is the bound for finding a deflagration wall. Only models
below the line α/αmax = 1 are allowed. 3.6
Bubble nucleation For yellow points the nucleation temperatures were rescaled
by κ = 0.7. See text for details. We used αmax = 0.38, corresponding to ξw ≈0.1. least one bubble per horizon volume is of order one. This condition can be written as S3(Tn)
Tn
= −log
3
4π
H(Tn)
Tn
4 2πTn
S3(Tn)
3/2! . (3.25) (3.25) We show the results of the nucleation-temperature calculation for our data set on the
left panel of figure 6. Obviously, a large number of points displayed in figure 4 are missing in
figure 6. The reason is that for these models, indicated by red dots in figure 4, no solution to
eq. (3.25) was found. In these cases, in the thin-wall approximation, the fields were trapped
in the false vacuum. Moreover, of the surviving models only four give large ηB. The situation is actually more dire than this: another implicit assumption in our baryon
asymmetry calculation is that the transition walls are subsonic deflagrations, which is required
for efficient diffusion of particle asymmetries across the bubble wall. However, with a large
latent heat release the walls tend to be supersonic detonations instead. A full microscopic
analysis of wall dynamics including a computation of the wall friction is beyond the scope of
this paper. However, a deflagration wall must necessarily satisfy a condition [42] α ≡∆V (Tn)
ρ(Tn)
< 1
3(1 −ξw)−13/10 ≡αmax ,
(3.26) (3.26) where ρ(Tn) is the radiation energy density in the symmetric phase. We show this condition
in the right panel of figure 6 (blue dots) for the set of models shown in the left panel. As ex-
pected, for the models with the lowest nucleation temperatures, deflagrations are not possible. This applies in particular to all four surviving models with a large baryon-to-entropy ratio. There are two issues that ameliorate the situation. First, the validity of the thin-wall
limit actually requires a small latent heat and/or a large surface tension, which is often not
the case here. When not applicable, thin-wall limit tends to overestimate the action S3(T),
and hence underestimate the nucleation rate and eventually Tn. Accurate calculation of the
nucleation rate is quite complicated in the full model, however, and we do not pursue it here. Instead, we compare the nucleation temperatures found in the thin-wall limit and in the full
calculation in the simpler, singlet extension of the SM, studied for example in [32, 33]. 3.6
Bubble nucleation In – 16 – 70
80
90
100
110
120
130
40
60
80
100
120
Tc(GeV)
Tn(GeV)
Figure 7. Shown are the nucleation temperatures in a scan over the parameters of the singlet
extension of the SM. Blue dots correspond to the thin-wall temperature and the yellow dots to a full
calculation. Gray lines connect pairs corresponding to the same physical parameters. The dotted line
corresponds to Tn = Tc. 70
80
90
100
110
120
130
40
60
80
100
120
Tc(GeV)
Tn(GeV) JCAP08(2016)057 Figure 7. Shown are the nucleation temperatures in a scan over the parameters of the singlet
extension of the SM. Blue dots correspond to the thin-wall temperature and the yellow dots to a full
calculation. Gray lines connect pairs corresponding to the same physical parameters. The dotted line
corresponds to Tn = Tc. practice we minimize the action practice we minimize the action S3(T) = 4π
Z
r2dr
1
2
dh
dr
2
+ 1
2
dS
dr
2
+ VSSM(h, S, T)
! ,
(3.27) (3.27) where h is the SM-Higgs field, and VSSM(h, S, T) is the singlet-model potential. The results of this analysis are shown in figure 7. The blue dots show the nucleation
temperature in the thin-wall approximation and the yellow dots the same quantity found
from the solving minimizing the action (3.27) . As expected, the thin-wall approximation
underestimates nucleation temperatures significantly. We find that the true nucleation tem-
perature Tn and the thin-wall value T tw
n
are related by Tn = Tc −κ (Tc −T tw
n ), where the
coefficient κ to some extent depends on MS and λS, but is less than 0.7. We anticipated this
result in the deflagration limit shown in the right panel of figure 6, where the yellow dots
were found by redefining all thin-wall nucleation temperatures by the above equation with
κ = 0.7. We believe that this scaling conservatively represents the effect of going beyond
thin-wall approximation in the full 2HDM and singlet model, and hence shows that most
parameter sets may in fact be deflagrations. We can also tune our search to prefer models with a higher critical temperatures. Indeed,
as is clearly seen from figure 7, the nucleation temperature approaches the critical tempera-
ture when Tc gets higher in the singlet extension of the SM and this feature persists also in the
full 2HDSM. 3.6
Bubble nucleation Hence, we made a new parametric scan, where we accept only models with Tc >
80 GeV. The result of this scan is seen in figure 8, where the left panel again shows the baryon-
to-entropy ratio and in the right panel the deflagration bound. We now found more points
with large asymmetry and, in particular after one rescales the thin-wall nucleation tempera-
tures as explained above, these points are now well below the deflagration bound eq. (3.26). 4
Conclusions and outlook We have studied the viability of a two-Higgs-doublet and inert-singlet model for EWBG and
for DM, taking into account also all existing observational and collider constraints. Our model – 17 – 0.5
0.6
0.7
0.8
0.9
-2.0
-1.5
-1.0
-0.5
0.0
Tn/Tc
log10(ηB/8.7×10-11)
0.5
0.6
0.7
0.8
0.9
-3
-2
-1
0
Tn/Tc
log10(α/αmax)
Figure 8. Left: shown is the baryon asymmetry ηB as a function of Tn/Tc for the new scan with
Tc > 80GeV (here Tn is the thin-wall nucleation temperature). Right: the deflagration bound for the
set displayed in the left plot as a function of Tn/Tc. We again used αmax = 0.38. 0.5
0.6
0.7
0.8
0.9
-2.0
-1.5
-1.0
-0.5
0.0
Tn/Tc
log10(ηB/8.7×10-11) 0.5
0.6
0.7
0.8
0.9
-3
-2
-1
0
Tn/Tc
log10(α/αmax) JCAP08(2016)057 Figure 8. Left: shown is the baryon asymmetry ηB as a function of Tn/Tc for the new scan with
Tc > 80GeV (here Tn is the thin-wall nucleation temperature). Right: the deflagration bound for the
set displayed in the left plot as a function of Tn/Tc. We again used αmax = 0.38. is based on the maximal GL(2,C) reparemetrization symmetry. This implies a universal
Yukawa-alignment scheme, where both Higgs fields couple similarly to all fermions and there
are no FCNCs. Exploiting the GL(2,C) symmetry, the the model can, in a particular basis, be
written with a type-I Yukawa sector combined with the most general CP-violating potential. Following [19–21] we implemented a novel way to construct potentials with a tree-
level stability and to study the symmetry breaking patterns at finite temperatures. This
construction was based on the Lorentz symmetry induced by the reprametrization symmetry
on bilinears formed from Higgs doublets. These techniques are applicable to all 2HDM
models, and they proved extremely useful when performing large-scale parametric scans over
the multidimensional phase space of the model. Dark matter and the strength of the electroweak phase transition in the model follow a
similar pattern to the pure singlet extension: in accordance with [33], we find that strong two-
step phase transitions are easily found, but they are consistent only with a subleading DM. Likewise, we find that experimental and observational constraints are fairly easy to avoid,
with the outstanding exception of the electron-EDM bound, which strongly constrains the
CP-violating parameters on the model. 4
Conclusions and outlook EDM constraints are particularly important because
creating a large baryon asymmetry during the electroweak phase transition requires large
CP-violating parameters; we found that the electron EDM indeed strongly constrains the
phase space consistent with baryogenesis. Yet the bounds are not as strong here as in the
pure 2HDM case [8], and we found a number of models consistent with all requirements. Finally, we observed that two-step transitions may suffer from an unexpected problem
of providing a too strong phase transition. We found that fields may get trapped in the
metastable minimum, and transition walls may not be subsonic as required by a successful
baryogenesis scenario. However, our analysis in the full model was restricted to the thin-wall
approximation. We then studied the bubble nucleation in full generality in the case of a pure
singlet extension of the SM. While the generic problem of too strong transitions remained,
we found that thin-wall limit tends to overestimate the strength of the transition. Based on
these results we argued that, when corrected for the thin-wall bias, the walls may well remain
subsonic in the full 2HDSM. In a revised scan concentrating to models with a large critical
temperature, we found many models potentially consistent with all available constraints. While our results are not a definite proof, they provide a strong indication of a success
of baryognesis in the 2HDM and an inert singlet model. Settling the issue beyond any doubt – 18 – would require two significant improvements. First is a detailed analysis of the bubble wall
dynamics, including a microscopic computation of the friction on the wall. Second is going
beyond the Tc-bounce solution, when solving the scalar field profiles over the bubble wall to
compute the top quark mass profile and eventually the baryogenesis source. These are both
very interesting topics that deserve to be studied in detail in the future. Acknowledgments We thank Jim Cline for useful comments and discussions and collaboration at the early stages
of this work. KK would like to thank Stephan Huber, Mikko Laine and Jose M. No for useful
discussions during the 2016 MIAPP programme “Why is there more Matter than Antimatter
in the Universe?”. This work was financially supported by the Academy of Finland projects
278722 and 267842. The CP3-Origins centre is partially funded by the Danish National
Research Foundation, grant number DNRF90. VV is supported by the Magnus Ehrnrooth
foundation. TA acknowledges partial funding from a Villum foundation grant. JCAP08(2016)057 A
Diagonalization of mass matrices In this appendix we show in detail the diagonalization of the scalar mass matrices. Follow-
ing [43], we write (A.1) rµ = φxΣµ
xyφy ,
(A.1) where φ =
H+
1I, H+
2I, H+
1R, H+
2R, H0
1I, H0
2I, H0
1R, H0
2R
,
(A.2) (A.2) where the subscripts R and I again denote the real and imaginary parts, respectively, and
the matrices Σµ are block diagonal matrices consisting of 4 × 4 elements where the subscripts R and I again denote the real and imaginary parts, respectively, and
the matrices Σµ are block diagonal matrices consisting of 4 × 4 elements Σ0 = 14 , Σ1 =
σ1 0
0 σ1
, Σ2 =
0 −iσ2
iσ2
0
, Σ3 =
σ3 0
0 σ3
. (A.3) (A.3) Now the (a, b) element of the mass matrix is Now the (a, b) element of the mass matrix is Now the (a, b) element of the mass matrix is Now the (a, b) element of the mass matrix is ∂2V
∂φa∂φb
φ=⟨φ⟩
= −Σµ
abMµ + Σµ
abλµν⟨rν⟩+ 2Σµ
ax⟨φx⟩λµν⟨φy⟩Σν
yb ,
(A.4) (A.4) and the minimum conditions are and the minimum conditions are ∂V
∂φa
φ=⟨φ⟩
= (−Σµ
axMµ + Σµ
axλµν⟨rν⟩) ⟨φx⟩= 0 . (A.5) (A.5) Let us consider the neutral part of the mass matrix. The eigenstate corresponding to eigen-
value zero is G0 = NGΣ5⟨φ0⟩, where Σ5 = Σ0Σ1Σ2Σ3 and NG is the normalization factor. Now Let us consider the neutral part of the mass matrix. The eigenstate corresponding to eigen-
value zero is G0 = NGΣ5⟨φ0⟩, where Σ5 = Σ0Σ1Σ2Σ3 and NG is the normalization factor. Now G0 = cos β cos θ H0
1R + sin β H0
1I −cos β sin θ H0
2R ,
(A.6) (A.6) where β = arctan v2
v1
. A
Diagonalization of mass matrices (A.7) (A.7) – 19 – – 19 – Hence we can diagonalize the neutral mass matrix by Hence we can diagonalize the neutral mass matrix by RN =
1
0
0
0
0
cycz
−cysz
sy
0 sxsycz + cxsz cxcz −sxsysz −sxcy
0 sxsz −cxsycz cxsysz + sxcz
cxcy
cβcθ
sβ −cβsθ 0
−sβcθ cβ
sβsθ
0
sθ
0
cθ
0
0
0
0
1
,
(A.8) (A.8) where we have used the short-hand notations sα ≡sin α, cα ≡cos α, and the neutral mass
eigenstates are where we have used the short-hand notations sα ≡sin α, cα ≡cos α, and the neutral mass
eigenstates are JCAP08(2016)057 JCAP08(2016)057
G0
A0
H0
h0
= RN
H0
1I
H0
2I
H0
1R
H0
2R
. (A.9) (A.9) On the charged sector there are two eigenstates with eigenvalue zero: G1 = NG1Σ5⟨φ0⟩
and G2 = NG2⟨φ0⟩. Now On the charged sector there are two eigenstates with eigenvalue zero: G1 = NG1Σ5⟨φ0⟩
and G2 = NG2⟨φ0⟩. Now G1 = cos β sin θ H+
1R + cos β cos θ H+
1I + sin β H+
2I ,
G2 = cos β cos θ H+
1R −cos β sin θ H+
1I + sin β H+
2R . (A.10) (A.10) Hence charged Goldstone bosons are G± = cos βe±iθH±
1 + sin βH±
2 . The charged mass
matrix in the basis {H+
1 , H+
2 } can be diagonalized by RC =
cβeiθ
sβ
−sβeiθ cβ
,
(A.11) (A.11) and charged mass eigenstates are and charged mass eigenstates are G+
H+
= RC
H+
1
H+
2
. A
Diagonalization of mass matrices (A.12) (A.12) B
1-loop beta functions for scalar couplings For completeness we show the 1-loop beta functions, βλi ≡
dλi
d ln µ,
(B.1) βλi ≡
dλi
d ln µ, (B.1) – 20 – we used to compute the perturbativity limits on the model parameters: 16π2βλ1 = 24λ2
1 + 2λ2
3 + 2λ3λ4 + λ2
4 + 4|λ5|2 + 12|λ6|2
+ 1
2λ2
S1 −3
3g2
L + g2
Y
λ1 + 3
8
3g4
L + 2g2
Lg2
Y + g4
Y
,
(B.2)
16π2βλ2 = 24λ2
2 + 2λ2
3 + 2λ3λ4 + λ2
4 + 4|λ5|2 + 12|λ7|2 + 1
2λ2
S2
−3
3g2
L + g2
Y
λ2 + 3
8
3g4
L + 2g2
Lg2
Y + g4
Y
+ 12y2
t λ2 −6y4
t ,
(B.3)
16π2βλ3 = (λ1 + λ2)(12λ3 + 4λ4) + 4λ2
3 + 2λ2
4 + 8|λ5|2 + 4|λ6|2
2 (B.2) (B.3) JCAP08(2016)057 (B.4) −3
3g2
L + g2
Y
λ3 + 3
4
3g4
L −2g2
Lg2
Y + g4
Y
+ 6y2
t λ3,
16π2βλ4 = 4(λ1 + λ2)λ4 + 8λ3λ4 + 4λ2
4 + 32|λ5|2 + 10
|λ6|2 + |λ7|2
+ 4Re (λ∗
6λ7) + |λS12|2 −3
3g2
L + g2
Y
λ4 + 3g2
Lg2
Y + 6y2
t λ4,
(B.5)
16π2βλ5 = (4λ1 + 4λ2 + 8λ3 + 12λ4)λ5 + 5λ2
6 + 2λ6λ7 + 5λ2
7
+ 1λ2
S12 −3
3g2
L + g2
Y
λ5 + 6y2
t λ5,
(B.6) 3
3gL + gY
λ3 + 4
3gL
2gLgY + gY
+ 6yt λ3,
16π2βλ4 = 4(λ1 + λ2)λ4 + 8λ3λ4 + 4λ2
4 + 32|λ5|2 + 10
|λ6|2 + |λ7|2
+ 4Re (λ∗
6λ7) + |λS12|2 −3
3g2
L + g2
Y
λ4 + 3g2
Lg2
Y + 6y2
t λ4,
(B.5)
16π2βλ5 = (4λ1 + 4λ2 + 8λ3 + 12λ4)λ5 + 5λ2
6 + 2λ6λ7 + 5λ2
7
(B 6) (B.5)
16π2βλ5 = (4λ1 + 4λ2 + 8λ3 + 12λ4)λ5 + 5λ2
6 + 2λ6λ7 + 5λ2
7
+ 1
2λ2
S12 −3
3g2
L + g2
Y
λ5 + 6y2
t λ5,
(B.6) + 1
2λ2
S12 −3
3g2
L + g2
Y
λ5 + 6y2
t λ5,
(B.6) 2
16π2βλ6 = (24λ1 + 6λ3 + 8λ4)λ6 + (6λ3 + 4λ4)λ7 + (20λ∗
6 + 4λ∗
7)λ5
+ λS1λS12 −3
3g2
L + g2
Y
λ6 + 3y2
t λ6,
(B.7) 2
16π2βλ6 = (24λ1 + 6λ3 + 8λ4)λ6 + (6λ3 + 4λ4)λ7 + (20λ∗
6 + 4λ∗
7)λ5
+ λS1λS12 −3
3g2
L + g2
Y
λ6 + 3y2
t λ6,
(B.7) 2
16π2βλ6 = (24λ1 + 6λ3 + 8λ4)λ6 + (6λ3 + 4λ4)λ7 + (20λ∗
6 + 4λ∗
7)λ5
+ λS1λS12 −3
3g2
L + g2
Y
λ6 + 3y2
t λ6,
(B.7) 16π2βλ7 = (6λ3 + 4λ4)λ6 + (24λ2 + 6λ3 + 8λ4)λ7 + (4λ∗
6 + 20λ∗
7)λ5
+ λS2λS12 −3
3g2
L + g2
Y
λ7 + 9y2
t λ7,
(B.8) 16π2βλ7 = (6λ3 + 4λ4)λ6 + (24λ2 + 6λ3 + 8λ4)λ7 + (4λ∗
6 + 20λ∗
7)λ5
+ λS2λS12 −3
3g2
L + g2
Y
λ7 + 9y2
t λ7,
(B.8) 16π2βλS = 18λ2
S + 2λ2
S1 + 2λ2
S2 + 4|λS12|2,
(B.9)
16π2βλS1 = (12λ1 + 6λS)λS1 + (4λ3 + 2λ4)λS2 + 4λ2
S1 + 12Re (λ∗
6λS12)
+ 4|λS12|2 −3
2
3g2
L + g2
Y
λS1,
(B.10)
16π2βλS2 = (4λ3 + 2λ4)λS1 + (12λ2 + 6λS)λS2 + 4λ2
S2 + 12Re (λ∗
7λS12)
+ 4|λS12|2 −3
2
3g2
L + g2
Y
λS2 + 6y2
t λS2,
(B.11)
16π2βλS12 = (2λ3 + 4λ4 + 6λS + 4λS1 + 4λS2)λS12 + 12λ5λ∗
S12
+ 6λ6λS1 + 6λ7λS2 −3
2
3g2
L + g2
Y
λS12 + 3y2
t λS12. A
Diagonalization of mass matrices (B.12) 18λS + 2λS1 + 2λS2 + 4|λS12| ,
(B.9)
= (12λ1 + 6λS)λS1 + (4λ3 + 2λ4)λS2 + 4λ2
S1 + 12Re (λ∗
6λS12)
+ 4|λS12|2 −3
2
3g2
L + g2
Y
λS1,
(B.10) C
Singlet scalar annihilation cross sections Here we give compact expressions for singlet pair annihilation rates used in the computation
of the DM abundances. In the mass eigenbasis, the relevant Lagrangian may be written as L =
X
j,k
c0S2HjHk +
X
i
cijkHiHjHk
! +
X
i
ciS2Hi +
X
i,f
yifHifRfL + h.c.
+
X
i
gWiW +W −Hi + gZiZZHi
,
(C.1) L =
X
j,k
c0S2HjHk +
X
i
cijkHiHjHk
! +
X
i
ciS2Hi +
X
i,f
yifHifRfL + h.c.
X +
(C.1) – 21 – where H = (A0, H0, h0, H+) . Now the singlet scalar annihilation cross section to scalars is
given by: where H = (A0, H0, h0, H+) . Now the singlet scalar annihilation cross section to scalars is
given by: σ(SS →HjHk) =
1
16πs2vs
A2
jkI0,j,k + 2AjkBjkI1,j,k + B2
jkI2,j,k
. C
Singlet scalar annihilation cross sections (C.2) (C.2) The rate into fermion-antifermion pairs is given by: The rate into fermion-antifermion pairs is given by: σ(SS →ff) =
I0,f,f
4πs2vs
X
i,j
cicj
(s −m2
i )(s −m2
j) ×
×
(yifRyjfR −yifIyjfI) s
2 −2yifRyjfRm2
f
,
(C.3) ×
(yifRyjfR −yifIyjfI) s
2 −2yifRyjfRm2
f
,
(C.3) (C.3) JCAP08(2016)057 nd the annihilation to vector bosons is: σ(SS →V V ) = I0,V,V
4πs2vs
X
i,j
cicjgV igV j
(s −m2
i )(s −m2
j)
3 + s(s −4M2
V )
4M2
V
1 −δV,Z
2
,
(C.4) (C.4) where I0,j,k =
q
s2 + (m2
j −m2
k)2 −2s(m2
j + m2
k) ,
I1,j,k =
4I0,j,k
m2
j + m2
k −s ,
I2,j,k =
2I0,j,k
m2
jm2
k −M2
S(2m2
j + 2m2
k −s) + M2s
s (m2
j −m2
k)2 +
8I0,j,k
(m2
j + m2
k −s)2 ,
(C.5) I0,j,k =
q
s2 + (m2
j −m2
k)2 −2s(m2
j + m2
k) ,
I1,j,k =
4I0,j,k
m2
j + m2
k −s ,
2I
8I
(C.5) ,j,
q
j
k
j
k
I1,j,k =
4I0,j,k
m2
j + m2
k −s ,
I2,j,k =
2I0,j,k
m2
jm2
k −M2
S(2m2
j + 2m2
k −s) + M2s
s (m2
j −m2
k)2 +
8I0,j,k
(m2
j + m2
k −s)2 ,
(C.5)
d
Ajk = 2(1 + δjk)c0 + 2
X
i
(1 + δij + δjk + δki + 2δijδjk) cicijk
s −m2
i
,
Bjk = 4cjck . (C.6) (C.5) j
k
=
2I0,j,k
m2
jm2
k −M2
S(2m2
j + 2m2
k −s) + M2s
s (m2
j −m2
k)2 +
8I0,j,k
(m2
j + m2
k −s)2 , and (C.6) References References [1] N. Turok and J. Zadrozny, Electroweak baryogenesis in the two doublet model, Nucl. Phys. B
358 (1991) 471 [INSPIRE]. 2] N. Turok and J. Zadrozny, Phase transitions in the two doublet model, Nucl. Phys. B 369
(1992) 729 [INSPIRE]. [2] N. Turok and J. Zadrozny, Phase transitions in the two doublet model, Nucl. Phys. B 369
(1992) 729 [INSPIRE]. [3] K. Funakubo, A. Kakuto and K. Takenaga, The effective potential of electroweak theory with
two massless Higgs doublets at finite temperature, Prog. Theor. Phys. 91 (1994) 341
[hep-ph/9310267] [INSPIRE]. [3] K. Funakubo, A. Kakuto and K. Takenaga, The effective potential of electroweak theory with
two massless Higgs doublets at finite temperature, Prog. Theor. Phys. 91 (1994) 341
[hep-ph/9310267] [INSPIRE]. [4] A.T. Davies, C.D. froggatt, G. Jenkins and R.G. Moorhouse, Baryogenesis constraints on two
Higgs doublet models, Phys. Lett. B 336 (1994) 464 [INSPIRE]. 4] A.T. Davies, C.D. froggatt, G. Jenkins and R.G. Moorhouse, Baryogenesis constraints on two
Higgs doublet models, Phys. Lett. B 336 (1994) 464 [INSPIRE]. [5] J.M. Cline, K. Kainulainen and A.P. Vischer, Dynamics of two Higgs doublet CP-violation and
baryogenesis at the electroweak phase transition, Phys. Rev. D 54 (1996) 2451
[hep-ph/9506284] [INSPIRE]. [6] M. Laine and K. Rummukainen, Two Higgs doublet dynamics at the electroweak phase
transition: a nonperturbative study, Nucl. Phys. B 597 (2001) 23 [hep-lat/0009025] [INSPIRE]. – 22 – [7] L. Fromme, S.J. Huber and M. Seniuch, Baryogenesis in the two-Higgs doublet model, JHEP
11 (2006) 038 [hep-ph/0605242] [INSPIRE]. [8] J.M. Cline, K. Kainulainen and M. Trott, Electroweak baryogenesis in two Higgs doublet models
and B meson anomalies, JHEP 11 (2011) 089 [arXiv:1107.3559] [INSPIRE]. [9] G.C. Dorsch, S.J. Huber and J.M. No, A strong electroweak phase transition in the 2HDM after
LHC8, JHEP 10 (2013) 029 [arXiv:1305.6610] [INSPIRE]. [10] A. Pich and P. Tuzon, Yukawa alignment in the two-Higgs-doublet model, Phys. Rev. D 80
(2009) 091702 [arXiv:0908.1554] [INSPIRE]. [11] E.H. Simmons, Phenomenology of a technicolor model with heavy scalar doublet, Nucl. Phys. B
312 (1989) 253 [INSPIRE]. JCAP08(2016)057 [12] A. Kagan and S. Samuel, Renormalization group aspects of bosonic technicolor, Phys. Lett. B
270 (1991) 37 [INSPIRE]. [13] M. Antola, M. Heikinheimo, F. Sannino and K. Tuominen, Unnatural origin of fermion masses
for technicolor, JHEP 03 (2010) 050 [arXiv:0910.3681] [INSPIRE]. [14] T. Alanne, S. Di Chiara and K. Tuominen, LHC data and aspects of new physics, JHEP 01
(2014) 041 [arXiv:1303.3615] [INSPIRE]. References [15] M. Geller, S. Bar-Shalom and A. Soni, Hybrid dynamical electroweak symmetry breaking with
heavy quarks and the 125 GeV Higgs boson, Phys. Rev. D 89 (2014) 035012 [arXiv:1302.2915]
[INSPIRE]. [16] M. Hashimoto and V.A. Miransky, Dynamical electroweak symmetry breaking with superheavy
quarks and 2 + 1 composite Higgs model, Phys. Rev. D 81 (2010) 055014 [arXiv:0912.4453]
[INSPIRE]. [17] H.S. Fukano and K. Tuominen, A hybrid 4th generation: technicolor with top-seesaw, Phys. Rev. D 85 (2012) 095025 [arXiv:1202.6296] [INSPIRE]. [18] H.S. Fukano and K. Tuominen, 126 GeV Higgs boson in the top-seesaw model, JHEP 09 (2013)
021 [arXiv:1306.0205] [INSPIRE]. [19] I.P. Ivanov, Minkowski space structure of the Higgs potential in 2HDM, Phys. Rev. D 75 (2007)
035001 [Erratum ibid. D 76 (2007) 039902] [hep-ph/0609018] [INSPIRE]. [20] I.P. Ivanov, Minkowski space structure of the Higgs potential in 2HDM II. Minima, symmetries
and topology, Phys. Rev. D 77 (2008) 015017 [arXiv:0710.3490] [INSPIRE]. [21] I.P. Ivanov, Thermal evolution of the ground state of the most general 2HDM, Acta Phys. Polon. B 40 (2009) 2789 [arXiv:0812.4984] [INSPIRE]. [22] ALEPH collaboration, A. Heister et al., Search for charged Higgs bosons in e+e−collisions at
energies up to √s = 209 GeV, Phys. Lett. B 543 (2002) 1 [hep-ex/0207054] [INSPIRE]. [23] ATLAS collaboration, Combined search for the Standard Model Higgs boson in pp collisions at
√s = 7 TeV with the ATLAS detector, Phys. Rev. D 86 (2012) 032003 [arXiv:1207.0319]
[INSPIRE]. [24] M.E. Peskin and T. Takeuchi, A new constraint on a strongly interacting Higgs sector, Phys. Rev. Lett. 65 (1990) 964 [INSPIRE]. [25] W. Grimus, L. Lavoura, O.M. Ogreid and P. Osland, The oblique parameters in
multi-Higgs-doublet models, Nucl. Phys. B 801 (2008) 81 [arXiv:0802.4353] [INSPIRE]. [26] Particle Data Group collaboration, K.A. Olive et al., Review of particle physics, Chin. Phys. C 38 (2014) 090001 [INSPIRE]. [27] CDF and D0 collaborations, T. Aaltonen et al., Higgs boson studies at the Tevatron, Phys. Rev. D 88 (2013) 052014 [arXiv:1303.6346] [INSPIRE]. – 23 – [28] CMS collaboration, Precise determination of the mass of the Higgs boson and tests of
compatibility of its couplings with the Standard Model predictions using proton collisions at 7
and 8 TeV, Eur. Phys. J. C 75 (2015) 212 [arXiv:1412.8662] [INSPIRE]. [29] ATLAS collaboration, Measurements of the Higgs boson production and decay rates and
coupling strengths using pp collision data at √s = 7 and 8 TeV in the ATLAS experiment, Eur. Phys. J. References C 76 (2016) 6 [arXiv:1507.04548] [INSPIRE]. [30] K. Kainulainen, S. Nurmi, T. Tenkanen, K. Tuominen and V. Vaskonen, Isocurvature
constraints on portal couplings, JCAP 06 (2016) 022 [arXiv:1601.07733] [INSPIRE]. [31] P. Gondolo and G. Gelmini, Cosmic abundances of stable particles: improved analysis, Nucl. Phys. B 360 (1991) 145 [INSPIRE]. JCAP08(2016)057 [32] J.M. Cline, K. Kainulainen, P. Scott and C. Weniger, Update on scalar singlet dark matter,
Phys. Rev. D 88 (2013) 055025 [arXiv:1306.4710] [INSPIRE]. [33] J.M. Cline and K. Kainulainen, Electroweak baryogenesis and dark matter from a singlet Higgs,
JCAP 01 (2013) 012 [arXiv:1210.4196] [INSPIRE]. [34] LUX collaboration, D.S. Akerib et al., Improved limits on scattering of weakly interacting
massive particles from reanalysis of 2013 LUX data, Phys. Rev. Lett. 116 (2016) 161301
[arXiv:1512.03506] [INSPIRE]. [35] J.R. Espinosa, T. Konstandin and F. Riva, Strong electroweak phase transitions in the
Standard Model with a singlet, Nucl. Phys. B 854 (2012) 592 [arXiv:1107.5441] [INSPIRE [36] S. Inoue, M.J. Ramsey-Musolf and Y. Zhang, CP-violating phenomenology of flavor conserving
two Higgs doublet models, Phys. Rev. D 89 (2014) 115023 [arXiv:1403.4257] [INSPIRE]. [37] ACME collaboration, J. Baron et al., Order of magnitude smaller limit on the electric dipole
moment of the electron, Science 343 (2014) 269 [arXiv:1310.7534] [INSPIRE]. [38] T. Abe, J. Hisano, T. Kitahara and K. Tobioka, Gauge invariant Barr-Zee type contributions
to fermionic EDMs in the two-Higgs doublet models, JHEP 01 (2014) 106 [Erratum ibid. 04
(2016) 161] [arXiv:1311.4704] [INSPIRE]. [39] L. Fromme and S.J. Huber, Top transport in electroweak baryogenesis, JHEP 03 (2007) 049
[hep-ph/0604159] [INSPIRE]. [39] L. Fromme and S.J. Huber, Top transport in electroweak baryogenesis, JHEP 03 (2007) 049
[hep-ph/0604159] [INSPIRE]. [40] Planck collaboration, P.A.R. Ade et al., Planck 2015 results. XIII. Cosmological parameters,
arXiv:1502.01589 [INSPIRE]. [40] Planck collaboration, P.A.R. Ade et al., Planck 2015 results. XIII. Cosmological parameters,
arXiv:1502.01589 [INSPIRE]. [41] A.D. Linde, Decay of the false vacuum at finite temperature, Nucl. Phys. B 216 (1983) 421
[Erratum ibid. B 223 (1983) 544] [INSPIRE]. [42] J.R. Espinosa, T. Konstandin, J.M. No and G. Servant, Energy budget of cosmological
first-order phase transitions, JCAP 06 (2010) 028 [arXiv:1004.4187] [INSPIRE]. [43] A. Degee and I.P. Ivanov, Higgs masses of the general 2HDM in the Minkowski-space
formalism, Phys. Rev. D 81 (2010) 015012 [arXiv:0910.4492] [INSPIRE]. – 24 –
|
https://openalex.org/W4243084734
|
https://peerj.com/articles/7002v0.3/submission
|
English
| null |
Peer Review #1 of "Mir-421 in plasma as a potential diagnostic biomarker for precancerous gastric lesions and early gastric cancer (v0.1)"
| null | 2,019
|
cc-by
| 10,899
|
Mir-421 in plasma as a potential diagnostic biomarker for
precancerous gastric lesions and early gastric cancer
Jianlin Chen
Corresp., Equal first author,
1 , Lihua Wu
Equal first author,
2 , Yifan Sun
Corresp., 1 , Qi Yin
1 , Xianhua Chen
1 , Siqun Liang
1 ,
Qingyan Meng
1 , Haihua Long
2 , Fangying Li
2 , Changjun Luo
3 , Xiaorong Xiao
4 1 Department of Clinical Laboratory, Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China
2 Department of Digestive internal medicine, Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China
3 Department of Internal Medicine-Cardiovascular, Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China
4 Department of Science and Education, Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China
Corresponding Authors: Jianlin Chen, Yifan Sun
Email address: cjl801900@163.com, sunyifan13@126.com Objective. MicroRNA (miR)-421 plays a key role in cancer progression. It has been
reported that circulating miR-421may be a potential tumor marker for the diagnosis of
several cancers. However, the role of miR-421 in plasma as a potential biomarker in the
diagnosis of precancerous gastric lesions (Pre) and early-stage gastric cancer (GC) remains
poorly understood. In this study, we investigated miR-421 in plasma as a novel potential
biomarker for the detection of precancerous gastric lesions and early-stage (GC). Objective. MicroRNA (miR)-421 plays a key role in cancer progression. It has been
reported that circulating miR-421may be a potential tumor marker for the diagnosis of
several cancers. However, the role of miR-421 in plasma as a potential biomarker in the
diagnosis of precancerous gastric lesions (Pre) and early-stage gastric cancer (GC) remains
poorly understood. In this study, we investigated miR-421 in plasma as a novel potential
biomarker for the detection of precancerous gastric lesions and early-stage (GC). Materials & Methods. The miRNA content was determined by quantitative real-time
polymerase chain reaction (qRT-PCR). MiR-421 content in all subjects was normalized by
endogenous miRNA (miR-16). The diagnostic value of miR-421 for Pre and GC was
assessed by comparing receiver operating characteristic (ROC) analysis with traditional
tumor markers, including CEA, CA125, CA153, CA211 and CA50. The correlation between
the expression of miR-421 and the pathological characteristics of Pre and GC was
analyzed. Results. Elevated expression of miR-421 in plasma can robustly distinguish the
normal population from Pre and GC cases, especially in the early stages of gastric cancer
cases (all p <0.05). The ROC analyses showed that the area under the ROC curve (AUC),
sensitivity, accuracy and Youden index of miR-421 were superior to traditional tumor
markers (CEA, CA125, CA153, CA211, and CA50) in GC diagnosis, while its specificity was
higher than CEA, CA153 and CA50 (all p <0.05). Manuscript to be reviewed Mir-421 in plasma as a potential diagnostic biomarker for
precancerous gastric lesions and early gastric cancer
Jianlin Chen
Corresp., Equal first author,
1 , Lihua Wu
Equal first author,
2 , Yifan Sun
Corresp., 1 , Qi Yin
1 , Xianhua Chen
1 , Siqun Liang
1 ,
Qingyan Meng
1 , Haihua Long
2 , Fangying Li
2 , Changjun Luo
3 , Xiaorong Xiao
4 MiR-421 in plasma had higher AUC value
than AFP, CA153, CA211 and CA50 in the diagnosis of Pre (all p<0.05), while specificity,
accuracy and Youden index of miR-421 was only lower than CA211. The efficiency of
miR-421 in the diagnosis of GC was significantly higher than that of CA211 and CA50, and
it was significantly higher than CA153, CA211 and CA50 in the diagnosis of Pre (all
p<0.05). In addition, up-regulation of miR-421 occurred initially in precancerous gastric
lesions as well as in the early stage of GC. Conclusions. Overexpression of plasma
PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) 2 precancerous gastric lesions and early gastric can 3
Jianlin Chen1,*, Lihua Wu2, Yifan Sun3, Qi Yin4, Xianhua Chen5, Siqun Liang6, Qingyan Meng7,
4
Haihua Long8, Fangying Li9, Changjun Luo10, Xiaorong Xiao11
5
Department of Clinical Laboratory1,3,4,5,6,7, Department of Digestive internal medicine2,8,9,
6
Department of Internal Medicine-Cardiovascular10, d Department of Science and Education11,
7
Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China
8
Corresponding Author: Jianlin Chen, Yifan Sun
9
14 Fei-e Road, Liuzhou, Guangxi, 545007, China
10
Email address: cjl801900@163.com; sunyifan13@126.com 3
Jianlin Chen1,*, Lihua Wu2, Yifan Sun3, Qi Yin4, Xianhua Chen5, Siqun Liang6, Qingyan Meng7,
4
Haihua Long8, Fangying Li9, Changjun Luo10, Xiaorong Xiao11
5
Department of Clinical Laboratory1,3,4,5,6,7, Department of Digestive internal medicine2,8,9,
6
Department of Internal Medicine-Cardiovascular10, d Department of Science and Education11,
7
Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China
8
Corresponding Author: Jianlin Chen, Yifan Sun
9
14 Fei-e Road, Liuzhou, Guangxi, 545007, China
10
Email address: cjl801900@163.com; sunyifan13@126.com 3
Jianlin Chen1,*, Lihua Wu2, Yifan Sun3, Qi Yin4, Xianhua Chen5, Siqun Liang6, Qingyan Meng7,
4
Haihua Long8, Fangying Li9, Changjun Luo10, Xiaorong Xiao11
5
Department of Clinical Laboratory1,3,4,5,6,7, Department of Digestive internal medicine2,8,9,
6
Department of Internal Medicine-Cardiovascular10, d Department of Science and Education11,
7
Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China
8
Corresponding Author: Jianlin Chen, Yifan Sun
9
14 Fei-e Road, Liuzhou, Guangxi, 545007, China
10
Email address: cjl801900@163.com; sunyifan13@126.com 10
Email address: cjl801900@163.com; sunyifan13@126.com Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed miR-421 is a novel biomarker for the detection of precancerous lesions and early gastric
cancer. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 30
CA211 and CA50 in the diagnosis of Pre (all p<0.05), while specificity, accuracy and Youden
31
index of miR-421 was only lower than CA211. The efficiency of miR-421 in the diagnosis of
32
GC was significantly higher than that of CA211 and CA50, and it was significantly higher than
33
CA153, CA211 and CA50 in the diagnosis of Pre (all p<0.05). In addition, up-regulation of miR-
34
421 occurred initially in precancerous gastric lesions as well as in the early stage of GC. 35
Conclusions. Overexpression of plasma miR-421 is a novel biomarker for the detection of
36
precancerous lesions and early gastric cancer. 30
CA211 and CA50 in the diagnosis of Pre (all p<0.05), while specificity, accuracy and Youden
31
index of miR-421 was only lower than CA211. The efficiency of miR-421 in the diagnosis of
32
GC was significantly higher than that of CA211 and CA50, and it was significantly higher than
33
CA153, CA211 and CA50 in the diagnosis of Pre (all p<0.05). In addition, up-regulation of miR-
34
421 occurred initially in precancerous gastric lesions as well as in the early stage of GC. 35
Conclusions. Overexpression of plasma miR-421 is a novel biomarker for the detection of
36
precancerous lesions and early gastric cancer. 33
CA153, CA211 and CA50 in the diagnosis of Pre (all p 0.05). In addition, up regulation of miR
34
421 occurred initially in precancerous gastric lesions as well as in the early stage of GC. 35
Conclusions. Overexpression of plasma miR-421 is a novel biomarker for the detection of
36
precancerous lesions and early gastric cancer. 37
INTRODUCTION
38
Gastric cancer (GC) is one of the most common malignant tumors in China(Zheng et al. 2019). 39
Due to the lack of tumor markers and specific symptoms and signs, most tumors have
40
metastasized by the time of diagnosis. Studies have shown that patients with advanced GC have
41
a poor prognosis with a 5-year survival rate of less than 25%(Chan et al. 2001). However, if
42
intervention and treatment are performed in the pre- or early stages of gastric cancer, the five-
43
year survival rate will rise to 95%(Craanen et al. 1991). Therefore, it is very important to detect
44
and treat gastric cancer at an early stage. 45
46
Gastric cancer originates from the malignant and aggressive proliferation of gastric mucosal
47
cells. 11
ABSTRACT 12
Objective. MicroRNA (miR)-421 plays a key role in cancer progression. It has been reported
13
that circulating miR-421may be a potential tumor marker for the diagnosis of several cancers. 14
However, the role of miR-421 in plasma as a potential biomarker in the diagnosis of
15
precancerous gastric lesions (Pre) and early-stage gastric cancer (GC) remains poorly
16
understood. In this study, we investigated miR-421 in plasma as a novel potential biomarker for
17
the detection of precancerous gastric lesions and early-stage (GC). 18
Materials & Methods. The miRNA content was determined by quantitative real-time
19
polymerase chain reaction (qRT-PCR). MiR-421 content in all subjects was normalized by
20
endogenous miRNA (miR-16). The diagnostic value of miR-421 for Pre and GC was assessed by
21
comparing receiver operating characteristic (ROC) analysis with traditional tumor markers,
22
including CEA, CA125, CA153, CA211 and CA50. The correlation between the expression of
23
miR-421 and the pathological characteristics of Pre and GC was analyzed. 24
Results. Elevated expression of miR-421 in plasma can robustly distinguish the normal
25
population from Pre and GC cases, especially in the early stages of gastric cancer cases (all p
26
<0.05). The ROC analyses showed that the area under the ROC curve (AUC), sensitivity,
27
accuracy and Youden index of miR-421 were superior to traditional tumor markers (CEA,
28
CA125, CA153, CA211, and CA50) in GC diagnosis, while its specificity was higher than CEA,
29
CA153 and CA50 (all p <0.05). MiR-421 in plasma had higher AUC value than AFP, CA153, 24
Results. Elevated expression of miR-421 in plasma can robustly distinguish the normal
25
population from Pre and GC cases, especially in the early stages of gastric cancer cases (all p
26
<0.05). The ROC analyses showed that the area under the ROC curve (AUC), sensitivity,
27
accuracy and Youden index of miR-421 were superior to traditional tumor markers (CEA,
28
CA125, CA153, CA211, and CA50) in GC diagnosis, while its specificity was higher than CEA,
29
CA153 and CA50 (all p <0.05). MiR-421 in plasma had higher AUC value than AFP, CA153, PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 62
MicroRNAs (miRNAs) are a class of endogenous non-coding RNA molecules (length of 18–25
63
nucleotides) that exert their roles by base pairing between the seed region of miRNA or 3′-un-
64
translated regions (3′-UTR) of the target gene(Lee & Ambros 2001). Studies have confirmed that
65
miRNAs’ expression are frequently de-regulated in human tumors(Hernando 2007; Lu et al. 66
2005). Recently, circulating miRNAs (miRNAs present in blood) have attracted attention due to
67
their stability and ease of measurement. Many circulating miRNAs have been quantified and
68
identified by qRT-PCR as tumor markers to detect GC, such as miR-223, miR-21, miR-218(Li et
69
al. 2012; Zhou et al. 2015), and miR-18a(Tsujiura et al. 2015) in plasma, and miR-20a(Yang et
70
al. 2017) in serum. However, there is limited research into circulating miRNA markers for the
71
diagnosis of precancerous gastric lesions and early-stage gastric cancer, and most of these studies
72
have not compared the value of these miRNAs with traditional tumor markers in the diagnosis of
73
gastric cancer and precancerous lesions. 75
MicroRNA-421 (miR-421) is a molecule that has been studied extensively. It has been
76
investigated and found to be expressed aberrantly in various types of cancer(Li et al. 2018; Wang
77
et al. 2018; Zhou et al. 2016). Previous studies showed that miR-421 rises notably in tissue and
78
gastric juice, as well as in blood mononuclear cells and serum of gastric cancer(Liu et al. 2015;
79
Zhang et al. 2012a; Zhao et al. 2015), suggesting that miR-421 seems to be an ideal biomarker
80
for gastric cancer. However, most of these studies failed to point out at which stage in the
81
evolution of gastric cancer miR-421 can be detected in the peripheral blood, and an optimal
82
circulating miRNA should have the ability to distinguish different phases in the course of cancer
83
development. Some critical issues remain unresolved. Firstly, the expression of miR-421 in the
84
plasma of precancerous patients has not been fully investigated. More importantly, the value and
85
significance of miR-421 in the early diagnosis of gastric cancer are still not clearly stated. The
86
present study aimed to clarify the diagnostic value of miR-421 in the early stage of gastric cancer
87
and in the precancerous lesions of gastric cancer and to explore its relation with the progression
88
of GC. Manuscript to be reviewed Most are developed and evolved by the chronic inflammation of gastric mucosa under the
48
influence of multiple factors, mainly in four stages (chronic superficial gastritis → chronic
49
atrophic gastritis → intestinal metaplasia and dysplasia → gastric cancer). Chronic atrophic
50
gastritis is currently recognized as a precancerous disease, intestinal metaplasia and dysplasia is a
51
precancerous lesion closely related to the development of gastric cancer. Currently, Gastroscopy
52
combined with biopsy is the gold standard for diagnosis of gastric cancer. However, it may not
53
be appropriate to carry out large-scale screening and early detection in most countries including
54
China due to acceptability, availability, cost and other reasons. GC related tumor markers such
55
as, cancer embryo antigen (CEA), pepsinogen (PG), carbohydrate antigen 199(CA199) and
56
carbohydrate antigen 724(CA724), gastrin-17(G17) have been used clinically for many years,
57
but many do not have sufficient sensitivity and specificity for GC screening(He et al. 2013;
58
Pectasides et al. 1997; Yang et al. 2014). Studies on their role in monitoring early gastric cancer
59
have rarely been reported. Therefore, there is a need for reliable and non-invasive biomarkers for
60
early detection and mass screening of GC. 61 61 PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) 90
Clinical specimens 91
Plasma samples from 90 GC patients, 89 patients with precancerous (Pre) lesions and 45 normal
92
healthy controls (NC) were collected at Affiliated Liutie Central Hospital of Guangxi Medical
93
University in Guangxi province, China and the levels of miR-421 were screened by qRT-PCR. 94
GC patients were confirmed by gastroscopy combined with biopsy. Tumor type and stage were PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 95
identified according to the Union of International Cancer Control (UICC) tumor-node-metastasis
96
(TNM) system, 7th edition. The histology of all patients was evaluated according to the World
97
Health Organization (WHO) criteria. Patients who had received Radiation therapy or
98
chemotherapy were excluded from the study. The pathological outcomes of GC and Pre patients
99
were obtained by two experienced pathologists. In addition, 45 healthy people without a previous
100
history of diabetes, heart disease, hypertension, and cancer were chosen as normal controls (NC). 101
We compared the plasma levels of miR-421 in patients with gastric cancer, and precancerous
102
lesions, and in normal controls to assess the feasibility of miR-421 as a novel non-invasive
103
biomarker for early GC detection. The expression levels of conventional tumor markers in GC,
104
Pre, and normal controls were analyzed to evaluate the specificity and sensitivity of miR-421 for
105
early diagnosis of gastric cancer. This study was subject to approval by the Ethics Committee of
106
the Affiliated Liutie Central Hospital of Guangxi Medical University and written informed
107
consent was issued by all study participants. 108
Plasma preparation and storage 109
8~10ml of venous blood of each participant was collected using ethylenediaminetetraacetic acid
110
(EDTA) anticoagulative tubes immediately. Cell-free plasma was separated within 2h after
111
collection by 3000rpm,5min centrifugation to prevent contamination by cellular nucleic acids. 112
Plasma transferred into RNase/DNase-free tubes was stored at -80℃ until MicroRNA extraction. 113
Plasma samples for conventional tumor markers and Helicobacter pylori antibody determination
114
were separated and kept at -20˚C until assayed 113
Plasma samples for conventional tumor markers and Helicobacter pylori antibody determination 114
were separated and kept at -20˚C until assayed. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 124
SDS 2.0 software (Applied Biosystems, Foster City, CA, USA). Samples with Ct values greater
125
than 30 were excluded. The levels of miR-421 in different samples were calculated by the 2−∆Ct
126
method, in which ∆Ct=Ct (miR-421)–Ct (miR-16). 124
SDS 2.0 software (Applied Biosystems, Foster City, CA, USA). Samples with Ct values greater
125
than 30 were excluded. The levels of miR-421 in different samples were calculated by the 2−∆Ct
126
method, in which ∆Ct=Ct (miR-421)–Ct (miR-16). 127
Conventional tumor markers and Helicobacter pylori antibodies 115
MicroRNA extraction and qRT-PCR assays 116
MicroRNAs were isolated from 200μl plasma utilizing Blood (Serum/Plasma) MicroRNA
117
Extraction and Purification Kit (spin column) (Novland Co., Ltd, Shanghai, China) following the
118
manufacturer’s protocol. The concentrations of miRNA from plasma samples were quantified
119
using a NanoQ Micro-volume Spectrophotometer, (CapitalBio, Beijing, China). The initial
120
template for qRT-PCR was 2ul. Circulating miRNAs expression was determined using one step
121
Stemaim-it miR qRT-PCR Kit Quantitation (Taqman probes) (Novland Co., Ltd, Shanghai,
122
China), while miR-16 served as a reference miRNA. Expression levels of target miRNAs were
123
performed on the ABI-7500 PCR system and calculated by the cycle threshold (Ct) values with 116
MicroRNAs were isolated from 200μl plasma utilizing Blood (Serum/Plasma) MicroRNA
117
Extraction and Purification Kit (spin column) (Novland Co., Ltd, Shanghai, China) following the
118
manufacturer’s protocol. The concentrations of miRNA from plasma samples were quantified
119
using a NanoQ Micro-volume Spectrophotometer, (CapitalBio, Beijing, China). The initial
120
template for qRT-PCR was 2ul. Circulating miRNAs expression was determined using one step
121
Stemaim-it miR qRT-PCR Kit Quantitation (Taqman probes) (Novland Co., Ltd, Shanghai,
122
China), while miR-16 served as a reference miRNA. Expression levels of target miRNAs were
123
performed on the ABI-7500 PCR system and calculated by the cycle threshold (Ct) values with PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 127
Conventional tumor markers and Helicobacter pylori antibodies 128
Conventional tumor markers were tested by electrochemiluminescence imm 128
Conventional tumor markers were tested by electrochemiluminescence immunoassay according
129
to the standard procedure of Roche Company's kit and Roche E170 automatic immunity
130
analyzer. Helicobacter pylori antibodies were assayed following the Anti-helicobacter pylori
131
antibody detection kit (ELISA) (Beier, biological engineering co. Ltd, Beijing, China). 128
Conventional tumor markers were tested by electrochemiluminescence immunoassay according
129
to the standard procedure of Roche Company's kit and Roche E170 automatic immunity
130
analyzer. Helicobacter pylori antibodies were assayed following the Anti-helicobacter pylori
131
antibody detection kit (ELISA) (Beier, biological engineering co. Ltd, Beijing, China). 132
Statistical analysis
133
All data were analyzed using MedCalc statistical software (v18.2.1) and GraphPad Prism 7.0. 134
The levels of miR-421and conventional tumor markers in plasma among Pre, GC patients, and
135
health cases were established by the Mann Whitney test. Receiver operating characteristic curves
136
(ROC) and the AUC were constructed to evaluate the diagnostic values of each tumor markers. 137
The optimal cut-off thresholds were determined by using the highest Youden index. The Chi-
138
squared test was utilized to analyze the associations between the expression level of miR-421
139
and the clinicopathological factors. All differences were examined statistically significant at p <
140
0.05. 141
Results
142
Expression of miR-421 and tumor markers in plasma of GC patients, Pre patients,
143
and normal controls
144
This study recruited 90 GC patients, 89 Pre patients, and 45 normal healthy subjects as controls. 145
Plasma miR-421 levels were detected by qRT-PCR and analyzed using miR-16 as a reference
146
miRNA. To verify whether miR-16 was adaptable to be a reference miRNA in our system, the
147
expression levels of miR-16 were detected. As shown in Fig. 1A, there was no statistical
148
difference in the expression of miR-16 between the normal and gastric cancer group, or the
149
normal and precancerous group, which implied that miR-16 was a suitable reference control for
150
detecting miR-421 in plasma samples. The qRT-PCR results showed that miR-421 was
151
significantly up-regulated in GC and Pre patients compared with the control group (all p<0.001,
152
Figs. 1B,1C). The concentrations of CEA, CA125, CA153, CA211, and CA50 in the GC group 157
miR-421 in plasma had higher diagnostic value as a non-invasive biomarker than
158
traditional tumor markers for gastric cancer 159
To further evaluate the diagnostic value of miR-421 for GC, receiver-operating characteristic
160
(ROC) curve analyses were performed (Fig. 2A and Table 2). According to the ROC curve,
161
when the cut-off of 3.23, the optimal sensitivity and specificity of miR-421 were 96.67% and
162
95.56% respectively [AUC =0.981 (0.942 - 0.997)]. To further clarify the diagnostic efficiency
163
of miR-421 in gastric cancer, the diagnostic values of traditional tumor markers were compared
164
to those of miR-421. Fig. 2B shows that the AUC, sensitivity, accuracy and Youden index of
165
miR-421 were higher than all five conventional biomarkers, while the specificity was higher than
166
CEA, CA153, and CA50. Table 3 shows the direct comparison of the ROC curves of circulating
167
miR-421 with five conventional tumor markers. The diagnostic efficacy of miR-421 was
168
significantly higher than CEA, CA153, CA211, and CA50 (all p<0.05). In order to determine
169
whether the plasma level of miR-421 has clinical value for GC diagnosis, the false positive rate
170
(FPR) and false negative rate (FNR) and diagnosis efficiency were calculated. As shown in
171
Table 2, the results indicate that the FNR of miR-421 was lower than all five conventional
172
biomarkers, while the FPR was lower than CEA, CA153, and CA50, which indicated that miR-
173
421 has a high diagnostic value for GC. These results indicate that the plasma miR-421 has
174
greater diagnostic value for GC than five conventional tumor markers and can be used as a non-
175
invasive diagnostic marker for gastric cancer. 142
Expression of miR-421 and tumor markers in plasma of GC patients, Pre patients, 144
This study recruited 90 GC patients, 89 Pre patients, and 45 normal healthy subjects as controls. 145
Plasma miR-421 levels were detected by qRT-PCR and analyzed using miR-16 as a reference
146
miRNA. To verify whether miR-16 was adaptable to be a reference miRNA in our system, the
147
expression levels of miR-16 were detected. As shown in Fig. 1A, there was no statistical
148
difference in the expression of miR-16 between the normal and gastric cancer group, or the
149
normal and precancerous group, which implied that miR-16 was a suitable reference control for
150
detecting miR-421 in plasma samples. The qRT-PCR results showed that miR-421 was
151
significantly up-regulated in GC and Pre patients compared with the control group (all p<0.001,
152
Figs. 1B,1C). The concentrations of CEA, CA125, CA153, CA211, and CA50 in the GC group PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 153
were significantly higher than in the healthy controls (all p<0.05, Table 1). Moreover, the
154
concentration of CA211 and CA50 in the Pre group was significantly higher than the healthy
155
controls (all p<0.05, Table 1). Of note, compared with the precancerous group, the concentration
156
of AFP and CA153 was significantly higher in the normal group (all p<0.05, Table 1). 157
miR-421 in plasma had higher diagnostic value as a non-invasive biomarker than
158
traditional tumor markers for gastric cancer
159
To further evaluate the diagnostic value of miR-421 for GC, receiver-operating characteristic
160
(ROC) curve analyses were performed (Fig. 2A and Table 2). According to the ROC curve,
161
when the cut-off of 3.23, the optimal sensitivity and specificity of miR-421 were 96.67% and
162
95.56% respectively [AUC =0.981 (0.942 - 0.997)]. To further clarify the diagnostic efficiency
163
of miR-421 in gastric cancer, the diagnostic values of traditional tumor markers were compared
164
to those of miR-421. Fig. 2B shows that the AUC, sensitivity, accuracy and Youden index of
165
miR-421 were higher than all five conventional biomarkers, while the specificity was higher than
166
CEA, CA153, and CA50. Table 3 shows the direct comparison of the ROC curves of circulating
167
miR-421 with five conventional tumor markers. The diagnostic efficacy of miR-421 was
168
significantly higher than CEA, CA153, CA211, and CA50 (all p<0.05). In order to determine
169
whether the plasma level of miR-421 has clinical value for GC diagnosis, the false positive rate
170
(FPR) and false negative rate (FNR) and diagnosis efficiency were calculated. As shown in
171
Table 2, the results indicate that the FNR of miR-421 was lower than all five conventional
172
biomarkers, while the FPR was lower than CEA, CA153, and CA50, which indicated that miR-
173
421 has a high diagnostic value for GC. These results indicate that the plasma miR-421 has
174
greater diagnostic value for GC than five conventional tumor markers and can be used as a non-
175
invasive diagnostic marker for gastric cancer. 176
miR-421 in plasma could be used to distinguish early-stage GC patients from
177
healthy controls 153
were significantly higher than in the healthy controls (all p<0.05, Table 1). Moreover, the
154
concentration of CA211 and CA50 in the Pre group was significantly higher than the healthy
155
controls (all p<0.05, Table 1). Manuscript to be reviewed Of note, compared with the precancerous group, the concentration
156
of AFP and CA153 was significantly higher in the normal group (all p<0.05, Table 1). 176
miR-421 in plasma could be used to distinguish early-stage GC 178
To evaluate the potential of the differentially expressed miR-421 to diagnose early-stage gastric
179
cancer patients, we further analyzed the expression levels of miR-421 in GC patients at an early
180
stage (TNM stages I-II). The results showed that the expression levels of circulating miR-421 in
181
healthy controls were significantly lower than those in early-stage gastric cancer patients PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 182
(p<0.0001, Fig.3A). The AUC values of miR-421 were 0.9907 (95% confidence interval (CI) =
183
0.931 to 1.0, sensitivity = 100% and specificity = 95.56%, Fig. 3B). These results indicate that
184
miR-421 could be invoked as a novel biomarker for early diagnosis of gastric cancer. 185
186
miR-421 had higher diagnostic value than traditional tumor markers for
187
precancerous gastric lesions
188
Based on the verified difference of miR-421 between the Pre and NC group (Fig. 1B,1C), ROC
189
curves were constructed to further assess the role of miR-421 expression in the diagnosis of
190
precancerous lesions. According to the different concentration of traditional tumor markers
191
between the Pre group and the NC group (Figs. 4A,4B), their diagnostic values with miR-
192
421were further compared. ROC analyses showed that up-regulation of plasma miR-421 could
193
discriminate Pre patients well from control subjects, with an AUC value of 0.873 (sensitivity =
194
66.29%, specificity = 95.56%, cutoff value: 3.23) (Fig.4C). Furthermore, as shown in table 4 and
195
Figs. 4C & 4D, the AUC of miR-421 was higher than CA153, CA211, and CA50, the sensitivity
196
was higher than CA50, while the specificity and Youden index were higher than CA153, and
197
CA50. Moreover, the FNR of miR-421 was lower than CA50, while the FPR was lower than
198
CA153, and CA50. According to the comparison of the ROC curves, the diagnostic efficacy of
199
miR-421 was considerably higher than CA153, CA211 and CA50 (all p<0.05, Table 5). These
200
results indicate that miR-421 has high diagnostic value for precancerous patients. 201
Up-regulation of miR-421 occurred initially in the precancerous diseases as well
202
as the early GC
203
We found that up-regulation of miR-421 not only distinguishes early-stage gastric cancer well,
204
but can also well distinguish precancerous lesions from normal controls. However, it is not clear
205
at what stage miR-421 begins to be up-regulated throughout the development of GC. Therefore,
206
expressions of miR-421 in precancerous disease, and different clinical stages (I, II, III and IV) 187
precancerous gastric lesions
188
Based on the verified difference of miR-421 between the Pre and NC group (Fig. 1B,1C), ROC
189
curves were constructed to further assess the role of miR-421 expression in the diagnosis of
190
precancerous lesions. According to the different concentration of traditional tumor markers
191
between the Pre group and the NC group (Figs. Manuscript to be reviewed 4A,4B), their diagnostic values with miR-
192
421were further compared. ROC analyses showed that up-regulation of plasma miR-421 could
193
discriminate Pre patients well from control subjects, with an AUC value of 0.873 (sensitivity =
194
66.29%, specificity = 95.56%, cutoff value: 3.23) (Fig.4C). Furthermore, as shown in table 4 and
195
Figs. 4C & 4D, the AUC of miR-421 was higher than CA153, CA211, and CA50, the sensitivity
196
was higher than CA50, while the specificity and Youden index were higher than CA153, and
197
CA50. Moreover, the FNR of miR-421 was lower than CA50, while the FPR was lower than
198
CA153, and CA50. According to the comparison of the ROC curves, the diagnostic efficacy of
199
miR-421 was considerably higher than CA153, CA211 and CA50 (all p<0.05, Table 5). These
200
results indicate that miR-421 has high diagnostic value for precancerous patients. Manuscript to be reviewed 211
gradually up-regulated from the NC to the Pre and stage I cases, and finally, the maximum
212
expression value appeared in stage I cases (Table 6). The marked up-regulation of miR-421
213
occurred initially in precancerous disease as well as early-stage GC. 211
gradually up-regulated from the NC to the Pre and stage I cases, and finally, the maximum
212
expression value appeared in stage I cases (Table 6). The marked up-regulation of miR-421
213
occurred initially in precancerous disease as well as early-stage GC. 214
Association of miR-421 with clinicopathological features 214
Association of miR-421 with clinicopathological features 215
In order to determine whether miR-421 levels in plasma were correlated with clinicopathological
216
features in patients with GC and precancerous lesions, the clinical data of 89 Pre and 90 GC-
217
proven cases were collected and analyzed. The high and low expression groups of each
218
parameter were calculated by the median of the relative quantity of miR-421. Correlation
219
analysis showed no statistically significant association of miR-421 expression with GC patients’
220
age (p = 0.0736), gender (p = 0.3265), TNM stage (p= 0.3531), differentiation degree (p=
221
0.5105), and histological type (p = 0.5079). Similarly, we found that there were no apparent
222
correlations of miR-421 expression with Pre patients’ age (p = 0.5896), gender (p = 0.5934),
223
histological type (p = 0.6939), and Helicobacter pylori infection status (p = 0.3095) (Table 7). 224
Discussion 201
Up-regulation of miR-421 occurred initially in the precancerous diseases as well
202
as the early GC 201
Up-regulation of miR-421 occurred initially in the precancerous diseases as well
202
as the early GC 203
We found that up-regulation of miR-421 not only distinguishes early-stage gastric cancer well,
204
but can also well distinguish precancerous lesions from normal controls. However, it is not clear
205
at what stage miR-421 begins to be up-regulated throughout the development of GC. Therefore,
206
expressions of miR-421 in precancerous disease, and different clinical stages (I, II, III and IV)
207
were further analyzed and compared. The results showed that the expressions of miR-421 were
208
statistically different between the Pre cases and GC stages I, II, III and IV (p<0.05, Fig.5). 209
However, there was no difference of miR-421 expression between the GC stages II, III and IV
210
and GC cases stage I. Moreover, compared to the expression in Pre cases, the biomarker was PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 240
diagnostic value in cancer. However, these researches are based on invasive, patients may not be
241
willing to accept, is bound to affect its promotion. Moreover, our study showed that miR-421
242
could achieve a satisfactory diagnostic efficiency in distinguishing GC patients from healthy
243
controls with an AUC of 0.981 (sensitivity = 96.67% and specificity = 95.56%), higher than
244
miR-21, miR-20a and miR-378 tumor markers in previous studies (Liu et al. 2012; Mirzaei et al. 245
2016). In our study, we compared its diagnostic efficiency with that of traditional markers,
246
further clarifying its diagnostic value as a new tumor marker. 247
248
It is particularly important to improve the early diagnosis and prompt treatment of patients with
249
gastric cancer. Researchers have illuminated the potential roles of several miRNAs in the early
250
detection of cancers, such as lung carcinoma(Pan et al. 2018), colorectal neoplasm(Liu et al. 251
2018), and early-stage breast carcinoma(An et al. 2018). However, to date only a few miRNAs
252
have been found effective as non-invasive biomarkers for the early detection of GC. Although
253
the study such as the five-miRNA panel presented a high diagnostic value for the early stage of
254
GC(Liu et al. 2012), the study did not include gastric precancerous lesions, and the subtle
255
changes and performances of the miRNAs were not further elaborated. The miR-17-92 cluster
256
was considered as a circulating biomarker for early detection of GC, but there is no ROC data to
257
support miRNA recognition of early stage cancer(Li et al. 2017). In our study, based on the
258
detailed TNM stage characteristics of the GC sample, we explored the diagnostic value of
259
plasma miR-421 in early gastric cancer. The results showed that the expression level of miR-421
260
could well distinguish early gastric cancer from normal controls, suggesting that miR-421 may
261
have significant clinical value in the early diagnosis of gastric cancer. 262
Precancerous changes in the stomach can increase the risk of gastric cancer six-fold. Previous
263
studies have reported that precancerous lesions are regulated by miRNAs(Li et al. 2014; Rotkrua
264
et al. 2011). 265
Due to the absence of clinical manifestations of gastrointestinal metaplasia and atypical
266
hyperplasia, there are few means for clinical non-invasive detection. Several circulating 240
diagnostic value in cancer. 224
Discussion 225
To date, there is no reliable non-invasive blood-based biomarker for the detection of
226
precancerous lesions and early-stage gastric cancer. We studied the diagnostic value of miR-421
227
in plasma in precancerous gastric lesions and GC patients. We found that the plasma expression
228
levels of miR-421 were statistically significantly higher in early-stage GC and precancerous
229
gastric lesions patients than in healthy controls. ROC analyses demonstrated that miR-421 has
230
high sensitivity and specificity in discriminating early-stage GC and precancerous gastric lesions
231
patients from healthy controls. Our results indicated that miR-421 in plasma presented
232
statistically significant diagnostic efficacy in detecting early GC as well as precancerous gastric
233
lesions patients. 234
235
High-throughput analysis showed that miR-421 is localized on human chromosome1p34.23, and
236
is one prominent member of the miR-200 family(Liu et al. 2015). Liu et al reported that miR-421
237
elevation in GC tissue(Liu et al. 2015; Zhao et al. 2015)and gastric juice(Zhang et al. 238
2012b)probably had implications in GC diagnosis. These results are consistent with our findings
239
that miR-421 was highly expressed in the plasma of gastric cancer might have the potential
PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) 235
High-throughput analysis showed that miR-421 is localized on human chromosome1p34.23, and
236
is one prominent member of the miR-200 family(Liu et al. 2015). Liu et al reported that miR-421
237
elevation in GC tissue(Liu et al. 2015; Zhao et al. 2015)and gastric juice(Zhang et al. 238
2012b)probably had implications in GC diagnosis. These results are consistent with our findings
239
that miR-421 was highly expressed in the plasma of gastric cancer might have the potential 238
2012b)probably had implications in GC diagnosis. These results are consistent with our findings
239
that miR-421 was highly expressed in the plasma of gastric cancer might have the potential PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed However, these researches are based on invasive, patients may not be
241
willing to accept, is bound to affect its promotion. Moreover, our study showed that miR-421
242
could achieve a satisfactory diagnostic efficiency in distinguishing GC patients from healthy
243
controls with an AUC of 0.981 (sensitivity = 96.67% and specificity = 95.56%), higher than
244
miR-21, miR-20a and miR-378 tumor markers in previous studies (Liu et al. 2012; Mirzaei et al. 245
2016). In our study, we compared its diagnostic efficiency with that of traditional markers,
246
further clarifying its diagnostic value as a new tumor marker. 248
It is particularly important to improve the early diagnosis and prompt treatment of patients with
249
gastric cancer. Researchers have illuminated the potential roles of several miRNAs in the early
250
detection of cancers, such as lung carcinoma(Pan et al. 2018), colorectal neoplasm(Liu et al. 251
2018), and early-stage breast carcinoma(An et al. 2018). However, to date only a few miRNAs
252
have been found effective as non-invasive biomarkers for the early detection of GC. Although
253
the study such as the five-miRNA panel presented a high diagnostic value for the early stage of
254
GC(Liu et al. 2012), the study did not include gastric precancerous lesions, and the subtle
255
changes and performances of the miRNAs were not further elaborated. The miR-17-92 cluster
256
was considered as a circulating biomarker for early detection of GC, but there is no ROC data to
257
support miRNA recognition of early stage cancer(Li et al. 2017). In our study, based on the
258
detailed TNM stage characteristics of the GC sample, we explored the diagnostic value of
259
plasma miR-421 in early gastric cancer. The results showed that the expression level of miR-421
260
could well distinguish early gastric cancer from normal controls, suggesting that miR-421 may
261
have significant clinical value in the early diagnosis of gastric cancer. 262
Precancerous changes in the stomach can increase the risk of gastric cancer six-fold. Previous
263
studies have reported that precancerous lesions are regulated by miRNAs(Li et al. 2014; Rotkrua
264
et al. 2011). 262
Precancerous changes in the stomach can increase the risk of gastric cancer six-fold. Previous
263
studies have reported that precancerous lesions are regulated by miRNAs(Li et al. 2014; Rotkrua
264
et al. 2011). Manuscript to be reviewed This study provides us with a new insight into the role of miR-
291
421 in gastric cancer These results could pave the way for the development of a novel biomarker 278
The occurrence and development of gastric cancer is a multi-stage process, and miR-421 may be
279
associated with a certain stage or the whole process. In this study, we further analyzed the trend
280
of the up-regulation of miR-421 with the progression of GC. Results showed that the expression
281
of plasma miR-421in GC cases at stage I is no different from stages II, II, and IV, but plasma
282
miR-421in precancerous cases can be distinctly distinguished from GC at each stage, indicating
283
that miR-421 occurred initially in precancerous lesions as well as early GC. Therefore, we
284
speculate that the up-regulation in the miR-421 may be triggered by risk factors, especially
285
chronic inflammation in the premalignant stomach, suggesting that a detailed investigation is
286
needed. Manuscript to be reviewed 269
and of GC progression. However, the comparative value of these miRNAs and traditional tumor
270
markers in the diagnosis of gastric cancer and precancerous lesions has not been clarified, and an
271
optimal circulating miRNA biomarker should have better specificity and sensitivity than
272
traditional tumor markers. In the current study, we found that plasma miR-421 could well
273
distinguish precancerous lesions of gastric cancer patients from healthy controls with an AUC of
274
0.872 (sensitivity = 66.29% and specificity = 95.56%). Furthermore, the diagnostic efficacy of
275
miR-421 was markedly higher than traditional tumor markers, such as CA153, CA211, and
276
CA50. 277 269
and of GC progression. However, the comparative value of these miRNAs and traditional tumor
270
markers in the diagnosis of gastric cancer and precancerous lesions has not been clarified, and an
271
optimal circulating miRNA biomarker should have better specificity and sensitivity than
272
traditional tumor markers. In the current study, we found that plasma miR-421 could well
273
distinguish precancerous lesions of gastric cancer patients from healthy controls with an AUC of
274
0.872 (sensitivity = 66.29% and specificity = 95.56%). Furthermore, the diagnostic efficacy of
275
miR-421 was markedly higher than traditional tumor markers, such as CA153, CA211, and
276
CA50. 277
278
The occurrence and development of gastric cancer is a multi-stage process, and miR-421 may be
279
associated with a certain stage or the whole process. In this study, we further analyzed the trend
280
of the up-regulation of miR-421 with the progression of GC. Results showed that the expression
281
of plasma miR-421in GC cases at stage I is no different from stages II, II, and IV, but plasma
282
miR-421in precancerous cases can be distinctly distinguished from GC at each stage, indicating
283
that miR-421 occurred initially in precancerous lesions as well as early GC. Therefore, we
284
speculate that the up-regulation in the miR-421 may be triggered by risk factors, especially
285
chronic inflammation in the premalignant stomach, suggesting that a detailed investigation is
286
needed. 287
288
Conclusions
289
Our findings demonstrate that plasma miR-421 may be a potential marker for precancerous
290
lesions and early gastric cancer. Manuscript to be reviewed 265
Due to the absence of clinical manifestations of gastrointestinal metaplasia and atypical
266
hyperplasia, there are few means for clinical non-invasive detection. Several circulating
267
miRNAs, especially microRNA-196a (Chen et al. 2018), miR-16-5p, and miR-19b-3p(Zhang et
268
al. 2015), have been screened out for early markers of gastric cancer and precancerous lesions 265
Due to the absence of clinical manifestations of gastrointestinal metaplasia and atypical
266
hyperplasia, there are few means for clinical non-invasive detection. Several circulating
267
miRNAs, especially microRNA-196a (Chen et al. 2018), miR-16-5p, and miR-19b-3p(Zhang et
268
al. 2015), have been screened out for early markers of gastric cancer and precancerous lesions PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) 298
REFERENCES 299
An X, Quan H, Lv J, Meng L, Wang C, Yu Z, and Han J. 2018. Serum microRNA as potential biomarker to detect
300
breast atypical hyperplasia and early-stage breast cancer. Future Oncol. 10.2217/fon-2018-0334 301
Chan AO, Wong BC, and Lam SK. 2001. Gastric cancer: past, present and future. Can J Gastroenterol 15:469-474. 302
Chen TH, Lee C, Chiu CT, Chu YY, Cheng HT, Hsu JT, Tsou YK, Wu RC, Chen TC, Chang NC, Yeh TS, and Lin
303
KH. 2018. Circulating microRNA-196a is an early gastric cancer biomarker. Oncotarget 9:10317-10323. 304
Craanen ME, Dekker W, Ferwerda J, Blok P, and Tytgat GN. 1991. Early gastric cancer: a clinicopathologic study. J
305
Clin Gastroenterol 13:274-283. 306
He CZ, Zhang KH, Li Q, Liu XH, Hong Y, and Lv NH. 2013. Combined use of AFP, CEA, CA125 and CAl9-9
307
improves the sensitivity for the diagnosis of gastric cancer. BMC Gastroenterol 13:87. 308
Hernando E. 2007. microRNAs and cancer: role in tumorigenesis, patient classification and therapy. Clin Transl Oncol
309
9:155-160. 310
Lee RC, and Ambros V. 2001. An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862-864. 311
Li BS, Zhao YL, Guo G, Li W, Zhu ED, Luo X, Mao XH, Zou QM, Yu PW, Zuo QF, Li N, Tang B, Liu KY, and
312
Xiao B. 2012. Plasma microRNAs, miR-223, miR-21 and miR-218, as novel potential biomarkers for gastric
313
cancer detection. PLoS One 7:e41629. 10.1371/journal.pone.0041629 314
Li H, Wu Q, Li T, Liu C, Xue L, Ding J, Shi Y, and Fan D. 2017. The miR-17-92 cluster as a potential biomarker for
315
the early diagnosis of gastric cancer: evidence and literature review. Oncotarget 8:45060-45071. 316
10.18632/oncotarget.15023 317
Li T, Lu YY, Zhao XD, Guo HQ, Liu CH, Li H, Zhou L, Han YN, Wu KC, Nie YZ, Shi YQ, and Fan DM. 2014. 318
MicroRNA-296-5p increases proliferation in gastric cancer through repression of Caudal-related homeobox
319
1. Oncogene 33:783-793. 10.1038/onc.2012.637 320
Li Y, Cui X, Li Y, Zhang T, and Li S. 2018. Upregulated expression of miR-421 is associated with poor prognosis in
321
non-small-cell lung cancer. Cancer Manag Res 10:2627-2633. 320
Li Y, Cui X, Li Y, Zhang T, and Li S. 2018. 288
Conclusions 289
Our findings demonstrate that plasma miR-421 may be a potential marker for precancerous
290
lesions and early gastric cancer. This study provides us with a new insight into the role of miR-
291
421 in gastric cancer. These results could pave the way for the development of a novel biomarker
292
for the early diagnosis of gastric cancer. Used this biomarker may allow a reliable indicator of
293
gastric cancer at a much lower cost than methods currently available. The limitations of this
294
study are that our normal control samples were relatively small, and most cancer samples were at
295
an advanced stage. The exact use of miR-421 as an early plasma biomarker in clinical diagnosis
296
of precancerous lesions and gastric cancer should be further explored in studies that use larger
297
samples and in clinical trials. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 338
Pectasides D, Mylonakis A, Kostopoulou M, Papadopoulou M, Triantafillis D, Varthalitis J, Dimitriades M, and
339
Athanassiou A. 1997. CEA, CA 19-9, and CA-50 in monitoring gastric carcinoma. Am J Clin Oncol 20:348-
340
353. 38
Pectasides D, Mylonakis A, Kostopoulou M, Papadopoulou M, Triantafillis D, Varthalitis ,
y
,
p
,
p
p
,
,
,
,
339
Athanassiou A. 1997. CEA, CA 19-9, and CA-50 in monitoring gastric carcinoma. Am J Clin Oncol 20:348-
340
353. 341
Rotkrua P, Akiyama Y, Hashimoto Y, Otsubo T, and Yuasa Y. 2011. MiR-9 downregulates CDX2 expression in
342
gastric cancer cells. Int J Cancer 129:2611-2620. 10.1002/ijc.25923 ,
y
,
,
,
g
p
342
gastric cancer cells. Int J Cancer 129:2611-2620. 10.1002/ijc.25923 43
Tsujiura M, Komatsu S, Ichikawa D, Shiozaki A, Konishi H, Takeshita H, Moriumura R, N 344
Hirajima S, Arita T, Fujiwara H, Okamoto K, and Otsuji E. 2015. Circulating miR-18a in plasma contributes 344
Hirajima S, Arita T, Fujiwara H, Okamoto K, and Otsuji E. 2015. Circulating miR-18a in plasma contributes
345
to cancer detection and monitoring in patients with gastric cancer. Gastric Cancer 18:271-279. 45
to cancer detection and monitoring in patients with gastric cancer. Gastric Cancer 18 346
Wang Y, Liu Z, and Shen J. 2018. MicroRNA-421-targeted PDCD4 regulates breast cancer cell proliferation. Int J
347
Mol Med. 348
Yang AP, Liu J, Lei HY, Zhang QW, Zhao L, and Yang GH. 2014. CA72-4 combined with CEA, CA125 and CAl9-
349
9 improves the sensitivity for the early diagnosis of gastric cancer. Clin Chim Acta 437:183-186. 350
10.1016/j.cca.2014.07.034 g
,
,
,
g Q
,
,
g
,
349
9 improves the sensitivity for the early diagnosis of gastric cancer. Clin Chim Acta 437:183-186. 350
10.1016/j.cca.2014.07.034 351
Yang R, Fu Y, Zeng Y, Xiang M, Yin Y, Li L, Xu H, Zhong J, and Zeng X. 2017. Serum miR-20a is a promising
352
biomarker for gastric cancer. Biomed Rep 6:429-434. 353
Zhang J, Song Y, Zhang C, Zhi X, Fu H, Ma Y, Chen Y, Pan F, Wang K, Ni J, Jin W, He X, Su H, and Cui D. 2015. 354
Circulating MiR-16-5p and MiR-19b-3p as Two Novel Potential Biomarkers to Indicate Progression of
355
Gastric Cancer. Theranostics 5:733-745. 355
Gastric Cancer. Theranostics 5:733-745. 298
REFERENCES Upregulated expression of miR-421 is associated with poor prognosis in
321
ll
ll l
C
M
R
10 2627 2633 322
Liu H, Gao Y, Song D, Liu T, and Feng Y. 2015. Correlation between microRNA-421 expression level and prognosis
323
of gastric cancer. Int J Clin Exp Pathol 8:15128-15132. 324
Liu H, Zhu L, Liu B, Yang L, Meng X, Zhang W, Ma Y, and Xiao H. 2012. Genome-wide microRNA profiles identify
325
miR-378 as a serum biomarker for early detection of gastric cancer. Cancer Lett 316:196-203. 326
Liu X, Xu T, Hu X, Chen X, Zeng K, Sun L, and Wang S. 2018. Elevated circulating miR-182 acts as a diagnostic
327
biomarker for early colorectal cancer. Cancer Manag Res 10:857-865. 10.2147/cmar.s158016 28
Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, 328
Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA,
329
Downing JR, Jacks T, Horvitz HR, and Golub TR. 2005. MicroRNA expression profiles classify human
330
cancers. Nature 435:834-838. 10.1038/nature03702 331
Mirzaei H, Khataminfar S, Mohammadparast S, Sales SS, Maftouh M, Mohammadi M, Simonian M, Parizadeh SM,
332
Hassanian SM, and Avan A. 2016. Circulating microRNAs as Potential Diagnostic Biomarkers and
333
Therapeutic Targets in Gastric Cancer: Current Status and Future Perspectives. Curr Med Chem 23:4135-
334
4150. 335
Pan J, Zhou C, Zhao X, He J, Tian H, Shen W, Han Y, Chen J, Fang S, Meng X, Jin X, and Gong Z. 2018. A two-
336
miRNA signature (miR-33a-5p and miR-128-3p) in whole blood as potential biomarker for early diagnosis
337
of lung cancer. Sci Rep 8:16699. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 356
Zhang X, Cui L, Ye G, Zheng T, Song H, Xia T, Yu X, Xiao B, Le Y, and Guo J. 2012a. Gastric juice microRNA-
357
421 is a new biomarker for screening gastric cancer. Tumour Biol 33:2349-2355. 10.1007/s13277-012-0497-
358
x 359
Zhang Y, Gong W, Dai S, Huang G, Shen X, Gao M, Xu Z, Zeng Y, and He F. 2012b. Downregulation of human
360
farnesoid X receptor by miR-421 promotes proliferation and migration of hepatocellular carcinoma cells. 361
Mol Cancer Res 10:516-522. 10.1158/1541-7786.mcr-11-0473 362
Zhao G, Xu L, Hui L, and Zhao J. 2015. Level of circulated microRNA-421 in gastric carcinoma and related
363
mechanisms. Int J Clin Exp Pathol 8:14252-14256. 364
Zheng RS, Sun KX, Zhang SW, Zeng HM, Zou XN, Chen R, Gu XY, Wei WW, and He J. 2019. Report of cancer
365
epidemiology in China, 2015. Zhonghua Zhong Liu Za Zhi 41:19-28. 366
Zhou S, Wang B, Hu J, Zhou Y, Jiang M, Wu M, Qin L, and Yang X. 2016. miR-421 is a diagnostic and prognostic
367
marker in patients with osteosarcoma. Tumour Biol. 368
Zhou X, Ji G, Chen H, Jin W, Yin C, and Zhang G. 2015. Clinical role of circulating miR-223 as a novel biomarker
369
in early diagnosis of cancer patients. Int J Clin Exp Med 8:16890-16898. 370 Manuscript to be reviewed PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Figure 1(on next page) MiR-421 expression level between different groups MiR-421 expression level between different groups (A) Differential expression levels of
reference miR-16(
#p >0.05). (B) The ΔCt values of miR-421 between different groups (****p
< 0.0001). (C) The levels (2
-ΔCt) of miR-421 between different groups (****p < 0.0001). The
ΔCt values denote the normalized Ct value by subtracting the Ct value of miR-16 from that of
miR-421. The lower ΔCt value means the higher level of miR-421 expression. The Mann-
Whitney U test was used to determine statistical significance at the level of p <0.05. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) GC
NC
Pre
0
10
20
30
#
#
Normalized Ct value
GC
NC
Pre
-16
-8
0
8
****
****
****
GC
NC
Pre
0.01
0.1
1
10
100
1000
10000
100000
The levels (2-ΔCt) of miR-421
****
****
****
A
B
C
PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019)
Manuscript to be reviewed GC
NC
Pre
#
#
Normalized Ct value
GC
NC
Pre
-16
-8
0
8
****
****
****
GC
NC
Pre
0.01
0.1
1
10
100
1000
10000
100000
The levels (2-ΔCt) of miR-421
****
****
****
B
C
PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019)
Manuscript to be reviewed GC
NC
Pre
0
10
20
30
The raw Ct value of miR-16
#
#
Normalized Ct value
GC
NC
Pre
-16
-8
0
8
****
****
****
GC
NC
0.01
0.1
1
10
100
1000
10000
100000
The levels (2-ΔCt) of miR-421
****
****
A
B
C
PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019)
Manuscript to be reviewed Normalized Ct value
GC
NC
Pre
-16
-8
0
8
****
****
****
GC
NC
Pre
0.01
0.1
1
10
100
1000
10000
100000
The levels (2-ΔCt) of miR-421
****
****
****
B
C
Manuscript to be reviewed Normalized Ct value
GC
NC
Pre
-16
-8
0
8
****
****
****
B
Manuscript to be reviewed GC
NC
Pre
0.01
0.1
1
10
100
1000
10000
100000
The levels (2-ΔCt) of miR-421
****
****
****
C GC
NC
Pre
0
10
20
30
The raw Ct value of miR-16
#
#
A 8B Normalized Ct value Figure 2 The ROC curves of miR-421 and traditional tumor markers for GC (A) The AUC of miR-421was 0.981 (p <0.05). (B)The AUC of CEA, CA50, CA125, CA153 and
CA211 were 0.672, 0.754, 0.645, 0.656 and 0.799, respectively (all p <0.05). Manuscript to be reviewed Figure 3 MiR-421 could distinguish early stage gastric cancer patients from healthy controls
(A) MiR-421 was up-regulated in early stages (TNM stage Ⅰ-Ⅱ) gastric cancer patients
compared to healthy controls (****p < 0.0001). (B) The ROC analysis for detection of early-
stages gastric cancer patients from health controls using miR-421. *Note: Auto Gamma Correction was used for the image. This only affects the reviewing manuscript. See original source image if needed for review. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Note: Auto Gamma Correction was used for the image. This only affects the reviewing manuscript. See original source *Note: Auto Gamma Correction was used for the image. This only affects the reviewing manuscript. See original source image if needed for review. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Figure 4 miR-421 had higher diagnostic value than traditional tumor markers for precancerous
patients. miR-421 had higher diagnostic value than traditional tumor markers for precancerous
patients. Differential expression levels of CA50(A) and CA211(B) between precancerous patients and
healthy controls (*p < 0.01, **** p < 0.0001). The ROC analysis for detection of precancerous
using miR-421(C) and traditional tumor markers CA50 and CA211(D). *Note: Auto Gamma Correction was used for the image. This only affects the reviewing manuscript. See original source image if needed for review. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Table 1(on next page) Table 1(on next page) Figure 5(on next page) Figure 5(on next page) MiR-421 expression levels in plasma of GC cases with different TNM stages, PLGC cases
and NC controls. Mann-Whitney U test was used to determine statistical significance, p < 0.05 was accepted
as statistically significant. * p < 0.05, * *** p < 0.0001, # p >0.05. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) NC
Pre
I
II
III,IV
0.01
0.1
1
10
100
1000
10000
100000
The levels (2-ΔCt) of miR-421
GC
****
*
****
****
#
#
PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019)
Manuscript to be reviewed 0.01
0.1
1
10
100
1000
10000
100000
The levels (2-ΔCt) of miR-421 GC Mann-Whitney U test was used to determine other categorical variables with statistical significance at the level of p <0.05. ann-Whitney U test was used to determine other categorical variables with statistical significan t For these categorical variables, the p value were calculated by Student’s t-test. used to determine other categorical variables with statistical significance at the level of p <0.05 Manuscript to be reviewed Manuscript to be reviewed Table 1(on next page) Differential expression levels of traditional tumor markers in plasma of GC, Pre and
health controls t For these categorical variables, the p value were calculated by Student’s t-test. Mann-
Whitney U test was used to determine other categorical variables with statistical significance
at the level of p <0.05. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Table 1. Differential expression levels of traditional tumor markers in plasma of GC, Pre and health con
GC
Pre
Pre vs NC
NC
GC vs NC
n
mean ± SD
n
mean ± SD
p value
n
mean ± SD
p value
AFP
85
(26.14±133.56)
66
(3.32±1.72)
0.0505
45
(3.69±1.25)
0.3002
CEA
85
(32.23±128.76)
66
(1.82±1.39)
0.3122t
45
(2.08±1.20)
0.0011
Ferritin
80
(281.08±383.42)
61
(270.11±302.39)
0.1270t
45
(185.67±237.46)
0.9908
CA125
68
(59.22±92.72)
53
(12.50±7.78)
0.9363
45
(11.07±4.05)
0.0091
CA153
67
(13.44±17.70)
50
(10.41±5.45)
0.0065t
45
(13.67±5.83)
0.0049
CA199
83
(107.03±251.90)
57
(13.95±12.37)
0.1071
45
(14.58±8.22)
0.0705
CA211
62
(16.15±44.84)
40
(2.96±1.40)
<0.0001
45
(2.09±0.48)
0.0002
CA242
66
(19.17±39.24)
45
(4.92±4.8)
0.2976t
36
(6.14±5.48)
0.1425
CA50
77
(38.01±99.02)
48
(8.18±5.97)
0.0101t
34
(4.91±4.46)
0.0089
CA724
75
(15.12±38.23)
44
(2.49±2.87)
0.2347t
26
(3.32±2.59)
0.2049
t For these categorical variables, the p value were calculated by Student’s t-test. Mann-Whitney U test was used to determine other categorical variables with statistical significance at the level of p <0.05. vels of traditional tumor markers in plasma of GC, Pre and health controls al expression levels of traditional tumor markers in plasma of GC, Pre and he Table 1. Differential expression levels of traditional tumor markers in plasma of GC, Pre and health controls Table 1. Differential expression levels of traditional tumor markers in plasma PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) mber of positive cases and negative cases. Manuscript to be reviewed Table 2(on next page) Table 2(on next page) The diagnostic value of miR-421 and tumor markers for GC AUC: Area under curve; FPR: False positive rate; FNR: False negative rate. Positive was
defined as >cut-off value; Negative was defined as < cut-off value; The sensitivity,
specificity, FPR, FNR, accuracy and Youden index were calculated based on the number of
positive cases and negative cases. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Table 2. The diagnostic value of miR-421 and tumor markers for GC. Cut-off
Sensitivity
(%)
Specificity
(%)
FPR
(%)
FNR
(%)
Accuracy
(%)
Youden
index (%)
AUC
p
miR-421
3.23
96.67
95.56
4.44
3.33
96.30
92.22
0.981(0.942 - 0.997)
<0.0001
CEA
1.94
67.06
66.67
33.33
32.94
66.93
33.73
0.672(0.584 - 0.752)
0.0002
CA125
18.75
45.59
100.00
0
54.41
67.26
45.59
0.645(0.549 - 0.732)
0.0055
CA153
11.31
68.66
62.22
37.78
31.34
66.07
30.88
0.656(0.561 - 0.743)
0.0034
CA211
2.2
80.65
95.65
4.35
19.35
86.96
76.30
0.799(0.711 - 0.871)
<0.0001
CA50
7.96
55.84
85.29
14.71
44.16
64.86
41.14
0.754(0.663 - 0.831)
<0.0001
AUC: Area under curve; FPR: False positive rate; FNR: False negative rate. Positive was defined as >cut-off value; Negative
was defined as < cut-off value; The sensitivity, specificity, FPR, FNR, accuracy and Youden index were calculated based on the
number of positive cases and negative cases. Table 2. The diagnostic value of miR-421 and tumor markers for GC. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Table 3(on next page) Table 3(on next page) The comparison of ROC curves between miR-421 and tumor markers
ROC curves comparisons were analyzed by using MedCalc statistical software with statistical
significance at the level Of p <0.05. ROC curves comparisons were analyzed by using MedCalc statistical software with statistical
significance at the level Of p <0.05. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1
Table 3. The comparison of ROC curves between miR-421 and tumor markers
2
ROC curves comparisons were analyzed by using MedCalc statistical software with statistical significance at
3
the level of p <0.05. CEA
CA125
CA153
CA211
CA50
ROC curves
comparison
Z
p
Z
p
Z
p
Z
p
Z
p
miR-421
2.418
0.0156
1.896
0.0580
2.021
0.0432
3.975
0.0001
3.343
0.0008 1
Table 3. The comparison of ROC curves between miR-421 and tumor markers 2
ROC curves comparisons were analyzed by using MedCalc statistical software with statistical significance at
3
the level of p <0.05. 3
the level of p <0.05. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Table 4(on next page) Table 4(on next page) The diagnostic value of miR-421 and tumor markers for Pre. The sensitivity, specificity, FPR, FNR, accuracy and Youden index were calculated based on
the number of positive cases and negative cases. AUC, FPR and FNR, Positive and Negative
as stated in Table 2. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Table 4. The diagnostic value of miR-421 and tumor markers for Pre. Cut-off
Sensitivity
(%)
Specificity
(%)
FPR
(%)
FNR
(%)
Accuracy
(%)
Youden
Index (%)
AUC
p
miR-421
3.23
66.29
95.56
4.44
33.71
76.37
61.85
0.872(0.804 - 0.924)
<0.0001
CA153
11.81
72.00
60.00
40.00
28.00
66.32
32.00
0.679(0.576 - 0.772)
0.0012
CA211
2.2
85.00
95.56
4.44
15.00
90.56
80.56
0.844(0.750 - 0.914)
<0.0001
CA50
7.26
52.08
82.35
17.65
47.92
64.63
34.44
0.677(0.564 - 0.776)
0.0034
The sensitivity, specificity, FPR, FNR, accuracy and Youden index were calculated based on the number of positive cases
and negative cases. AUC, FPR and FNR, Positive and Negative as stated in Table 2. Table 4. The diagnostic value of miR-421 and tumor markers for Pre. and negative cases. AUC, FPR and FNR, Positive and Negative as stated in Table 2. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Table 5(on next page) Table 5(on next page) The comparison of ROC curves among miR-421 and tumor markers for Pre
ROC curves comparisons were analyzed by using MedCalc statistical software with statistical
significance at the level of p <0.05. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Table 6(on next page) Manuscript to be reviewed 1
Table 5. The comparison of ROC curves among miR-421 and tumor markers for Pre
2
3
4
5
ROC curves comparisons were analyzed by using MedCalc statistical software with
6
statistical significance at the level of p <0.05. 7
8
CA153
CA211
CA50
ROC
curves
comparison
Z
p
Z
p
Z
p
miR-421
2.211
0.027
4.414
< 0.0001
1.997
0.0458 Table 5. The comparison of ROC curves among miR-421 and tumor markers for Pre PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Table 6(on next page) The levels (2
-ΔCt)of miR-421 between different groups ap represents the comparison of Pre between GC with Ⅰ,Ⅱ,ⅢandⅥ, respectively. bp represents
the comparison of Ⅰ between GC withⅡ,ⅢandⅥ, respectively PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed 1
Table 6 The levels (2-ΔCt)of miR-421 between different groups
2
3
4
5
6
7
8
9
10
aP represents the comparison of Pre between GC with Ⅰ,Ⅱ,ⅢandⅥ, respectively. 11
bP represents the comparison of Ⅰ between GC withⅡ,ⅢandⅥ, respectively
n
miR-421 in plasma
Sd
aP value
bP value
NC
45
0.45(0.16,37.0)
5.50
Pre
89
7.57(0.06,1674.25)
229.36
Ⅰ
15
59.97(8.54,702.0)
187.37
<0.0001
Ⅱ
9
53.60(3.3,1577.80)
510.76
0.0277
0.3786
ⅢandⅥ
63
40.72(0.95,22511.89)
3020.8
<0.0001
0.3325 1
Table 6 The levels (2-ΔCt)of miR-421 between different groups 10
aP represents the comparison of Pre between GC with Ⅰ,Ⅱ,ⅢandⅥ, respectively. 11
bP represents the comparison of Ⅰ between GC withⅡ,ⅢandⅥ, respectively 10
aP represents the comparison of Pre between GC with Ⅰ,Ⅱ,ⅢandⅥ, respectively. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Table 7(on next page) Table 7(on next page) Table 7(on next page) The relationships between the expression levels of miR-421(2
−ΔCt) in plasma and
clinicopathological factors of patients with precancerous gastric lesions and gastric
cancer PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1
Table 7 The relationships between the expression levels of miR-421(2−ΔCt) in plasma and
2
clinicopathological factors of patients with precancerous gastric lesions and gastric cancer
3
4
miR-421
level (2−ΔCt)
Characteristics
Case No. High
Low
χ2 test
P
value
GC Total cases
90
45
45
Age(years)
<60
≥60
30
60
11
34
19
26
3.2
0.0736
Gender
Male
Female
68
22
32
13
36
9
0.9626
0.3265
TNM stage
Ⅰ
Ⅱ
Ⅲ
Ⅵ
16
9
23
40
11
5
12
17
5
4
11
23
3.261
0.3531
Diferentiation degree
High and moderate
Poor
25
46
11
24
14
22
0.433
0.5105
Histological type
Adenocarcinoma(A)
Mucinous carcinoma(M)
Signet ring cell carcinoma(S)
A with S
65
2
5
11
31
0
3
6
34
2
2
5
2.324
0.5079
Pre Total cases
89
45
44
Age(years)
<60
≥60
41
48
22
23
19
25
0.2916
0.5892
Gender
Male
Female
46
43
22
23
24
20
0.2851
0.5934
Histological type
Intestinal metaplasia
Atypical hyperplasia and other type
82
7
41
4
41
3
0.1316
0.6939
HP infection status
HP (-)
HP (+)
38
16
20
6
18
10
1.033
0.3095
Manuscript to Table 7 The relationships between the expression levels of miR-421(2−ΔCt) in plasma and
2
clinicopathological factors of patients with precancerous gastric lesions and gastric cancer 1
Table 7 The relationships between the expression levels of miR-421(2−ΔCt) in plas PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019)
|
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.